Population Pharmacokinetic Modeling of Free Plasma and Free Brain Concentrations of Ceftaroline in Healthy and Methicillin-Resistant Staphylococcus aureus-Infected Wistar Rats.

A population pharmacokinetic model was developed to describe alterations in ceftaroline brain disposition caused by meningitis in healthy and methicillin-resistant Staphylococcus aureus (MRSA)-infected rats. Blood and brain microdialysate samples were obtained after a single bolus dose of ceftaroline fosamil (20 mg/kg) administered intravenously. Plasma data were modeled as one compartment, and brain data were added to the model as a second compartment, with bidirectional drug transport between plasma and brain (Qin and Qout). The cardiac output (CO) of the animals showed a significant correlation with the relative recovery (RR) of plasma microdialysis probes, with animals with greater CO presenting lower RR values. The Qin was approximately 60% higher in infected animals, leading to greater brain exposure to ceftaroline. Ceftaroline brain penetration was influenced by MRSA infection, increasing from 17% (Qin/Qout) in healthy animals to 27% in infected animals. Simulations of a 2-h intravenous infusion of 50 mg/kg every 8 h achieved >90% probability of target attainment (PTA) in plasma and brain for the modal MRSA MIC (0.25 mg/L), suggesting that the drug should be considered an option for treating central nervous system infections.

Lisdexamfetamine and amphetamine pharmacokinetics in oral fluid, plasma, and urine after controlled oral administration of lisdexamfetamine.

Lisdexamfetamine (LDX) is a long-acting prodrug stimulant indicated for the treatment of attention-deficit/hyperactivity disorder (ADHD) and binge-eating disorder (BED) symptoms. In vivo hydrolysis of the LDX amide bond releases the therapeutically active d-amphetamine (d-AMPH). This study aims to describe the pharmacokinetics of LDX and its major metabolite d-AMPH in human oral fluid, urine and plasma after a single 70 mg oral dose of LDX dimesylate. Six volunteers participated in the study. Oral fluid and blood samples were collected for up to 72 h and urine for up to 120 h post-drug administration for the pharmacokinetic evaluation of intact LDX and d-AMPH. Samples were analyzed by LC-MS/MS. Regarding noncompartmental analysis, d-AMPH reached the maximum concentration at 3.8 and 4 h post-administration in plasma and oral fluid, respectively, with a mean peak concentration value almost six-fold higher in oral fluid. LDX reached maximum concentration at 1.2 and 1.8 h post-administration in plasma and oral fluid, respectively, with a mean peak concentration value almost three-fold higher in plasma. Intact LDX and d-AMPH were detected in the three matrices. The best fit of compartmental analysis was found in the one-compartment model for both analytes in plasma and oral fluid. There was a correlation between oral fluid and plasma d-AMPH concentrations and between parent to metabolite concentration ratios over time in plasma as well as in oral fluid.

Quantification of pyrrolizidine alkaloids in Senecio brasiliensis, beehive pollen, and honey by LC-MS/MS.

This article presents the determination of eight pyrrolizidine alkaloids (PAs) by LC-MS/MS in honeys, pollen, and Senecio brasiliensis (Asteraceae) samples, all from Santa Catarina state, Brazil. In addition, the Box-Behnken design was used to perform an optimized sample preparation on pollens and S. brasiliensis parts. Senecionine and its N-oxide, besides retrorsine N-oxide, were determined in six of the seven honeys samples. Pollen from species of the Asteraceae, Fabaceae, and Boraginaceae families were found with greater predominance in three of the seven honeys samples. In these three honeys samples were also found the highest PAs levels. In beehive pollen, flower, and leaf of S. brasiliensis, the total levels of PAs and their N-oxides reached 221, 14.1 × 104, and 14.8 × 104 mg kg-1, respectively. In honeys, these compounds are chemical contaminants and therefore undesirable when the sum exceeds 71 µg kg-1, according to EFSA. On the other hand, although PAs are naturally present in plant and pollen of some species (Senecio, Crotalaria, Bacharis, Ecchium, Mimosa scabrella, Vernonia), it is important to monitor their levels in plants but also in honeys, and other beehive products since these compounds are transferred to the final product.

