Sensitizing methicillin-resistant Staphylococcus aureus (MRSA) to cefuroxime: the synergic effect of bicarbonate and the wall teichoic acid inhibitor ticlopidine.

Methicillin-resistant Staphylococcus aureus (MRSA) strains are a major challenge for clinicians due, in part, to their resistance to most ß-lactams, the first-line treatment for methicillin-susceptible S. aureus. A phenotype termed "NaHCO3-responsiveness" has been identified, wherein many clinical MRSA isolates are rendered susceptible to standard-of-care ß-lactams in the presence of physiologically relevant concentrations of NaHCO3, in vitro and ex vivo; moreover, such "NaHCO3-responsive" isolates can be effectively cleared by ß-lactams from target tissues in experimental infective endocarditis (IE). One mechanistic impact of NaHCO3 exposure on NaHCO3-responsive MRSA is to repress WTA synthesis. This NaHCO3 effect mimics the phenotype of tarO-deficient MRSA, including sensitization to the PBP2-targeting ß-lactam, cefuroxime (CFX). Herein, we further investigated the impacts of NaHCO3 exposure on CFX susceptibility in the presence and absence of a WTA synthesis inhibitor, ticlopidine (TCP), in a collection of clinical MRSA isolates from skin and soft tissue infections (SSTI) and bloodstream infections (BSI). NaHCO3 and/or TCP enhanced susceptibility to CFX in vitro, by both minimum inhibitor concentration (MIC) and time-kill assays, as well as in an ex vivo simulated endocarditis vegetations (SEV) model, in NaHCO3-responsive MRSA. Furthermore, in experimental IE (presumably in the presence of endogenous NaHCO3), pre-exposure to TCP prior to infection sensitized the NaHCO3-responsive MRSA strain (but not the non-responsive strain) to enhanced clearances by CFX in target tissues. These data support the notion that NaHCO3 is acting similarly to WTA synthesis inhibitors, and that such inhibitors have potential translational applications in the treatment of certain MRSA strains in conjunction with specific ß-lactam agents.

Genomic investigation and clinical correlates of the in vitro ß-lactam: NaHCO3 responsiveness phenotype among methicillin-resistant Staphylococcus aureus isolates from a randomized clinical trial.

NaHCO3 responsiveness is a novel phenotype where some methicillin-resistant Staphylococcus aureus (MRSA) isolates exhibit significantly lower minimal inhibitory concentrations (MIC) to oxacillin and/or cefazolin in the presence of NaHCO3. NaHCO3 responsiveness correlated with treatment response to ß-lactams in an endocarditis animal model. We investigated whether treatment of NaHCO3-responsive strains with ß-lactams was associated with faster clearance of bacteremia. The CAMERA2 trial (Combination Antibiotics for Methicillin-Resistant Staphylococcus aureus) randomly assigned participants with MRSA bloodstream infections to standard therapy, or to standard therapy plus an anti-staphylococcal ß-lactam (combination therapy). For 117 CAMERA2 MRSA isolates, we determined by broth microdilution the MIC of cefazolin and oxacillin, with and without 44 mM of NaHCO3. Isolates exhibiting ≥4-fold decrease in the MIC to cefazolin or oxacillin in the presence of NaHCO3 were considered "NaHCO3-responsive" to that agent. We compared the rate of persistent bacteremia among participants who had infections caused by NaHCO3-responsive and non-responsive strains, and that were assigned to combination treatment with a ß-lactam. Thirty-one percent (36/117) and 25% (21/85) of MRSA isolates were NaHCO3-responsive to cefazolin and oxacillin, respectively. The NaHCO3-responsive phenotype was significantly associated with sequence type 93, SCCmec type IVa, and mecA alleles with substitutions in positions -7 and -38 in the regulatory region. Among participants treated with a ß-lactam, there was no association between the NaHCO3-responsive phenotype and persistent bacteremia (cefazolin, P = 0.82; oxacillin, P = 0.81). In patients from a randomized clinical trial with MRSA bloodstream infection, isolates with an in vitro ß-lactam-NaHCO3-responsive phenotype were associated with distinctive genetic signatures, but not with a shorter duration of bacteremia among those treated with a ß-lactam.

