Cognitive performance in relation to MIND and MEPA III dietary pattern accordance of NHANES participants.

BACKGROUND: There is growing evidence that Mediterranean-DASH Intervention for Neurodegenerative Delay (MIND) and Mediterranean-like diets are associated with better cognitive performance. METHODS: In this cross-sectional sample from two NHANES cycles (2011-2014), scores for the MIND dietary pattern (maximum score = 14) and for the Mediterranean Eating Pattern for Americans (MEPA) III (maximum score = 22) were calculated based on the reported foods consumed on two nonconsecutive 24-h dietary recalls. Only adults with two completed recalls and cognitive testing were studied (n = 2598). Cognitive assessments included the word learning and recall components from the Consortium to Establish a Registry for Alzheimer's Disease (CERAD), Animal Fluency test (AFT) and Digit Symbol Substitution Test. RESULTS: The ages of participants were (mean ± SD) 69.2 ± 0.3 years, with almost equal proportions of men and women. MIND score was 5.0 ± 0.0, and MEPA III score was 8.6 ± 2.1. Positive associations between continuous MIND scores and education-dependent standardised cognitive scores for each test and global cognition were observed, unadjusted or adjusted for covariates; no such associations were observed for MEPA III. Compared to adults in the lowest MIND tertile, those in the highest were less likely to exhibit low cognitive performance on the AFT [0.45 (0.29-0.69)], CERAD Delayed Recall [0.52 (0.32-0.83)] and global cognition [0.50 (0.27-0.94)]. Similar observations were noted with MEPA III with AFT [0.58 (0.43-0.79)] and CERAD Delayed Recall [0.66 (0.46-0.94)]. CONCLUSIONS: Older Americans were generally non-accordant to MIND and MEPA III patterns. However, those who reported greater MIND or MEPA III diet accordance exhibited better cognitive performance.

Meldrum's acid derivates are MepA efflux pump inhibitors: In vitro and in silico essays.

Efflux pumps are proteins capable of expelling antibiotics from bacterial cells, have emerged as a major mechanism of bacterial resistance. In the ongoing pursuit to overcome and reduce bacterial resistance, novel substances are being explored as potential efflux pump inhibitors. Meldrum's acid, a synthetic molecule widely studied for its role in synthesizing bioactive compounds, holds promise in this regard. Therefore, the objective of this study is to evaluate the antibacterial activity of three derivatives of Meldrum's acid and assess their ability to inhibit efflux mechanisms, employing both in silico and in vitro approaches. The antibacterial activity of the derivatives was assessed using a broth microdilution testing method. Surprisingly, the derivatives did not exhibit direct antibacterial activity on their own. However, they displayed a significant effect in enhancing the efficacy of antibiotics, suggesting a potential role in potentiating their effects. Furthermore, fluorescence emission assays using ethidium bromide indicated that the derivatives could potentially block efflux pumps, as they exhibited fluorescence levels comparable to the positive control. To further investigate their inhibitory capacity, molecular docking studies were conducted in silico, revealing binding interactions similar to ciprofloxacin and carbonyl cyanide 3-chlorophenylhydrazone, known efflux pump inhibitors. These findings highlight the potential of Meldrum's acid derivatives as effective inhibitors of efflux pumps. By targeting these mechanisms, the derivatives offer a promising avenue to enhance the effectiveness of antibiotics and combat bacterial resistance. This study underscores the importance of exploring novel strategies in the fight against bacterial resistance and provides valuable insights into the potential of Meldrum's acid derivatives as efflux pump inhibitors. Further research and exploration in this field are warranted to fully exploit their therapeutic potential.

In vitro and in silico evidences about the inhibition of MepA efflux pump by coumarin derivatives.

The discovery of antibiotics has significantly transformed the outcomes of bacterial infections in the last decades. However, the development of antibiotic resistance mechanisms has allowed an increasing number of bacterial strains to overcome the action of antibiotics, decreasing their effectiveness against infections they were developed to treat. This study aimed to evaluate the antibacterial activity of synthetic coumarins Staphylococcus aureus in vitro and analyze their interaction with the MepA efflux pump in silico. The Minimum Inhibitory Concentration (MIC) determination showed that none of the test compounds have antibacterial activity. However, all coumarin derivatives decreased the MIC of the standard efflux inhibitor ethidium bromide, indicating antibacterial synergism. On the other hand, the C14 derivative potentiated the antibacterial activity of ciprofloxacin against the resistant strain. In silico analysis showed that C9, C11, and C13 coumarins showed the most favorable interaction with the MepA efflux pump. Nevertheless, due to the present in silico and in vitro investigation limitations, further experimental research is required to confirm the therapeutic potential of these compounds in vivo.

