Multiclonal human origin and global expansion of an endemic bacterial pathogen of livestock.

Most new pathogens of humans and animals arise via switching events from distinct host species. However, our understanding of the evolutionary and ecological drivers of successful host adaptation, expansion, and dissemination are limited. Staphylococcus aureus is a major bacterial pathogen of humans and a leading cause of mastitis in dairy cows worldwide. Here we trace the evolutionary history of bovine S. aureus using a global dataset of 10,254 S. aureus genomes including 1,896 bovine isolates from 32 countries in 6 continents. We identified 7 major contemporary endemic clones of S. aureus causing bovine mastitis around the world and traced them back to 4 independent host-jump events from humans that occurred up to 2,500 y ago. Individual clones emerged and underwent clonal expansion from the mid-19th to late 20th century coinciding with the commercialization and industrialization of dairy farming, and older lineages have become globally distributed via established cattle trade links. Importantly, we identified lineage-dependent differences in the frequency of host transmission events between humans and cows in both directions revealing high risk clones threatening veterinary and human health. Finally, pangenome network analysis revealed that some bovine S. aureus lineages contained distinct sets of bovine-associated genes, consistent with multiple trajectories to host adaptation via gene acquisition. Taken together, we have dissected the evolutionary history of a major endemic pathogen of livestock providing a comprehensive temporal, geographic, and gene-level perspective of its remarkable success.

MALDI-TOF bacterial subtyping for rapid detection of biomarkers in Staphylococcus aureus from subclinical bovine mastitis.

AIMS: This study aimed to evaluate matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) bacterial subtyping for the rapid detection of biomarkers in Staphylococcus aureus from subclinical bovine mastitis. METHODS AND RESULTS: A total of 229 S. aureus isolates were obtained from milk samples collected from cows with subclinical mastitis using microbiological culture. Staphylococcus aureus isolates were also submitted to PCR analysis targeting the mecA and mecC genes, which are indicative of methicillin resistance. Confirmation of the species was achieved through MALDI-TOF MS analysis. To analyze antimicrobial resistance patterns, the MALDI BioTyper Compass Explorer and ClinProTools Bruker software were employed, and dendrograms were generated using Bionumerics software. CONCLUSIONS: MALDI-TOF MS successfully identified S. aureus at the species level, but no methicillin resistance was observed. Moreover, spectral typing displayed limited similarity when compared to pulsed-field gel electrophoresis (PFGE).

Bioimaging approaches for quantification of individual cell behavior during cell fate decisions.

Tracking individual cells has allowed a new understanding of cellular behavior in human health and disease by adding a dynamic component to the already complex heterogeneity of single cells. Technically, despite countless advances, numerous experimental variables can affect data collection and interpretation and need to be considered. In this review, we discuss the main technical aspects and biological findings in the analysis of the behavior of individual cells. We discuss the most relevant contributions provided by these approaches in clinically relevant human conditions like embryo development, stem cells biology, inflammation, cancer and microbiology, along with the cellular mechanisms and molecular pathways underlying these conditions. We also discuss the key technical aspects to be considered when planning and performing experiments involving the analysis of individual cells over long periods. Despite the challenges in automatic detection, features extraction and long-term tracking that need to be tackled, the potential impact of single-cell bioimaging is enormous in understanding the pathogenesis and development of new therapies in human pathophysiology.

Species diversity and antimicrobial resistance patterns of Enterococcus spp. isolated from mastitis cases, milking machine and the environment of dairy cows.