Impact of Bicarbonate on PBP2a Production, Maturation, and Functionality in Methicillin-Resistant Staphylococcus aureus (MRSA).

Certain methicillin-resistant Staphylococcus aureus (MRSA) strains exhibit ß-lactam-susceptibility in vitro, ex vivo and in vivo in the presence of NaHCO3 (NaHCO3-responsive MRSA). Herein, we investigate the impact of NaHCO3 on factors required for PBP2a functionality. Prototype NaHCO3-responsive and -nonresponsive MRSA strains (as defined in vitro) were assessed for the impact of NaHCO3 on: expression of genes involved in PBP2a production-maturation pathways (mecA, blaZ, pbp4, vraSR, prsA, sigB, and floA); membrane PBP2a and PrsA protein content; and membrane carotenoid content. Following NaHCO3 exposure in NaHCO3-responsive (vs - nonresponsive) MRSA, there was significantly reduced expression of: i) mecA and blaZ; ii) the vraSR-prsA gene axis; and iii) pbp4 Carotenoid production was reduced, while floA expression was increased by NaHCO3 exposure in all MRSA strains. This work underscores the distinct regulatory impact of NaHCO3 on a cadre of genes encoding factors required for maintenance of the MRSA phenotype through PBP2a functionality and maturation.

The NaHCO3-Responsive Phenotype in Methicillin-Resistant Staphylococcus aureus (MRSA) Is Influenced by mecA Genotype.

Methicillin-resistant Staphylococcus aureus (MRSA) strains are a leading cause of many invasive clinical syndromes, and pose treatment difficulties due to their in vitro resistance to most ß-lactams on standard laboratory testing. A novel phenotype frequently identified in MRSA strains, termed 'NaHCO3-responsiveness', is a property whereby strains are susceptible in vitro to many ß-lactams in the presence of NaHCO3. Specific mecA genotypes, repression of mecA/PBP2a expression and perturbed maturation of PBP2a by NaHCO3 have all been associated with this phenotype. The aim of this study was to define the relationship between specific mecA genotypes and PBP2a substitutions, on the one hand, with NaHCO3-responsiveness in vitro. Mutations were made in the mecA ribosomal binding site (RBS -7) and at amino acid position 246 of its coding region in parental strains MW2 (NaHCO3-responsive) and C36 (NaHCO3- nonresponsive) to generate 'swap' variants, each harboring the other's mecA-RBS/coding region genotypes. Successful swaps were confirmed by both sequencing, as well as predicted swap of in vitro penicillin-clavulanate susceptibility phenotypes. MW2 swap variants harboring the nonresponsive mecA genotypes became NaHCO3-nonresponsive (resistant to the ß-lactam, oxacillin [OXA]), in the presence of NaHCO3. Moreover, these swap variants had lost NaHCO3-mediated repression of mecA/PBP2a expression. In contrast, C36 swap variants harboring the NaHCO3-responsive mecA genotypes remained NaHCO3-nonresponsive phenotypically, and still exhibited nonrepressible mecA/PBP2a expression. These data demonstrate that in addition to the mecA genotype, NaHCO3-responsiveness may also depend on strain-specific genetic backgrounds.

Pollutant removal from cheese processing effluent using effective indigenous natural scavengers.

The goal of this study was to come up with an efficient method for treating cheese production wastewater. Because the effluent has a higher concentration of organic and inorganic materials, the indigenous microbial treatment process was used to effectively remove total dissolved solids (TDS), chemical oxygen demand (COD), and color without the addition of any nutrients. The indigenous microorganisms were tested for color, TDS, and COD elimination by growing them in "nutrient broth medium" loaded with different amounts of cheese effluent. The isolates were identified by 16S rRNA sequencing, and the results revealed that strain 1 was Enterobacter cloacae, strain 2 was Lactococcus garvieae, and strains 3 and 4 were Bacillus cereus and Bacillus mycoides, respectively. After 36 h of incubation, the data were evaluated. Among all the microbes, E. cloacae reduced TDS and COD from the effluent the most (80 ± 0.2% and 87 ± 0.4% COD, respectively). When compared to individual species, consortia were more efficient (86 ± 0.2% TDS and 90 ± 0.3% COD). On treatment, the correlation coefficient "r" for TDS and COD elimination was found to be 1, resulting in a positive linear connection. The current study suggests that microbial therapies are both effective and environmentally beneficial.