A liquid chromatography coupled to quadrupole-time-of-flight-mass spectrometry (LC-QTOF-MS) method for identification analysis of saponins from Quillaja saponaria bark extracts in foot-and-mouth disease vaccines: Development, validation and applicability.

Saponins from Quillaja saponaria have been commonly used as immunomodulatory adjuvants in foot-and-mouth disease vaccines (FMDVs). However, due to the lack of consensus over the possible exacerbation of local inflammatory responses in cattle and its economic impacts, their use has been discouraged by Brazilian authorities. A qualitative method intended to determine the presence of saponins from Q. saponaria bark extracts in FMDVs was developed and validated. Instrumental analysis was performed using an liquid chromatography (LC) coupled to a quadrupole-time-of-flight-mass spectrometry (TOF-MS) system. The method was validated according to the International Conference on Harmonization Harmonized Tripartite Guideline Q2 (R1) and Brazilian Ministry of Agriculture, Livestock and Food Supply Analytical Quality Assurance Guidelines. Validation parameters were determined and considered suitable to the established criteria. The validated method has been applied in routine analysis in the National Agricultural Laboratory at Rio Grande do Sul (LANAGRO-RS). All results obtained were in agreement with the vaccine's composition described by the manufacturer. The method is easy and adequate for analysis in routine laboratories. To the best of the authors' knowledge, this is the first report of a method which intends to investigate the presence of saponins from Q. saponaria bark extracts in veterinary vaccines.

The effect of breed, sex, and drug concentration on the pharmacokinetic profile of ivermectin in cattle.

Ivermectin (IVM) is one of the most widely used antiparasitic drugs worldwide and has become the drug of choice for anthelmintic and tick treatment in beef cattle production. It is known that pharmacokinetic parameters are fundamental to the rational use of a drug and food safety and these parameters are influenced by different factors. The aim of this study was to evaluate the pharmacokinetic profile of IVM in Bos indicus, Bos taurus, and crossbreed cattle (B. indicus × B. taurus) kept under same field conditions and the possible impacts of sex and IVM formulation (1% and 3.15%). It was observed that IVM concentration was significantly affected by breed. The plasma concentrations of IVM, AUC, Cmax , and t1/2ß were significantly higher in B. indicus compared to B. taurus. Crossbreed animals showed an intermediate profile between European and Indian cattle. No alteration in pharmacokinetics parameters was detected when comparing different gender. Concerning the pharmacokinetic data of IVM formulation, it was verified that Tmax , AUC, and t1/2ß were higher in 3.15% IVM animals than those from 1% IVM formulation. The results clearly indicated that the IVM plasma concentrations in B. indicus were higher than that in B. taurus.

Retention of vitamin A after goat milk processing into cheese: a nutritional strategy.

The deficiency in micronutrients is a public health problem, principally in lower-middle-income countries. Vitamin A (VA) is considered a micronutrient fundamental to the maintenance and development of different tissues in the organism. Therefore, it is an essential micronutrient in the human diet. In these terms, goat milk is the leading food consumed to provide nutritional support in innumerous lower-middle-income countries. Here our work aimed to produce goat cheese studying strategies to promote the retention of VA. Our experiment design also explores the use of the salting process to evaluate the levels of VA retention. The level of VA in goat cheese was determined using LC-MS/MS analysis. Additionally, the redox status of the goat cheese in terms of lipid peroxidation and protein oxidation was determined. The texture analysis was also evaluated to verify if the redox status and salting process influence the texture profile. The results showed that the salting process during goat cheese production improves the retention of VA in goat cheese. Moreover, the salting process also is related to alterations in the status redox of the goat cheese and texture parameters. Therefore, our results show that goat cheese production can be an alternative to produced dairy derivates with recognized concentrations of VA for human nutrition.

Effects of methylmercury and retinol palmitate co-administration in rats during pregnancy and breastfeeding: Metabolic and redox parameters in dams and their offspring.