The objectives were to study the diversity of Enterococcus spp. isolated from mastitis cases, milking equipment and the environment of dairy cows; and to determine in vitro resistance of isolates to antimicrobials that are relevant to human and animal health. Ten dairy farms were visited to collect samples from mastitis cases, faeces, bedding, aisles, water and milking equipment. Identification of Enterococcus at the species level and antimicrobial resistance testing was performed by MALDI-TOF and the disk-diffusion method, respectively. Of 365 isolates, Enterococcus hirae was the most prevalent, being more likely to be isolated from faeces than from milk (odds ratio (OR) = 39·2), liners (OR = 5·4) or bedding (OR = 2·2). Enterococcus saccharolyticus was the most prevalent in milk samples. The chances of isolating Enterococcus faecalis from milk were higher than from aisles (OR = 12·5), faeces (OR = 5·3), bedding (OR = 3·6) or liners (OR = 3·0). The odds of isolating Enterococcus faecium from faeces were higher than from liners (OR = 7·3), bedding (OR = 2·5) or aisles (OR = 2·4). Of 360 tested isolates, 1·9, 0·3 and 0·6% were resistant to penicillin, vancomycin and teicoplanin, respectively. Our results suggest that Enterococcus species can occupy specific ecological niches on dairy farms and pose a risk to public and animal health.

Herd-level associations between somatic cell counts and economic performance indicators in Brazilian dairy herds.

The aims of the present study were to provide a portrait of the techno-economic status of dairy herds in Minas Gerais, Brazil, particularly with respect to bulk-tank somatic cell count (BTSCC) data, and to examine the herd-level associations of BTSCC with various economic performance indicators (EPI). Data from 543 herds, 1,052 herd-year records in total, spread over 3 years (2015-2017), from the South and Southwest mesoregions of Minas Gerais State were provided by the Brazilian Support Agency to Micro and Small Companies Division Minas Gerais (SEBRAE). Herds had an average of 82 lactating cows per herd, milk yield of 17 L/cow per day, and availability of financial information via routine monthly economic surveys. The EPI data (revenue, gross margin, GM; net margin, NM; profit; break-even point; and operational profitability) of each herd was measured monthly by SEBRAE personnel, and herd-year averages of all variables were computed. Bulk-tank data (SCC, total bacterial count, content of crude protein and fat) taken by producers or dairy processors were recorded by SEBRAE personal; and corresponding herd-year averages were calculated and included in the SEBRAE database. There were 209 selected herds, which passed all edit checks, and which had data for all 3 years. The EPI (all expressed on a per-cow basis, $/cow per year) were analyzed, including the effects of region, year, log (ln) BTSCC, production level, and herd size, together with the random effect of herd nested within region. A high proportion of herds (94.6%) presented data records (herd-years) with an average BTSCC > 200 × 103 cells/mL: 37.8% of herd-year records had BTSCC between >200 and ≤400, 14.5% with BTSCC between >400 and ≤500, 25% with BTSCC between >500 and ≤750, and 17.3% with BTSCC >750. For each unit increase in ln BTSCC, revenue declined by $228.5/cow per year, GM by $155.6/cow per year, and profit by $138.6/cow per year. Herds with cows of lower production (<14 kg/d) presented lower GM ($286.8/cow per year) compared with herds containing cows producing ≥14 kg/d (≥14 and <19 kg/d = $446.5, and ≥19 kg/d = $601.9). The small-scale milk producers (<39 lactating cows) presented lower revenue ($1,914.9/cow per year) and GM ($274.5/cow per year) and consequently a negative profit (-$224.1/cow per year) compared with other herd size categories (≥39 lactating cows). The reduction in milk yield was 641 L/cow per lactation for each unit increase in ln BTSCC; this represented 9.4% of the milk yield per lactation, assuming an average milk production of 6,843.3 L/cow per lactation of cows from herds that had BTSCC ≤ 200 × 103 cells/mL. Consequently, we found a negative association of BTSCC with profit; profit declining from $227.0 to -53.1/cow per year when the BTSCC increased from 100 to 750 × 103 cell/mL. In short, the lower the BTSCC, the greater the revenue, GM and NM, profit, and operational profitability of the herds. The reduction of milk yield was the main factor associated with higher BTSCC.

Association between antimicrobial use and antimicrobial resistance of Streptococcus uberis causing clinical mastitis.