Ability of Bicarbonate Supplementation To Sensitize Selected Methicillin-Resistant Staphylococcus aureus Strains to ß-Lactam Antibiotics in an Ex Vivo Simulated Endocardial Vegetation Model.

Supplementation of standard growth media (cation-adjusted Mueller-Hinton Broth [CAMHB]) with bicarbonate (NaHCO3) increases ß-lactam susceptibility of selected methicillin-resistant Staphylococcus aureus (MRSA) strains ("NaHCO3 responsive"). This "sensitization" phenomenon translated to enhanced ß-lactam efficacy in a rabbit model of endocarditis. The present study evaluated NaHCO3-mediated ß-lactam MRSA sensitization using an ex vivo pharmacodynamic model, featuring simulated endocardial vegetations (SEVs), to more closely mimic the host microenvironment. Four previously described MRSA strains were used: two each exhibiting in vitro NaHCO3-responsive or NaHCO3-nonresponsive phenotypes. Cefazolin (CFZ) and oxacillin (OXA) were evaluated in CAMHB with or without NaHCO3 Intra-SEV MRSA killing was determined over 72-h exposures. In both "responsive" strains, supplementation with 25 mM or 44 mM NaHCO3 significantly reduced ß-lactam MICs to below the OXA susceptibility breakpoint (≤4 mg/liter) and resulted in bactericidal activity (≥3-log killing) in the model for both OXA and CFZ. In contrast, neither in vitro-defined nonresponsive MRSA strain showed significant sensitization in the SEV model to either ß-lactam. At both NaHCO3 concentrations, the fractional time above MIC was >50% for both CFZ and OXA in the responsive MRSA strains. Also, in media containing RPMI plus 10% Luria-Bertani broth (proposed as a more host-mimicking microenvironment and containing 25 mM NaHCO3), both CFZ and OXA exhibited enhanced bactericidal activity against NaHCO3-responsive strains in the SEV model. Neither CFZ nor OXA exposures selected for emergence of high-level ß-lactam-resistant mutants within SEVs. Thus, in this ex vivo model of endocarditis, in the presence of NaHCO3 supplementation, both CFZ and OXA are highly active against MRSA strains that demonstrate similar enhanced susceptibility in NaHCO3-supplemented media in vitro.

Scope and Predictive Genetic/Phenotypic Signatures of Bicarbonate (NaHCO3) Responsiveness and ß-Lactam Sensitization in Methicillin-Resistant Staphylococcus aureus.

Addition of sodium bicarbonate (NaHCO3) to standard antimicrobial susceptibility testing medium reveals certain methicillin-resistant Staphylococcus aureus (MRSA) strains to be highly susceptible to ß-lactams. We investigated the prevalence of this phenotype (NaHCO3 responsiveness) to two ß-lactams among 58 clinical MRSA bloodstream isolates. Of note, ∼75% and ∼36% of isolates displayed the NaHCO3 responsiveness phenotype to cefazolin (CFZ) and oxacillin (OXA), respectively. Neither intrinsic ß-lactam MICs in standard Mueller-Hinton broth (MHB) nor population analysis profiles were predictive of this phenotype. Several genotypic markers (clonal complex 8 [CC8]; agr I and spa t008) were associated with NaHCO3 responsiveness for OXA.

Bicarbonate Resensitization of Methicillin-Resistant Staphylococcus aureus to ß-Lactam Antibiotics.