Ubiquitous low-dose methylmercury (MeHg) exposure through an increased fish consumption represents a global public health problem, especially among pregnant women. A plethora of micronutrients presented in fish affects MeHg uptake/distribution, but limited data is available. Vitamin A (VitA), another fish micronutrient is used in nutritional supplementation, especially during pregnancy. However, there is no information about the health effects arising from their combined exposure. Therefore, the present study aimed to examine the effects of both MeHg and retinyl palmitate administered on pregnant and lactating rats in metabolic and redox parameters from dams and their offspring. Thirty Wistar female rats were orally supplemented with MeHg (0,5 mg/kg/day) and retinyl palmitate (7500 µg RAE/kg/day) via gavage, either individually or in combination from the gestational day 0 to weaning. For dams (150 days old) and their offspring (31 days old), glycogen accumulation (hepatic and cardiac) and retinoid contents (plasma and liver) were analyzed. Hg deposition in liver tissue was quantified. Redox parameters (liver, kidney, and heart) were evaluated for both animals. Cytogenetic damage was analyzed with micronucleus test. Our results showed no general toxic or metabolic alterations in dams and their offspring by MeHg-VitA co-administration during pregnancy and lactation. However, increased lipoperoxidation in maternal liver and a disrupted pro-oxidant response in the heart of male pups was encountered, with apparently no particular effects in the antioxidant response in female offspring. GST activity in dam kidney was altered leading to possible redox disruption of this tissue with no alterations in offspring. Finally, the genomic damage was exacerbated in both male and female pups. In conclusion, low-dose MeHg exposure and retinyl palmitate supplementation during gestation and lactation produced a potentiated pro-oxidant effect, which was tissue-specific. Although this is a pre-clinical approach, we recommend precaution for pregnant women regarding food consumption, and we encourage more epidemiological studies to assess possible modulations effects of MeHg-VitA co-administration at safe or inadvertently used doses in humans, which may be related to specific pathologies in mothers and their children.

Method validation and determination of lisdexamfetamine and amphetamine in oral fluid, plasma and urine by LC-MS/MS.

Lisdexamfetamine (LDX) is a long-acting prodrug stimulant indicated for the treatment of attention-deficit/hyperactivity disorder and binge-eating disorder symptoms. In vivo hydrolysis of LDX amide bond releases the therapeutically active d-amphetamine (d-AMPH). Since toxicological tests in biological samples can detect AMPH from the use of some legal medications, efficient methods are needed in order to correctly interpret the results. The aim of this study was to develop and validate an LC-MS/MS method for the simultaneous quantification of LDX and its main biotransformation product AMPH in human oral fluid, plasma and urine. Calibration curve range for both analytes was 1-128 ng/mL in oral fluid and plasma and 4-256 ng/mL in urine, being the lowest concentration the limit of quantification. Accuracy of the determined values of the target analytes for the five control levels ranged from 94.8 to 111.7% for oral fluid, from 91.3 to 100.2% for plasma and from 94.8 to 109.8% for urine. Imprecision for the five control levels did not exceeded 12.8% for oral fluid, 16.2% for plasma and 17.1% for urine. The method developed for the three matrices was validated and was also successfully applied to assess real samples, showing for the first time the detection of LDX in oral fluid.

Methodology for trace analysis of 17 pyrethroids and chlorpyrifos in foodstuff by gas chromatography-tandem mass spectrometry.

This study aimed to develop an efficient, sensitive, and reliable analytical method for trace analysis of 17 different pyrethroids and chlorpyrifos in the fatty content of animal products, including beef, chicken, eggs, fish, and milk. The method developed is based on an ultrasound extraction using lyophilized samples, a solid phase extraction cleanup with basic alumina and C18 cartridges in tandem, and analysis by gas chromatography coupled to tandem mass spectrometry in negative chemical ionization mode. Recovery values were in the range of 27-128 % with relative standard deviation always below 25 %, and chiral analysis of recovery data showed predominance of isomers of cis form over trans. Limits of detection (LODs) ranged from 0.002 to 6.43 ng g-1 lipid weight (lw), and limits of quantification (LOQs) ranged between 0.006 and 21.4 ng g-1 lw. The developed methodology was used for the analysis of 25 samples of fatty foods. All samples were positive for at least one of the pesticides, chlorpyrifos, bifenthrin, cyhalothrin, permethrin, cypermethrin, or deltamethrin, with mass fraction levels ranging from 0.03 to 270 ng g-1 lw. Graphical Abstract ᅟ.