It is unknown whether overuse of antimicrobials against clinical mastitis (CM) from Streptococcus uberis is associated with increased antimicrobial resistance (AMR). Therefore, the aim of this study was to evaluate the association between antimicrobial use (AMU) and AMR in relation to the Strep. uberis causing CM in dairy herds. A total of 83 Strep. uberis isolates were selected from a collection created during a previous study evaluating the epidemiology of CM in dairy herds (n = 17) of southeastern Brazil. For each case of CM identified on farm, the following information was recorded: cow's identification number, affected mammary quarter, date of CM diagnosis, antimicrobial commercial names, number of administrations, and descriptions of protocol changes during the treatment. Streptococcus uberis isolates were confirmed by conventional culture, MALDI-TOF mass spectrometry, and quantitative multiplex PCR analyses. Thus, a total of 8 antimicrobials commonly used for CM treatment were evaluated for antimicrobial activity against Strep. uberis isolates. The minimum inhibitory levels of antimicrobials were determined at the lowest concentrations able to inhibit 50 and 90%, respectively, of Strep. uberis isolates. Data related to the antibiotics used for treatment of CM was used to calculate the frequency of administered antimicrobials as the number of defined daily doses (DDD). The highest frequencies of resistant Strep. uberis were observed for erythromycin (80.7% resistant, R), tetracycline (R = 59%), and penicillin G (R = 57.8%), whereas against ceftiofur only 10.8% of Strep. uberis isolates were resistant, and only 1.2% of the Strep. uberis isolates were resistant to enrofloxacin. Regarding the evaluation of resistance for antimicrobial classes, the highest frequency was observed for macrolides (R = 80.7%; 19.3% susceptible, S). Additionally, a frequency of 18.7% of Strep. uberis isolates were resistant to cephalosporins (S = 81.3%), respectively. Further, 94% of Strep. uberis isolates were multiresistant; all these isolates presented resistance to at least 3 different antimicrobial classes. The overall monthly average of antimicrobial treatment incidence (ATI) among the 17 herds enrolled in the study was 23.7 DDD per 1,000 lactating dairy cows [standard deviation (SD) = 13.9], ranging from 5.0 to 55.4 DDD per 1,000 cows in lactation-day. Cephalosporins and penicillins were the most commonly used antimicrobial classes among the evaluated herds (n = 16; 94.1%), followed by tetracyclines (n = 15 herds; 88.2%), fluoroquinolones (n = 14; 82.3%), and sulfonamides (n = 14; 82.3%). The tetracycline class had the highest ATI mean (5.0 DDD per 1,000 lactating cow-days, SD = 5.8), followed by fluoroquinolones (4.7 DDD per 1,000 lactating cow-days, SD = 6.0) and cephalosporins (3.8 DDD per 1,000 lactating cow-days, SD = 6.0). The overall use of antimicrobials was associated with the resistance of Strep. uberis to the antimicrobial tetracycline.

Short communication: Association between the accessory gene regulator (agr) group and the severity of bovine mastitis caused by Staphylococcus aureus.

Staphylococcus aureus can elicit mild to more severe degrees of mastitis in cattle, depending on the response of the host's immune system and the virulence factors of the specific isolate. Several virulence factors are controlled by a global regulatory system, designated accessory gene regulator (agr). Thus, the objective was to examine associations between different capsular and agr types and the severity of bovine mastitis caused by S. aureus. All isolates were obtained from bovine subclinical (n = 50), mild clinical (n = 73), and moderate clinical mastitis cases (n = 28). Isolates containing the agrI gene and lacking the agr locus (agr-) were more prevalent among subclinical than clinical mastitis cases, whereas isolates containing the agrII and agrIII genes were more prevalent among clinical mastitis cases. The capsular types 5 (cap5) and 8 (cap8) were found in 42 and 44%, respectively, of the isolates obtained from subclinical cases and in 38.6 and 58.4%, respectively, of those isolated from clinical mastitis cases. Capsular type was not associated with type of mastitis (subclinical, mild clinical, or moderate clinical). We found a strong association between agr type and type of mastitis, suggesting that knowledge of S. aureus genetic profiles could be an additional tool to control this disease.