Endovascular infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are a major health care concern, especially infective endocarditis (IE). Standard antimicrobial susceptibility testing (AST) defines most MRSA strains as "resistant" to ß-lactams, often leading to the use of costly and/or toxic treatment regimens. In this investigation, five prototype MRSA strains, representing the range of genotypes in current clinical circulation, were studied. We identified two distinct MRSA phenotypes upon AST using standard media, with or without sodium bicarbonate (NaHCO3) supplementation: one highly susceptible to the antistaphylococcal ß-lactams oxacillin and cefazolin (NaHCO3 responsive) and one resistant to such agents (NaHCO3 nonresponsive). These phenotypes accurately predicted clearance profiles of MRSA from target tissues in experimental MRSA IE treated with each ß-lactam. Mechanistically, NaHCO3 reduced the expression of two key genes involved in the MRSA phenotype, mecA and sarA, leading to decreased production of penicillin-binding protein 2a (that mediates methicillin resistance), in NaHCO3-responsive (but not in NaHCO3-nonresponsive) strains. Moreover, both cefazolin and oxacillin synergistically killed NaHCO3-responsive strains in the presence of the host defense antimicrobial peptide (LL-37) in NaHCO3-supplemented media. These findings suggest that AST of MRSA strains in NaHCO3-containing media may potentially identify infections caused by NaHCO3-responsive strains that are appropriate for ß-lactam therapy.

A Novel Enhanced Gray Scale Adaptive Method for Prediction of Breast Cancer.

Breast cancer is the important problem across the globe in which, most of the women are suffering without knowing the causes and effects of the cancer cells. Mammographic is the most powerful tool for the diagnosis of the Breast cancer. The analysis of this mammogram images proves to be more vital in terms of diagnosis but the accuracy level still needs improvisation. Several intelligent techniques are suggested for the detection of Microcalcification, Clusters, Masses, Spiculate lesions, Asymmetry and Architectural distortions in the mammograms. But the prediction of the cancer levels needs more research light. For the determination of the higher level of accuracy and prediction, the proposed algorithm called Enhanced Gray Scale Adaptive Method (EGAM) which works on the principle of combination of K-GLCM and Extreme Fuzzy Learning Machines (EFLM). The proposed algorithm has achieved 99% accuracy and less computation time in terms of classification, detection and prediction when compared with the existing intelligent algorithms.

Dissipation kinetics of beta-cyfluthrin and imidacloprid in tea and their transfer from processed tea to infusion.

Dissipation kinetics of mixed formulation consisting beta-cyfluthrin and imidacloprid in tea crop under an open field ecosystem was investigated. The mixed formulation was applied on tea plant at recommended (27 + 63) and double the recommended (54 + 126g a.i./ha) dose and residues were determined using gas chromatography-electron capture detector and high performance liquid chromatography-photodiode array detector for beta-cyfluthrin and imidacloprid, respectively. The limit of quantification of analytical method was 0.05µg/g and the average recoveries were ranged from 88.36% to 103.49% with relative standard deviations of less than 6% at three spiked levels. The experimental results showed that in the green tea leaves imidacloprid dissipated faster than beta-cyfluthrin with the half-life ranging between 1.20-1.39 and 2.89-3.15days, respectively. The beta-cyfluthrin residues present in the processed tea not transferred into the tea infusion during the infusion process and imidacloprid transferred in the range 43.12-49.7%. On the basis of the transfer of residues from processed tea to infusion, a waiting period of 17 days for tea plucking after pesticide application at recommended dose may be suggested.

Persistence and dissipation of flubendiamide and its risk assessment on gherkin (Cucumis sativus L.).

A supervised open field trial was conducted to evaluate the dissipation pattern and risk assessment of flubendiamide in gherkin fruits following foliar application of Fame 480 SC at 60 and 120 g a.i. ha(-1). Samples of gherkin fruits were drawn at different time intervals and quantified by HPLC-DAD. The maximum initial deposits of flubendiamide on gherkin were found to be 0.79 and 1.52 mg kg(-1), respectively, at recommended and double the recommended doses. The dissipation pattern of flubendiamide followed a first-order kinetics with half-lives of 1.87 to 2.16 days at 60 and 120 g a.i. ha(-1), respectively. The limit of quantification of flubendiamide and desiodo flubendiamide was observed to be 0.01 mg kg(-1) for gherkin fruit and soil substrates. Theoretical maximum residue contribution (TMRC) for flubendiamide was calculated and found to be well below the maximum permissible intake (MPI) on gherkin fruits. Thus, the application of flubendiamide at the recommended dose on gherkin fruits presents no human health risks and safe to consumers.