Staphylococcus aureus Efflux Pumps and Tolerance to Ciprofloxacin and Chlorhexidine following Induction by Mupirocin.

Mupirocin induced expression of genes encoding efflux pumps NorA and MepA as well as a yellow fluorescent protein (YFP) fluorescence reporter of NorA. Mupirocin exposure also produced reduced susceptibility to pump substrates ciprofloxacin and chlorhexidine, a change that was dependent on intact norA and mepA, respectively.

Antibacterial and antibiotic modifying activity of chalcone (2E)-1-(4'-aminophenyl)-3-(4-methoxyphenyl)-prop-2-en-1-one in strains of Staphylococcus aureus carrying NorA and MepA efflux pumps: In vitro and in silico approaches.

A large number of infections are caused by multi-resistant bacteria worldwide, increasing to around 700,000 deaths per year. Because of that, many strategies are being developed to combat the resistance of microorganisms to drugs, and recently, chalcones have been studied for this purpose. Chalcones are known as α, ß-unsaturated ketones, characterized by having the presence of two aromatic rings that are joined by a three-carbon chain. They are a class of compounds considered an exceptional model due to chemical simplicity and a wide variety of biological activities, including anticancer, anti-inflammatory, antioxidants, antimicrobials, anti-tuberculosis, anti-HIV, antimalarial, anti-allergic, antifungal, antibacterial, and antileishmaniasis. The objective of this work was to evaluate the antibacterial and antibiotic modifying activity of chalcone (2E)-1-(4'-aminophenyl)-3-(4-methoxyphenyl)-prop-2-en-1-one against the bacteria Staphylococcus aureus carrying a NorA and MepA efflux pump. The results showed that chalcone showed no toxicity on macrophage cells and was able to synergistically modulate the action of Norfloxacin and Ethidium Bromide against the bacteria Staphylococcus aureus 1199B and K2068, respectively. Furthermore, the theoretical physicochemical and pharmacokinetic properties of chalcone showed that it did not present a severe risk of toxicity such as genetic mutation or cardiotoxicity, constituting an excellent pharmacological active ingredient.

In vitro and in silico studies of chalcones derived from natural acetophenone inhibitors of NorA and MepA multidrug efflux pumps in Staphylococcus aureus.

Bacterial resistance induced by efflux pumps is a frequent concern in clinical treatments involving multi-resistant bacteria. Staphylococcus aureus is a microorganism responsible for several types of infections and has several strains carrying efflux pumps, among them are the strain 1199B (NorA overexpresser), and the strain K2068 (MepA overexpresser). In this work, four chalcones derived from Croton anisodontus with modifications in the B ring in their structures were tested regarding their ability to inhibit NorA and MepA efflux pumps. The efflux pump inhibition mechanism was tested with the ethidium bromide substrate in the presence and absence of standard efflux pump inhibitors. The minimum inhibitory concentration values were also compared to those of strains that do not overexpress these efflux pumps. In order to gain some insights about the efflux pump mechanisms of these chalcones, two homology models were created (NorA and MepA) for a docking procedure. In addition, the ADME properties (absorption, distribution, metabolism and excretion) were also evaluated. The tested chalcones promoted synergism of the norfloxacin antibiotic by inhibiting associated efflux pumps. All four tested chalcones appear to bind to the binding sites of the efflux pump models in the same fashion as other chalcones with efflux pump inhibition capabilities. It was also verified that the chalcones 1-4 are well absorbed in the intestine, but with a decrease in their bioavailability, resulting in a low volume of distribution in the blood plasma, in addition to having a mild CNS activity. However, the chalcone 3 and 4 were not toxic due to metabolic activation. Whereas the chalcones 1 and 2 present a mutagenic risk, depending on the oral dose administered. The tested chalcones have not antibacterial activity; however, they are capable of inhibiting efflux pumps for the 1199B and K2068 strains. They promoted synergism of the norfloxacin antibiotic by inhibiting associated efflux pumps, as well as other associated mechanisms.

Mutations at the Ribosomal S10 Gene in Clinical Strains of Staphylococcus aureus with Reduced Susceptibility to Tigecycline.