Lipase production by microorganisms isolated from the Serra de Ouro Branco State Park.

Lipases are hydrolases used in various sectors such as the food, pharmaceutical and chemical synthesis industries. In this study, epiphytic microorganisms were isolated from the Serra of Ouro Branco State Park (Minas Gerais, Brazil) and were subsequently evaluated for their ability to produce extracellular lipases. Among the 46 isolated strains, 25 presented positive results for lipase production in the agar plate screening assay. Two of these strains that expressed the highest diffusion halos, were genetically identified as Serratia marcescens and Pseudomonas fluorescens and catalogued in the Tropical Cultures Collection from the André Tosello Foundation/Brazil as CCT 7796 and CCT 7797, respectively. The fermentation growth kinetics indicated that the maximum extracellular lipase activities were achieved between 96 and 120h of cultivation. The highest lipolytic activity for both strains was observed at an optimum temperature and pH of 37°C and 7.0, respectively. At these conditions, the lipase activity detected in the crude enzymatic extract of both strains was close to 15.0 U/mL. We consider that these species are promising lipase producers for industrial applications.

First investigation of Staphylococcus argenteus in a Brazilian collections of S. aureus isolated from bovine mastitis.

BACKGROUND: Staphylococcus argenteus is a new specie positive coagulase staphylococci. We investigate the presence of S. argenteus in isolates previously classified as S. aureus, obtained from the milk of cows with mastitis in Brazil. RESULTS: Among 856 S. aureus tested in chocolate agar, tryptone soya agar and salt egg yolk agar, white or colorless colonies were observed in 185 (21.6%) isolates. Regarding the ctrOPQMN operon, 111 (60%) presented the complete cluster. Despite some missing genes in this cluster, the remaining strains (74) were confirmed as S. aureus using the nrps gene. CONCLUSIONS: As far as we know, this is the first review of S. aureus collection in Brazil and S. argenteus does not appear to be a significant problem in Brazilian herds.

Effects of thermal shock on the survival and reproduction of Stratiolaelaps scimitus (Mesostigmata: Laelapidae).

The responses of arthropods to thermal stress are vital in ecological studies in order to understand survival, development, and reproduction. However, this subject is poorly addressed. In the order Mesostigmata, an abundance of species lives in the soil. Among these species, Stratiolaelaps scimitus (Womersley) is a predator used in the control of pest organisms that live in the soil. Mites of this species are commercialized in several countries, including Brazil, presenting efficiency in pest control in several crops. The objective of this study was to evaluate the effect of thermal shock on S. scimitus females, as well as to monitor the temperature variation in the environment. For each temperature, 80 experimental units were assembled for different periods (0.5, 1, 2, and 4 h). Experimental units were maintained at 25 °C, after exposure of the mites. Mortality and oviposition were evaluated. The results showed a 40% reduction in the survival of mites exposed to 37 °C for 4 h, compared to the control treatment (25 °C). Oviposition was less affected at 1 h exposure to temperatures of 19 and 12 °C and thermic fluctuation was observed in the greenhouse, especially inside the slabs. Understanding temperature effects in mites and the thermic fluctuation in the environment is essential to achieve satisfactory results in biological control. It is important to observe the scenario in which predatory mites will be released as these aspects are decisive in predatory activity.

A randomized trial of amlodipine in addition to standard chelation therapy in patients with thalassemia major.