Residue and dissipation kinetics of novel nematicide fluopyram in rice assessed by QuEChERS-liquid chromatography-tandem mass spectrometry.

Supervised field trial was conducted to study the dissipation pattern of fluopyram in rice plant after application of fluopyram 400 g/L SC(Velum Prime) as soil drenching at the time of sowing in nursery bed at X (500 g a.i. ha-1) and 1.25X (625.2 g a.i. ha-1) doses. Samples of rice plant were collected on 0, 1, 3, 7, 10, 15, and 20 days after transplanting. QuEChERS-based extraction method was validated and adopted to determine the residues of fluopyram in rice seedlings, whole rice grains (with husk), polished rice grain, husk, straw, and soil using LC-MS/MS (liquid chromatography-tandem mass spectrometry).The initial deposit of fluopyram in rice plant recorded were 0.27 and 0.41 mg kg-1in X and 1.25X doses, respectively. Fluopyram residues dissipated following first-order kinetics with half-life of 2.53 and 2.57 days at X and 1.25X doses, respectively. Residues were detected in seedlings up to 15 days after transplanting and were at below LOQ in whole rice grains (with husk), polished rice grain, husk, straw, and soil collected at harvest. Monitoring study revealed that application of novel nematicide fluopyram for the management of nematodes in rice does not pose any risks to consumers.

Transfer learned deep feature based crack detection using support vector machine: a comparative study.

Technology offers a lot of potential that is being used to improve the integrity and efficiency of infrastructures. Crack is one of the major concerns that can affect the integrity or usability of any structure. Oftentimes, the use of manual inspection methods leads to delays which can worsen the situation. Automated crack detection has become very necessary for efficient management and inspection of critical infrastructures. Previous research in crack detection employed classification and localization-based models using Deep Convolutional Neural Networks (DCNNs). This study suggests and compares the effectiveness of transfer learned DCNNs for crack detection as a classification model and as a feature extractor to overcome this restriction. The main objective of this paper is to present various methods of crack detection on surfaces and compare their performance over 3 different datasets. Experiments conducted in this work are threefold: initially, the effectiveness of 12 transfer learned DCNN models for crack detection is analyzed on three publicly available datasets: SDNET, CCIC and BSD. With an accuracy of 53.40%, ResNet101 outperformed other models on the SDNET dataset. EfficientNetB0 was the most accurate (98.8%) model on the BSD dataset, and ResNet50 performed better with an accuracy of 99.8% on the CCIC dataset. Secondly, two image enhancement methods are employed to enhance the images and are transferred learned on the 12 DCNNs in pursuance of improving the performance of the SDNET dataset. The results from the experiments show that the enhanced images improved the accuracy of transfer-learned crack detection models significantly. Furthermore, deep features extracted from the last fully connected layer of the DCNNs are used to train the Support Vector Machine (SVM). The integration of deep features with SVM enhanced the detection accuracy across all the DCNN-dataset combinations, according to analysis in terms of accuracy, precision, recall, and F1-score.

Epidemiology and outcomes of infections during extracorporeal membrane oxygenation in adult patients with COVID-19 ARDS- A single center study.