Commercial propolis extracts from Apis mellifera, Scaptotrigona bipunctata and Melipona quadrifasciata: quality control, antioxidant and antiglycemic potential.

Quality control of propolis plays a pivotal role in ensuring the appropriate concentrations of active compounds, limiting unwanted substances, verifying authenticity, and adhering to regulatory standards. This study aimed to assess the identity and quality standards, the individual phenolic composition (LC-ESI-MS/MS), and the antioxidant and antiglycemic potential of commercial propolis extracts (CPEs) from Apis mellifera, Scaptotrigona bipunctata, and Melipona quadrifasciata bees. CPEs met wax content and oxidation activity criteria, surpassing minimum thresholds for total phenolic content (TPC) and flavonoid content (TFC), although stingless bee CPE did not test positive for 10% lead acetate. CPEs exhibited antioxidant and potential antiglycemic activities. Epicatechin among the thirty-three identified phenolics, showed significant correlation with TPC, DPPH, ABTS, and EC50 values of α-amylase enzyme. These promising attributes underscore the potential health benefits of commercial propolis extracts from Apis mellifera and stingless bees for further medicinal and nutritional applications.

Pharmacokinetic/pharmacodynamic modeling of cortical dopamine concentrations after quetiapine lipid core nanocapsules administration to schizophrenia phenotyped rats.

Schizophrenia (SCZ) response to pharmacological treatment is highly variable. Quetiapine (QTP) administered as QTP lipid core nanocapsules (QLNC) has been shown to modulate drug delivery to the brain of SCZ phenotyped rats (SPR). In the present study, we describe the brain concentration-effect relationship after administrations of QTP as a solution or QLNC to SPR and naïve animals. A semimechanistic pharmacokinetic (PK) model describing free QTP concentrations in the brain was linked to a pharmacodynamic (PD) model to correlate the drug kinetics to changes in dopamine (DA) medial prefrontal cortex extracellular concentrations determined by intracerebral microdialysis. Different structural models were investigated to fit DA concentrations after QTP dosing, and the final model describes the synthesis, release, and elimination of DA using a pool compartment. The results show that nanoparticles increase QTP brain concentrations and DA peak after drug dosing to SPR. To the best of our knowledge, this is the first study that combines microdialysis and PK/PD modeling in a neurodevelopmental model of SCZ to investigate how a nanocarrier can modulate drug PK and PD, contributing to the development of new treatment strategies for SCZ.

Development and validation of an LC-MS/MS method to quantify ceftaroline in microdialysate samples from plasma and brain: Application to a preclinical pharmacokinetic investigation.

A bioanalytical LC-MS/MS method was developed and validated to determine ceftaroline in microdialysate samples from plasma and brain. Ceftaroline was separated using a C18 column and a mobile phase consisting of water and acetonitrile, both with 5 mM of ammonium formate and acid formic 0.1%, eluted as gradient. Ceftaroline was monitored using electrospray ionization operating on positive mode (ESI+) monitoring the transition 604.89 > 209.3 m/z. The method showed linearity in the concentration range of 0.5-500 ng/mL for brain microdialysate and 0.5-2500 ng/mL for plasma microdialysate with coefficients of determination ≥0.997. The inter-and intra-day precision, the accuracy, and the stability of the drug in different conditions were in accordance with the acceptable limits determined by international guidelines. Plasma pharmacokinetics and brain distribution of the drug were carried out after intravenous administration of 20 mg/kg of ceftaroline to male Wistar rats. The estimated geometric mean (geometric coefficient of variation) area under the curve (AUC0-∞) was 4.68 (45.8%) mg·h/L and 1.20 (54.2%) mg·h/L for plasma and brain, respectively, resulting in a brain exposure of about 33% (AUCfree brain/AUCfree plasma). The results indicate that ceftaroline presents good penetration in the brain when considering free plasma and free brain concentrations.