Cardiovascular disease resulting from iron accumulation is still a major cause of death in patients with thalassemia major (TM). Voltage-gated calcium-channel blockade prevents iron entry into cardiomyocytes and may provide an adjuvant treatment to chelation, reducing myocardial iron uptake. We evaluated whether addition of amlodipine to chelation strategies would reduce myocardial iron overload in TM patients compared with placebo. In a multicenter, double-blind, randomized, placebo-controlled trial, 62 patients were allocated to receive oral amlodipine 5 mg/day or placebo in addition to their current chelation regimen. The main outcome was change in myocardial iron concentration (MIC) determined by magnetic resonance imaging at 12 months, with patients stratified into reduction or prevention groups according to their initial T2* below or above the normal human threshold of 35 ms (MIC, 0.59 mg/g dry weight). At 12 months, patients in the reduction group receiving amlodipine (n = 15) had a significant decrease in MIC compared with patients receiving placebo (n = 15) with a median of -0.26 mg/g (95% confidence interval, -1.02 to -0.01) vs 0.01 mg/g (95% confidence interval, -0.13 to 0.23), P = .02. No significant changes were observed in the prevention group (treatment-effect interaction with P = .005). The same findings were observed in the subgroup of patients with T2* <20 ms. Amlodipine treatment did not cause any serious adverse events. Thus, in TM patients with cardiac siderosis, amlodipine combined with chelation therapy reduced cardiac iron more effectively than chelation therapy alone. Because this conclusion is based on subgroup analyses, it needs to be confirmed in ad hoc clinical trials. This trial was registered at www.clinicaltrials.gov identifier as #NCT01395199.

Milk losses associated with somatic cell counts by parity and stage of lactation.

The reduction of milk production caused by subclinical mastitis in dairy cows was evaluated through the regression of test-day milk yield on log-transformed somatic cell counts (LnSCC). Official test-day records (n = 1,688,054) of Holstein cows (n = 87,695) were obtained from 719 herds from January 2010 to December 2015. Editing was performed to ensure both reliability and consistency for the statistical analysis, and the final data set comprised 232,937 test-day records from 31,692 Holstein cows in 243 herds. A segmented regression was fitted to estimate the cutoff point in the LnSCC scale where milk yield started to be affected by mastitis. The statistical model used to explain daily milk yield included the effect of herd as a random effect and days in milk and LnSCC as fixed effects regressions, and analyses were performed by parity and stage of lactation. The cutoff point where milk yield starts to be affected by changes in LnSCC was estimated to be around 2.52 (the average of all estimates of approximately 12,400 cells/mL) for Holsteins cows from Brazilian herds. For first-lactation cows, milk losses per unit increase of LnSCC had estimates around 0.68 kg/d in the beginning of the lactation [5 to 19 d in milk (DIM)], 0.55 kg/d in mid-lactation (110 to 124 DIM), and 0.97 kg/d at the end of the lactation (289 to 304 DIM). For second-lactation cows, milk losses per unit increase of LnSCC had estimates around 1.47 kg/d in the beginning of the lactation (5 to 19 DIM), 1.09 kg/d in mid-lactation (110 to 124 DIM), and 2.45 kg/d at the end of the lactation (289 to 304 DIM). For third-lactation cows, milk losses per unit increase of LnSCC had estimates around 2.22 kg/d in the beginning of the lactation (5 to 19 DIM), 1.13 kg/d in mid-lactation (140 to 154 DIM), and 2.65 kg/d at the end of the lactation (289 to 304 DIM). Daily milk losses caused by increased LnSCC were dependent on parity and stage of lactation, and these factors should be considered when estimating losses associated with subclinical mastitis.

Central role for hydrogen peroxide in P2Y1 ADP receptor-mediated cellular responses in vascular endothelium.