BACKGROUND: There is a scarcity of data regarding nosocomial infections in patients with COVID-19 treated with ECMO. This observational study from India aims to describe the epidemiology and microbiology of infections in patients with COVID-19 associated ECMO. METHODS: This is an ambi-directional observational study of COVID-19 ECMO patients admitted from April 2021 to June 2022 in a tertiary care hospital. The total number of sepsis episodes for each patient was recorded and were categorized as bloodstream infections (BSI), pneumonias, skin and soft tissue infections (SSTI), invasive candidiasis (IC), catheter associated urinary tract infection (CAUTI), intra-abdominal infections (IAI), and Clostridioides difficile infections. Details regarding each infection including the microbiological profile and outcomes were recorded. RESULTS: 29 patients who received ECMO for COVID-19 pneumonia during the study period were identified. Of the 29 patients, there were a total of 185 septic episodes. The incidence of septic episodes was 72.4 per 1000 ECMO days. Of the 185 sepsis events, 82 (44.3%) were BSI, 72 (39%) were pneumonia, 19 (10.3%) were SSTI, 7 (3.8%) were CAUTI and 5 (2.7%) were IAIs. Of these 29 patients, 16 (55.2%) patients were discharged and 13 (44.8%) died. CONCLUSIONS: The most common infections in our patients were bloodstream infections followed by pneumonia. High rates of gram negative infections, including those caused by carbapenem resistant bacteria, reflect the Indian critical care unit epidemiology in general. Despite these high infection rates with antimicrobial resistant set of micro-organisms, we had a successful outcome in 55.2% of patients.

Cyclic AMP-Rap1A signaling mediates cell surface translocation of microvascular smooth muscle α2C-adrenoceptors through the actin-binding protein filamin-2.

The second messenger cyclic AMP (cAMP) plays a vital role in vascular physiology, including vasodilation of large blood vessels. We recently demonstrated cAMP activation of Epac-Rap1A and RhoA-Rho-associated kinase (ROCK)-F-actin signaling in arteriolar-derived smooth muscle cells increases expression and cell surface translocation of functional α2C-adrenoceptors (α2C-ARs) that mediate vasoconstriction in small blood vessels (arterioles). The Ras-related small GTPAse Rap1A increased expression of α2C-ARs and also increased translocation of perinuclear α2C-ARs to intracellular F-actin and to the plasma membrane. This study examined the mechanism of translocation to better understand the role of these newly discovered mediators of blood flow control, potentially activated in peripheral vascular disorders. We utilized a yeast two-hybrid screen with human microvascular smooth muscle cells (microVSM) cDNA library and the α2C-AR COOH terminus to identify a novel interaction with the actin cross-linker filamin-2. Yeast α-galactosidase assays, site-directed mutagenesis, and coimmunoprecipitation experiments in heterologous human embryonic kidney (HEK) 293 cells and in human microVSM demonstrated that α2C-ARs, but not α2A-AR subtype, interacted with filamin. In Rap1-stimulated human microVSM, α2C-ARs colocalized with filamin on intracellular filaments and at the plasma membrane. Small interfering RNA-mediated knockdown of filamin-2 inhibited Rap1-induced redistribution of α2C-ARs to the cell surface and inhibited receptor function. The studies suggest that cAMP-Rap1-Rho-ROCK signaling facilitates receptor translocation and function via phosphorylation of filamin-2 Ser(2113). Together, these studies extend our previous findings to show that functional rescue of α2C-ARs is mediated through Rap1-filamin signaling. Perturbation of this signaling pathway may lead to alterations in α2C-AR trafficking and physiological function.

Electric vehicle parameter identification and state of charge estimation of Li-ion​ batteries: Hybrid WSO-HDLNN method.

This manuscript proposes a hybrid method for measuring the battery's dynamic electrical response as it is compressed by an external-force. The proposed hybrid technique is the wrapper of the War Strategy Optimization algorithm and Hierarchical Deep Learning Neural Network, commonly called as WSO-HDLNN technique. The main aim of the proposed method is to lessen the battery-voltage error. The War Strategy Optimization method detects the parameters of the battery method. The Hierarchical Deep Learning Neural Network is used to predict the dynamic-electrical-response of the battery when it deforms during external-force. By using the proposed method, the estimated voltage and measured voltage error are reduced, and identifies the parameter effectively. Finally, the proposed method is done in the MATLAB platform and it is compared with different existing approaches. The error of the proposed method is 4 mV, the Jellyfish Search Optimizer method error is 6 mV, the Heap-based Optimizer method error is 12 mV, and the Grey Wolf Optimizer method error is 14 mV. The proposed method time is 0.7 s The proposed method shows better results in all methods, like Jellyfish Search Optimizer, Heap-based Optimizer, and Grey Wolf Optimizer, The proposed method provides less computation time and error than the existing one is proved from the simulation outcome.