Validation of a MALDI-TOF MS Method for SARS-CoV-2 Detection on the Bruker Biotyper and Nasopharyngeal Swabs: A Brazil-UK Collaborative Study.

We developed a MALDI-TOF mass spectrometry method for the detection of the SARS-CoV-2 virus in saliva-gargle samples using Shimadzu MALDI-TOF mass spectrometers in the UK. This was validated in the USA to CLIA-LDT standards for asymptomatic infection detection remotely via sharing protocols, shipping key reagents, video conferencing, and data exchange. In Brazil, more so than in the UK and USA, there is a need to develop non-PCR-dependent, rapid, and affordable SARS-CoV-2 infection screening tests that also identify variant SARS-CoV-2 and other virus infections. In addition, travel restrictions necessitated remote collaboration with validation on the available clinical MALDI-TOF-the Bruker Biotyper (microflex® LT/SH)-and on nasopharyngeal swab samples, as salivary gargle samples were not available. The Bruker Biotyper was shown to be almost log103 more sensitive at the detection of high molecular weight spike proteins. A protocol for saline swab soaks out was developed, and duplicate swab samples collected in Brazil were analyzed by MALDI-TOF MS. The swab collected sample spectra that varied from that of saliva-gargle in three additional mass peaks in the mass region expected for IgG heavy chains and human serum albumin. A subset of clinical samples with additional high mass, probably spike-related proteins, were also found. Further, spectral data comparisons and analysis, subjected to machine learning algorithms in order to resolve RT-qPCR positive from RT-qPCR negative swab samples, showed 56-62% sensitivity, 87-91% specificity, and a 78% agreement with RT-qPCR scoring for SARS-CoV-2 infection.

Multi-virulence of Campylobacter jejuni carried by chicken meat in Brazil.

Campylobacter jejuni is the most frequent cause of bacterial gastroenteritis; therefore, the characteristics of its epidemiology must be continuously investigated to support possible mitigating measures. This is particularly important when evaluating representative strains from the world's leading chicken meat exporter, Brazil. We evaluated a panel of 14 virulence genes in 359 strains of C. jejuni isolated from chilled broiler carcasses in Brazil. The genes were classified into five virulence categories (B: biofilm/motility; SS: secretion/cytotoxicity system; CI: invasion/colonization; GB: Guillain-Barré; and AE: adaptation to stress). The percentage of strains with stress adaptation genes (86.07%) indicates the ability to survive in unfavorable environments; in addition, the strains showed a risk of causing infections in humans due to the frequency of the hcp gene (97.77%). Genes related to Guillain-Barre syndrome (GBS) in 77.44% of strains are an additional concern, which must be monitored. The gene panel showed the presence of 124 virulence profiles. Individual analyses by carcass, slaughter establishment, and municipalities in which they were located showed high index variabilities (I.Var.) of 0.82, 0.87, and 0.78, respectively. Georeferencing indicated the state of Paraná as a hotspot for virulent strains. Higher levels of isolation and multi-virulence were identified in the summer, which is hot and humid in Brazil. Together, our results showed that the studied strains are a potential danger to public health and that there is an urgent need for their surveillance and the adoption of control measures, especially in the state of Paraná.

Multi-residue methodology for quantification of antiparasitics in hen eggs by LC-MS/MS: development, validation and application to 348 samples from Brazil.

Analytical methodology for quantification of 15 antiparasitic drugs and their respective metabolites in laying hen eggs was optimized and validated. The method uses acetonitrile as solvent extraction, sodium chloride for salting-out, low-temperature purification and analysis by LC-MS/MS. A total of 348 egg samples were collected in 11 states of Brazil and 50% of the total samples presented antiparasitic residues, which were albendazole, fipronil, fenbendazole, ivermectin, oxibendazole and mebendazole. A total of 12.4% of the samples were considered non-compliant, and residues quantified in these samples were albendazole, fipronil, and mebendazole. Albendazole was always identified as albendazole sulfone. Only one sample presented fipronil and fipronil sulfone; all others exclusively the sulfone metabolite. Fenbendazole was characterized by the presence of both metabolites: sulfone and sulfoxide. Maximum limits adopted are based on the Normative Instruction 51/2019 of the Brazilian Health Regulatory Agency (ANVISA), but albendazole, fipronil, oxibendazole, ivermectin, and mebendazole do not have their maximum residue level established. In addition, metabolites of albendazole, fipronil and fenbendazole in eggs are not considered in this Instruction.