ADP activates a family of cell surface receptors that modulate signaling pathways in a broad range of cells. ADP receptor antagonists are widely used to treat cardiovascular disease states. These studies identify a critical role for the stable reactive oxygen species hydrogen peroxide (H2O2) in mediating cellular responses activated by the G protein-coupled P2Y1 receptor for ADP. We found that ADP-dependent phosphorylation of key endothelial signaling proteins--including endothelial nitric oxide synthase, AMP-activated protein kinase, and the actin-binding MARCKS protein--was blocked by preincubation with PEG-catalase, which degrades H2O2. ADP treatment promoted the H2O2-dependent phosphorylation of c-Abl, a nonreceptor tyrosine kinase that modulates the actin cytoskeleton. Cellular imaging experiments using fluorescence resonance energy transfer-based biosensors revealed that ADP-stimulated activation of the cytoskeleton-associated small GTPase Rac1 was independent of H2O2. However, Rac1-dependent activation of AMP-activated protein kinase, the signaling phospholipid phosphatidylinositol-(4, 5)-bisphosphate, and the c-Abl-interacting protein CrkII are mediated by H2O2. We transfected endothelial cells with differentially targeted HyPer2 H2O2 biosensors and found that ADP promoted a marked increase in H2O2 levels in the cytosol and caveolae, and a smaller increase in mitochondria. We performed a screen for P2Y1 receptor-mediated receptor tyrosine kinase transactivation and discovered that ADP transactivates Fms-like tyrosine kinase 3 (Flt3), a receptor tyrosine kinase expressed in these cells. Our observation that P2Y1 receptor-mediated responses involve Flt3 transactivation may identify a unique mechanism whereby cancer chemotherapy with receptor tyrosine kinase inhibitors promotes vascular dysfunction. Taken together, these findings establish a critical role for endogenous H2O2 in control of ADP-mediated signaling responses in the vascular wall.

Novel role for retinol-binding protein 4 in the regulation of blood pressure.

Elevated levels of serum retinol-binding protein 4 (RBP4) contribute to insulin resistance and correlate with increased prevalence of hypertension and myocardial infarction. We sought to determine whether lowering RBP4 would improve blood pressure (BP) and protect against obesity- or angiotensin (Ang)-II-induced hypertension. Systolic and diastolic BP were lower in the RBP4-knockout (RBP4-KO) mice and higher in the RBP4-overexpressing (RBP4-Tg) mice compared with BP in the wild-type (WT) littermates. Carbachol-induced vasodilatation was increased in arteries from the RBP4-KO compared with the WT mice and was impaired in the RBP4-Tg mice. Aortic eNOS(Ser1177) phosphorylation was enhanced ∼50% in the RBP4-KO mice, with no change in total eNOS protein. Feeding a high-fat diet increased BP in the RBP4-KO mice only to the level in the WT mice fed chow and had no effect on aortic eNOS(Ser1177) phosphorylation. Ang-II infusion resulted in 22 mmHg lower systolic BP in the RBP4-KO than in the WT mice, although the relative BP increase over saline infusion was ∼30% in both. Ang-II treatment decreased aortic eNOS(Ser1177) phosphorylation in the WT and RBP4-KO mice, but phosphorylation remained higher in the RBP4-KO mice. Cardiac hypertrophy with Ang-II treatment was diminished by 56% in the RBP4-KO mice. Thus, elevated serum RBP4 raises BP and lack of RBP4 reduces it, with commensurate changes in aortic eNOS(Ser1177) phosphorylation. Lowering RBP4 may reduce BP through enhanced eNOS-mediated vasodilatation and may be a novel therapeutic approach for hypertension.

Genetic structure from the oldest Jatropha germplasm bank of Brazil and contribution for the genetic improvement.

Jatropha is a potential oilseed crop, which requires mitigating factors such as the low genetic variability of the species. The solution runs through the research of Brazilian germplasm. Attention should be given to the germplasm of jatropha the north of Minas Gerais, because this is the oldest national collection and because this region may be a regions of jatropha diversity due to selection pressure arising from environmental adversities. Therefore, the objective of this study was to investigate the genetic diversity of 48 accessions of collection from Empresa de Pesquisa Agropecuária de Minas Gerais (EPAMIG), using SSR and ISSR markers. The results showed low genetic diversity, but some individuals stood out as J. mollissima (48), J. podagrica (47), Mexican accessions (42, 43, 44 and 45) and some national accessions (28, 29, 41 and 46). Therefore, aiming to increase the genetic variability and improve the effectiveness of jatropha breeding programs, it is suggested to explore such as parental accessions to generate commercial hybrids. This fact implies the possibility to support future production of jatropha, since this culture may be an important source of income, especially for small farmers living in semiarid regions of Brazil.