Role of the NaHCO3 Transporter MpsABC in the NaHCO3-ß-Lactam-Responsive Phenotype in Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) infections are an increasing concern due to their intrinsic resistance to most standard-of-care ß-lactam antibiotics. Recent studies of clinical isolates have documented a novel phenotype, termed NaHCO3 responsiveness, in which a substantial proportion of MRSA strains exhibit enhanced susceptibility to ß-lactams such as cefazolin and oxacillin in the presence of NaHCO3. A bicarbonate transporter, MpsAB (membrane potential-generating system), was recently found in S. aureus, where it plays a role in concentrating NaHCO3 for anaplerotic pathways. Here, we investigated the role of MpsAB in mediating the NaHCO3 responsiveness phenotype. Radiolabeled NaH14CO3 uptake profiling revealed significantly higher accumulation in NaHCO3-responsive vs nonresponsive MRSA strains when grown in ambient air. In contrast, under 5% CO2 conditions, NaHCO3-responsive (but not nonresponsive) strains exhibited repressed uptake. Oxacillin MICs were measured in four prototype strains and their mpsABC deletion mutants in the presence of NaHCO3 supplementation under 5% CO2 conditions. NaHCO3-mediated reductions in oxacillin MICs were observed in the responsive parental strains but not in mpsABC deletion mutants. No significant impact on oxacillin MICs was observed in the nonresponsive strains under the same conditions. Transcriptional and translational studies were carried out using both quantitative reverse transcription-PCR (qRT-PCR) and mpsA-green fluorescent protein (GFP) fusion constructs; these investigations showed that mpsA expression and translation were significantly upregulated during mid-exponential-phase growth in oxacillin-NaHCO3-supplemented medium in responsive versus nonresponsive strains. Taken together, these data show that the NaHCO3 transporter MpsABC is a key contributor to the NaHCO3-ß-lactam responsiveness phenotype in MRSA. IMPORTANCE MRSA infections are increasingly difficult to treat, due in part to their resistance to most ß-lactam antibiotics. A novel and relatively common phenotype, termed NaHCO3 responsiveness, has been identified in which MRSA strains show increased susceptibility in vitro and in vivo to ß-lactams in the presence of NaHCO3. A recently described S. aureus NaHCO3 transporter, MpsAB, is involved in intracellular NaHCO3 concentration for anaplerotic pathways. We investigated the role of MpsAB in mediating the NaHCO3 responsiveness phenotype in four prototype MRSA strains (two responsive and two nonresponsive). We demonstrated that MpsABC is an important contributor to the NaHCO3-ß-lactam responsiveness phenotype. Our study adds to the growing body of well-defined characteristics of this novel phenotype, which could potentially translate to alternative targets for MRSA treatment using ß-lactams.

Cyclic AMP-Rap1A signaling activates RhoA to induce α(2c)-adrenoceptor translocation to the cell surface of microvascular smooth muscle cells.

Intracellular signaling by the second messenger cyclic AMP (cAMP) activates the Ras-related small GTPase Rap1 through the guanine exchange factor Epac. This activation leads to effector protein interactions, activation, and biological responses in the vasculature, including vasorelaxation. In vascular smooth muscle cells derived from human dermal arterioles (microVSM), Rap1 selectively regulates expression of G protein-coupled α(2C)-adrenoceptors (α(2C)-ARs) through JNK-c-jun nuclear signaling. The α(2C)-ARs are generally retained in the trans-Golgi compartment and mobilize to the cell surface and elicit vasoconstriction in response to cellular stress. The present study used human microVSM to examine the role of Rap1 in receptor localization. Complementary approaches included murine microVSM derived from tail arteries of C57BL6 mice that express functional α(2C)-ARs and mice deficient in Rap1A (Rap1A-null). In human microVSM, increasing intracellular cAMP by direct activation of adenylyl cyclase by forskolin (10 µM) or selectively activating Epac-Rap signaling by the cAMP analog 8-pCPT-2'-O-Me-cAMP (100 µM) activated RhoA, increased α(2C)-AR expression, and reorganized the actin cytoskeleton, increasing F-actin. The α(2C)-ARs mobilized from the perinuclear region to intracellular filamentous structures and to the plasma membrane. Similar results were obtained in murine wild-type microVSM, coupling Rap1-Rho-actin dynamics to receptor relocalization. This signaling was impaired in Rap1A-null murine microVSM and was rescued by delivery of constitutively active (CA) mutant of Rap1A. When tested in heterologous HEK293 cells, Rap1A-CA or Rho-kinase (ROCK-CA) caused translocation of functional α(2C)-ARs to the cell surface (~4- to 6-fold increase, respectively). Together, these studies support vascular bed-specific physiological role of Rap1 and suggest a role in vasoconstriction in microVSM.