Probability of Target Attainment of Tobramycin Treatment in Acute and Chronic Pseudomonas aeruginosa Lung Infection Based on Preclinical Population Pharmacokinetic Modeling.

Biofilms and infectious process may alter free antimicrobial concentrations at the site of infection. Tobramycin (TOB), an aminoglycoside used to treat lung infections caused by Pseudomonas aeruginosa, binds to alginate present in biofilm extracellular matrix increasing its minimum inhibitory concentration (MIC). This work aimed to investigate the impact of biofilm-forming P. aeruginosa infection on TOB lung and epithelial lining fluid (ELF) penetration, using microdialysis, and to develop a population pharmacokinetic (popPK) model to evaluate the probability of therapeutic target attainment of current dosing regimens employed in fibrocystic and non-fibrocystic patients. The popPK model developed has three compartments including the lung. The ELF concentrations were described by a penetration factor derived from the lung compartment. Infection was a covariate in lung volume (V3) and only chronic infection was a covariate in central volume (V1) and total clearance (CL). Simulations of the recommended treatments for acute and chronic infection achieved >90% probability of target attainment (PTA) in the lung with 4.5 mg/kg q24h and 11 mg/kg q24h, respectively, for the most prevalent P. aeruginosa MIC (0.5 mg/mL). The popPK model was successfully applied to evaluate the PTA of current TOB dosing regimens used in the clinic, indicating the need to investigate alternative posology.

Residues of Veterinary Drugs in Animal Products Commercialized in the Border Region of Brazil, Argentina, and Uruguay.

ABSTRACT: The traffic in international animal products can become a public health hazard when legal import sanitary procedures are not followed. In Brazil, due to its extensive border area, the importation of animal products is a common practice in many areas, especially in Rio Grande do Sul, a state that borders Argentina and Uruguay. The objective of this study was to evaluate the presence of veterinary drug residues (antibiotics and antiparasitics) in animal products consumed in Rio Grande do Sul. The presence of residues of veterinary antibiotics and antiparasitics was assessed in 189 meat (beef, pork, and chicken), processed dairy, and meat product samples bought in Argentina (n = 90) and Uruguay (n = 99). Residues of these veterinary drugs were detected in 50 (26.45%) of the samples; 28 samples (14.81%) had antibiotic residues, and 22 samples (11.64%) had antiparasitic residues. Of the 50 positive samples, 40% (15 from Argentina and 5 from Uruguay) had residues above the maximum residue limits (MRLs). Of these 20 samples, 12 had antiparasitic residues above the MRLs (11 beef samples had ivermectin and 1 pork sample had ivermectin and doramectin) and 8 had antibiotic residues above the MRLs (2 pork and 2 sausage samples had doxycycline, 2 cheese samples had doxycycline and chlortetracycline, 1 poultry meat sample had chloramphenicol, and 1 cheese sample had monensin). Because of the potential toxic effects on humans and the potential for pathogens to develop antibiotic resistance, the presence of these residues above the MRLs is a potential risk to public health. The negative impact of consumption of imported animal products can be reduced by implementation of an effective surveillance system and educational campaigns for the general population.

Dermatopharmacokinetic and pharmacodynamic evaluation of a novel nanostructured formulation containing capsaicinoids for treating neuropathic pain.