Mutations on the tip of the extended loop of the ribosomal S10 protein have been associated to tigecycline (TGC) resistance in passaged mutants of different bacteria species. This study described the first two clinical TGC-resistant Staphylococcus aureus isolates with these mutations. One strain (TGC MIC = 2 mg/liter) had a 12-nucleotide deletion affecting residues 56 to 59 (HKYK) of the S10 protein. The second strain (TGC MIC = 1 mg/liter) had amino acid substitutions (K57M and Y58F) previously described in S. aureus passaged mutants.

Thiazine-derived compounds in inhibiting efflux pump in Staphylococcus aureus K2068, mepA gene expression, and membrane permeability alteration.

Staphylococcus aureus is a bacterium responsible for resistance to multiple drugs and the efflux system is widely studied among the resistance mechanisms developed by this species. The present study evaluates the inhibition of the MepA efflux pump by thiadiazine-derived compounds. For this purpose, thiadiazine-derived compounds (IJ-14 to IJ-20) were tested against S. aureus K2068 strains. Microdilution tests were initially conducted to assess the Minimum Inhibitory Concentration (MIC) of the compounds and their efflux pump inhibition activity. In addition, fluorimetry tests were performed using BrEt emission and tests were conducted to inhibit the expression of the mepA gene. This involved comparing the bacterial gene expression with the antibiotic alone to the gene expression after combining compounds (IJ-17 and IJ-20) with the antibiotic. Furthermore, membrane permeability assessment tests and in silico molecular docking tests were performed. It was observed that the IJ17 and IJ20 compounds exhibited direct activity against the tested strain. The IJ17 compound produced significant results in the gene inhibition tests, which was also evidenced through the membrane permeability alteration test. These findings suggest that thiadiazine-derived compounds have promising effects against one of the main resistance mechanisms, with the IJ17 compound presenting observable mechanisms of action.

The murein endopeptidase MepA regulated by MtrAB and MprAB participate in cell wall homeostasis.

The complete genome of Corynebacterium glutamicum contain a gene encoding murein endopeptidase MepA which maintain cell wall homeostasis by regulating peptidoglycan biosynthesis. In this study, we investigate the physiological function, localization and regulator of MepA. The result shows that mepA overexpression lead to peptidoglycan degradation and the defects in cell division. MepA-EGFP was shown to localizes exclusively at the cell cell septum. In addition, mepA overexpression increased cell permeability and reduced the resistance of cells to isoniazid, an antibiotic used to treat Mycobacterium tuberculosis infection. Furthermore, transcription analysis showed that mepA affected cell division and membrane transport pathways, and was coordinately regulated by the two-component systems MtrAB and MprAB(CgtS/R2).

Tigecycline Resistance-Associated Mutations in the MepA Efflux Pump in Staphylococcus aureus.

Tigecycline is an important antibacterial drug for treating infection by clinical multidrug-resistant bacteria, and tigecycline-resistant Staphylococcus aureus (TRSA) has been increasingly reported in recent years. Notably, only rpsJ and mepA are associated with the tigecycline resistance of S. aureus. The mepA gene encodes MepA efflux pumps, and the overexpression of mepA has been confirmed to be directly related to tigecycline resistance. Although the mutations of MepA widely occur, the associations between TRSA and mutations of MepA are still unclear. In this study, we explored mutations in the mepA genes from various sources. Then, tigecycline resistance-associated mutations T29I, E287G, and T29I+E287G in MepA were identified, and their effects were evaluated through mutant deletion and complementation, tigecycline accumulation assay, and molecular docking experiments. Results showed that the MICs of tigecycline, gentamicin, and amikacin increased in special complementary transformants and recovered after the addition of the efflux pump inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP). The tigecycline accumulation assay of the mepA-deleted mutant strain and its complementary transformants showed that T29I, E287G, and T29I+E287G mutations promoted tigecycline efflux, and molecular docking showed that mutations T29I, E287G, and T29I+E287G decreased the binding energy and contributed to ligand binding. Moreover, we inferred the evolutionary trajectory of S. aureus under the selective pressure of tigecycline in vitro. Overall, our study indicated that mutations in MepA play important roles in tigecycline resistance in S. aureus. IMPORTANCE Previous analysis has shown that overexpression of MepA is an exact mechanism involved in tigecycline resistance apart from the rpsJ mutation and is usually dependent on the mutant mepR. However, no research has evaluated the effects of diverse mutations discovered in TRSA in MepA. This study demonstrates that the mutations in MepA confer resistance to tigecycline without overexpression and provides genotypic references for identifying TRSA. Although tigecycline resistance-associated mutations in MepA identified in this study have not been observed in clinical isolates, the mechanism should be explored given that S. aureus strains are prevalent in the environment. Measures should be implemented to contain TRSA within the time window before tigecycline resistance-associated mutations in MepA are prevalent.