Multiresidue analysis of organochlorine pesticides in milk, egg and meat by GC-ECD and confirmation by GC-MS.

A multiresidue method was developed and optimized for the quantification of organochlorine pesticides (OCPs) in milk, egg and meat samples. Sample extraction was performed by adopting QuEChERS principle and the extracts were cleaned-up dispersive solid-phase extraction with primary secondary amine after salting out with NaCl and MgSO(4). Analysis was carried out by gas chromatography coupled with electron capture detector and confirmation by gas chromatography-mass spectrometry. The performance of the method was investigated in terms of linearity, accuracy, precision, detection limit and quantification limit (LOQ). Good linearity was obtained, with correlation coefficients (r(2)) higher than 0.992. Mean recoveries were found in the ranges 72%-108%, 74%-101% and 75.27%-104.56% for the milk, egg and meat, respectively, RSD % turned out to range from 0.28% to 10.05%. The method developed was successfully tested on commercial milk, egg, and meat samples from the markets of Tamil Nadu (India), proving to be a useful tool in routine analysis of OCPs for monitoring purposes. None of the compounds of interest were observed above their respective LOQ.

Impacts of NaHCO3 on ß-Lactam Binding to PBP2a Protein Variants Associated with the NaHCO3-Responsive versus NaHCO3-Non-Responsive Phenotypes.

Methicillin-resistant Staphylococcus aureus (MRSA) regulates resistance to ß-lactams via preferential production of an alternative penicillin-binding protein (PBP), PBP2a. PBP2a binds many ß-lactam antibiotics with less affinity than PBPs which are predominant in methicillin-susceptible (MSSA) strains. A novel, rather frequent in vitro phenotype was recently identified among clinical MRSA bloodstream isolates, termed "NaHCO3-responsiveness". This phenotype features ß-lactam susceptibility of certain MRSA strains only in the presence of NaHCO3. Two distinct PBP2a variants, 246G and 246E, have been linked to the NaHCO3-responsive and NaHCO3-non-responsive MRSA phenotypes, respectively. To determine the mechanistic impact of PBP2a variants on ß-lactam susceptibility, binding profiles of a fluorescent penicillin probe (Bocillin-FL) to each purified PBP2a variant were assessed and compared to whole-cell binding profiles characterized by flow cytometry in the presence vs. absence of NaHCO3. These investigations revealed that NaHCO3 differentially influenced the binding of the fluorescent penicillin, Bocillin-FL, to the PBP2a variants, with binding intensity and rate of binding significantly enhanced in the 246G compared to the 246E variant. Of note, the NaHCO3-ß-lactam (oxacillin)-responsive JE2 strain, which natively harbors the 246G variant, had enhanced Bocillin-FL whole-cell binding following exposure to NaHCO3. This NaHCO3-mediated increase in whole-cell Bocillin-FL binding was not observed in the NaHCO3-non-responsive parental strain, COL, which contains the 246E PBP2a variant. Surprisingly, genetic swaps of the mecA coding sites between JE2 and COL did not alter the NaHCO3-enhanced binding seen in JE2 vs. COL. These data suggest that the non-coding regions of mecA may be involved in NaHCO3-responsiveness. This investigation also provides strong evidence that the NaHCO3-responsive phenotype in MRSA may involve NaHCO3-mediated increases in both initial cell surface ß-lactam binding, as well as ultimate PBP2a binding of ß-lactams.