The in vivo skin penetration by dermal microdialysis and the pharmacological efficacy of a chitosan hydrogel containing capsaicinoids-loaded nanocapsules (CHNCCaps) was evaluated in this study. Such gel has previously been proven to control capsaicinoids release and decrease the drugs side effects in humans. The nanocapsules containing capsaicinoids had an average size around 150 nm, with a low polydispersity index, positive zeta potential, and high encapsulation efficiency of the drugs. The CHNCCaps showed intact nanocapsules, a slightly acid pH value, and a pseudoplastic behavior suitable for topical application. Microdialysis experiments showed a 1.6-fold increase in the concentration of capsaicinoids in the dermis (after 12 h of its application) when CHNCCaps was administered compared to a chitosan hydrogel containing capsaicinoids in hydroethanolic solution (CHETCaps) and the commercial cream. The CHNCCaps showed antiallodynic and antihyperalgesic effects from 6 h to 96 h after treatment initiation, whereas CHETCaps and the commercial cream showed antiallodynic and antihyperalgesic effects only at 48 h and 96 h after treatment initiation, respectively. CHNCCaps and the commercial cream maintained antihyperalgesic activity for 6 days after treatment interruption. For mechanical allodynia, the antinociceptive effect was maintained for 48 h after treatment interruption only with CHNCCaps. In conclusion, CHNCCaps is a promising formulation for treating peripheral neuropathic pain.

Lipoteichoic acid from Staphylococcus aureus increases matrix metalloproteinase 9 expression in RAW 264.7 macrophages: modulation by A2A and A2B adenosine receptors.

Peptidoglycan (PEG) and lipoteichoic acid (LTA) are the main constituents of Gram-positive bacteria cell wall and are described to modulate immune functions. Increased levels of matrix metalloproteinases (MMPs) were described in endotoxemia, suggesting that they participate to tecidual damage, multiple organs failure and vascular disfunction. Staphylococcus aureus PEG is described to increase MMPs 2 and 9 levels in plasma from rat and MMP 9 secretion by human neutrophils, however, the effect of LTA on MMPs is unknown. In this work, was evaluated the modulation of MMPs 2 and 9 expression and secretion in RAW 264.7 macrophages by LTA from S. aureus. The role of A2A and A2B adenosine receptors was also investigated. LTA increased MMP 9 expression and secretion at 12h of treatment. The modulation of MMP 9 secretion was dose dependent, with maximal effect above 1microg/ml. The inhibitor of mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway (U0126, 10microM) prevented LTA stimulation of MMP 9 secretion; however, the inhibitors of p38 (SB203580, 10microM) and Jun N-terminal kinase (JNK; SP600125, 10microM) presented any effect. A2A and A2B adenosine receptors pharmacological blockade or gene knockdown resulted in exacerbated MMP 9 secretion, while an adenosine receptors agonist inhibited LTA-stimulated MMP 9 secretion. These results suggest that LTA increased MMP 9 secretion in macrophages could be involved in complications associated to S. aureus infections. Moreover, LTA modulation of MMP 9 is dependent on MEK/ERK pathway and is regulated by A2A and A2B adenosine receptors.

High-performance method to detection of Klebsiella pneumoniae Carbapenemase in Enterobacterales by LC-MS/MS.

Carbapenem-resistant Enterobacterales (CREs) have been recognized as an important threat to global health. CRE cause the majority of the difficult-to-treat infections in health-care settings and are associated with high mortality. Klebsiella pneumoniae carbapenemase (KPC)-producing CREs, in particular Klebsiella pneumoniae, are globally disseminated and responsible for a large number of outbreaks. Development of rapid methods for KPC detection can provide great clinical and epidemiological benefits to prevent KPC dissemination. The aim of this study was to standardize and validate a LC-MS/MS method to detect KPC. This method was also tested against a broad variety of species, including CRE with other carbapenemase genes and the recently reported mcr-1. For validation, 111 isolates with reduced susceptibility to carbapenems were selected (49 KPC-positive and 62 KPC-negative). The presence of four tryptic peptides related to the KPC enzyme was evaluated, and the identification of at least two of them classified the isolate as "KPC-positive." The LTLGSALAAPQR and LALEGLGVNGQ peptides were both detected in 47 of 49 isolates with the blaKPC gene. The other two peptides, GFLAAAVLAR and APIVLAVYTR, were detected in 46 and 19 isolates with the blaKPC gene, respectively. The method correctly classified 47 of 49 KPC-positive and all KPC-negative isolates yielding 96.07% of sensitivity and 100% of specificity. In conclusion, our results demonstrate that the KPC peptide markers were robustly detected by the method which presented high sensitivity and full specificity and therefore can be used as a reliable method to identify this resistance mechanism.