Genetic changes associated with tigecycline resistance in Staphylococcus aureus in vitro-selected mutants belonging to different lineages.

Tigecycline (TGC) resistance remains rare in Staphylococcus aureus worldwide. In this study, 12 TGC-resistant S. aureus mutants (TRSAm) were obtained displaying an increase in efflux activity. The isolates belonged to seven different genetic lineages, with a predominance of clonal complex 5 (CC5). Diverse genetic changes in mepA and mepR genes were found producing alterations in the amino acid sequences of the corresponding proteins (MepA and MepR, respectively). The most frequent amino acid change in MepA was Glu287Gly. All of the TRSAm exhibited different single nucleotide polymorphisms (SNPs) or insertions/deletions (InDels) in mepR causing premature stop codons or amino acid changes in MepR. Expression of mepA was significantly increased in TRSAm with different mutations in mepA and mepR. Of the 12 TRSAm, 6 also harboured mutations in rpsJ that resulted in amino acid changes in the S10 ribosomal protein, with Lys57 being the most frequently mutated site. Our findings demonstrate that these acquired mechanisms of TGC resistance are not restricted to a single type of genotypic background and that different lineages might have the same plasticity to develop TGC resistance. The impact of TGC selective pressure assessed by whole-genome sequencing in four selected strain pairs revealed mutations in other singular genes and IS256 mobilisation.

Chemical synthesis, molecular docking and MepA efflux pump inhibitory effect by 1,8-naphthyridines sulfonamides.

This study aimed to evaluate the antibacterial activity and to verify, in silico and in vitro, the inhibition of efflux mechanisms using a series of synthesized 1,8-naphthyridines sulfonamides against Staphylococcus aureus strains carrying MepA efflux pumps. The chemical synthesis occurred through the thermolysis of the Meldrum's acid adduct. The sulfonamide derivatives were obtained by the sulfonylation of 2-amino-5­chloro-1,8-naphthyridine with commercial benzenesulfonyl chloride. Antibacterial activity was assessed by the broth microdilution test. Efflux pump inhibitory capacity was evaluated in silico by molecular docking and in vitro by analyzing synergistic effects on ciprofloxacin and ethidium bromide (EtBr) and by EtBr fluorescence emission assays. The following 1,8-naphthyridines were synthesized: 4-methyl-N-(5­chloro-1,8-naphthyridin-2-yl)-benzenesulfonamide (Compound 10a); 2,5-dichloro-N-(5­chloro-1,8-naphthyridin-2-yl)-benzenesulfonamide (Compound 10b); 4-fluoro-N-(5­chloro-1,8-naphthyridin-2-yl)-benzenesulfonamide (Compound 10c); 2,3,4-trifluoro-N-(5­chloro-1,8-naphthyridin-2-yl)-benzenesulfonamide (Compound 10d); 3-trifluoromethyl-N-(5­chloro-1,8-naphthyridin-2-yl)-benzenesulfonamide (Compound 10e); 4­bromo-2,5-difluoro-N-(5­chloro-1,8-naphthyridin-2-yl)-benzenesulfonamide (Compound 10f). The 1,8-naphthyridines derivatives associated with sulfonamides did not show antibacterial activity. However, they showed a favorable pharmacokinetic profile with possible MepA efflux pump inhibitory action, demonstrated in molecular docking. In addition to the promising results in reducing the concentration of intracellular EtBr. 1,8-naphthyridines act as putative agents in the inhibitory action of the MepA efflux pump.

Mutations in the MepRAB efflux system contribute to the in vitro development of tigecycline resistance in Staphylococcus aureus.

OBJECTIVE: To characterize the evolutionary pathways of tigecycline (TGC) resistance and alterations in the biological characteristics of hospital-derived Staphylococcus aureus isolates under selective pressure. METHODS: Three clinical S. aureus strains and one standard S. aureus strain, ATCC 29213, were used for the in vitro selection of TGC-resistant S. aureus variants with gradient concentrations of TGC. Changes in drug resistance and genetic alterations in resistance-related genes (operon mepRAB and rpsJ) in mutant strains were determined. The efflux inhibitor assay for MepA and the fitness cost, determined by comparing the growth and virulence of parental and mutant strains, were also investigated. RESULTS: Mutants induced in vitro showed a 64- to 128-fold increase in the minimum inhibitory concentration (MIC) of TGC. Substitution mutations were detected in the transcriptional repressor mepR and the efflux pump gene mepA. A K57M amino acid substitution occurred in the ribosomal S10 protein-encoding gene rpsJ. The MICs of TGC in the final mutants were significantly decreased in the presence of efflux pump inhibitors. It was worth noting that growth was unaffected by TGC resistance selection in vitro, with the exception of one strain, and the MICs of other antibiotics and virulence were also unaffected. CONCLUSIONS: The evolution of TGC resistance in S. aureus in vitro is associated with a loss-of-function mutation in the efflux pump transcriptional repressor mepR and a missense mutation in the efflux pump-encoding gene mepA. Our work further validated the resistance mechanisms of S. aureus to TGC and reported previously undiscovered mutations.

Virulence factors and antimicrobial resistance in Staphylococcus aureus isolated from bovine mastitis in Brazil.

This study aimed to evaluate virulence factors and genetic markers of antimicrobial resistance in 400 Staphylococcus aureus strains isolated from bovine mastitis in four Brazilian states, as well as to assess the association between these characteristics and field information. Virulence factors and drug resistance genes were identified by PCR screening. Biofilm-forming and hemolytic phenotype were detected using Congo red Tryptic Soy Broth and defibrinated sheep blood agar, respectively. Of all isolates, 83.5% were biofilm-forming and 98.5% strains exhibited biofilm gene icaAD, and a significant association between phenotype and genotype for biofilm was observed (P = 0.0005). Hemolysin genes were observed in 82.85% (hla+hlb+), 16.5% (hla+) and 0.75% (hlb+) isolates, whereas the hemolytic phenotype exhibited was complete and incomplete hemolysis in 64.25%, complete in 28.25%, incomplete in 4.75%, and negative in 2.75% of the strains. Virulence factors genes luk, seb, sec, sed, and tst were observed in 3.5%, 0.5%, 1%, 0.25%, and 0.74% isolates, respectively. The gene blaZ was detected in 82.03% of penicillin-resistant isolates, whereas tetK and aac(6')-Ie-aph(2')-Ia were observed in 33.87% and 45.15% of the tetracycline and aminoglycosides-resistant isolates, respectively. Fluoroquinolone resistance gene mepA was detected for the first time in S. aureus from bovine mastitis. Resistance genes tetM (3.22%), tetL (1.61%), ermA (14.29%), ermB (14.29%), ermC (33.3%), ermT (9.52%), ermY (4.76%), msrA (9.52%), and mphC (9.52%) were also detected among resistant isolates. No association between virulence factors or antimicrobial-resistant genes and year of isolation, geographic origin, or antimicrobial resistance profile was observed. Our results showed that S. aureus strains isolated from bovine mastitis in the four Brazilian states sampled are mainly biofilm-forming and hemolytic, whereas virulence genes associated with enterotoxins, luk and tst, were less frequently observed. Moreover, a wide variety of resistance genes that confer resistance to almost all classes of antimicrobial agents approved for use in animals and humans were found. Overall, the data point to a great pathogenic potential of S. aureus associated with bovine mastitis and to the non-negligible risks to public health of staphylococcal infections from animal origin.

Resistance in In Vitro Selected Tigecycline-Resistant Methicillin-Resistant Staphylococcus aureus Sequence Type 5 Is Driven by Mutations in mepR and mepA Genes.

A tigecycline-susceptible (TGC-S) Sequence Type (ST) 5 clinical methicillin-resistant Staphylococcus aureus (MRSA) strain was cultured in escalating levels of tigecycline, yielding mutants eightfold more resistant. Their genomes were sequenced to identify genetic alterations, resulting in resistance. Alterations in rpsJ, commonly related to tigecycline resistance, were also investigated. Tigecycline resistance was mediated by loss-of-function mutations in the transcriptional repressor mepR, resulting in derepression of the efflux pump mepA. Increased levels of resistance were obtained by successive mutations in mepA itself. No alterations in RpsJ were observed in selected strains, but we observed a K57M substitution, previously correlated with resistance, among TGC-S clinical strains. Thus, the pathway to tigecycline resistance in CC5 MRSA in vitro appears to be derepression of mep operon as the result of mepR loss-of-function mutation, followed by alterations in MepA efflux pump. This shows that other evolutionary pathways, besides mutation of rpsJ, are available for evolving tigecycline resistance in CC5 MRSA.