Hydrogen peroxide differentially modulates cardiac myocyte nitric oxide synthesis.

Nitric oxide (NO) and hydrogen peroxide (H(2)O(2)) are synthesized within cardiac myocytes and play key roles in modulating cardiovascular signaling. Cardiac myocytes contain both the endothelial (eNOS) and neuronal (nNOS) NO synthases, but the differential roles of these NOS isoforms and the interplay of reactive oxygen species and reactive nitrogen species in cardiac signaling pathways are poorly understood. Using a recently developed NO chemical sensor [Cu(2)(FL2E)] to study adult cardiac myocytes from wild-type, eNOS(null), and nNOS(null) mice, we discovered that physiological concentrations of H(2)O(2) activate eNOS but not nNOS. H(2)O(2)-stimulated eNOS activation depends on phosphorylation of both the AMP-activated protein kinase and kinase Akt, and leads to the robust phosphorylation of eNOS. Cardiac myocytes isolated from mice infected with lentivirus expressing the recently developed H(2)O(2) biosensor HyPer2 show marked H(2)O(2) synthesis when stimulated by angiotensin II, but not following ß-adrenergic receptor activation. We discovered that the angiotensin-II-promoted increase in cardiac myocyte contractility is dependent on H(2)O(2), whereas ß-adrenergic contractile responses occur independently of H(2)O(2) signaling. These studies establish differential roles for H(2)O(2) in control of cardiac contractility and receptor-dependent NOS activation in the heart, and they identify new points for modulation of NO signaling responses by oxidant stress.

Comparison of Minimum Inhibitory Concentrations of Selected Antimicrobials for Non-Aureus Staphylococci, Enterococci, Lactococci, and Streptococci Isolated from Milk Samples of Cows with Clinical Mastitis.

The objective of this study was to compare the minimum inhibitory concentrations of antimicrobials included in a commercial broth microdilution panel among Gram-positive pathogens that caused non-severe clinical mastitis on three Michigan dairy farms. Duplicate quarter milk samples were collected from eligible quarters of cows enrolled in a randomized clinical trial, cultured in a university laboratory, and identified using MALDI-TOF. Etiologies were grouped by genus as Enterococcus species (n = 11), Lactococcus species (n = 44), non-aureus Staphylococcus species (n = 39), or Streptococcus species (n = 25). Minimum inhibitory concentrations (MICs) were determined using the mastitis panel of a commercially available broth microdilution test. In vitro susceptibility was determined using approved guidelines and included breakpoints for mastitis pathogens, or when not available, breakpoints from other species. Most isolates were inhibited at or below breakpoints that demonstrated in vitro susceptibility. The proportions of susceptible isolates varied among pathogens for pirlimycin, penicillin, and tetracycline. The greatest proportion of resistance was observed for pirlimycin, tetracycline, and sulfadimethoxine. Survival analysis was performed to evaluate differences in MICs among pathogen groups. MIC values varied among pathogens for ceftiofur, cephalothin, erythromycin, penicillin, pirlimycin, and tetracycline. However, nearly all isolates were susceptible to ceftiofur and cephalothin, indicating that pathogen differences in MIC are not likely clinically relevant, as these are the two most commonly administered mastitis treatments in the United States. While differences in vitro susceptibility were observed for some antimicrobials, susceptibility was high to cephalosporin-based IMM treatments that are most commonly used and did not vary among pathogens.

Biofilm Formation and Antimicrobial Resistance of Staphylococcus aureus and Streptococcus uberis Isolates from Bovine Mastitis.

This study aimed to assess (a) the biofilm producer ability and antimicrobial resistance profiles of Staphylococcus (Staph.) aureus and Streptococcus (Strep.) uberis isolated from cows with clinical mastitis (CM) and subclinical mastitis (SCM), and (b) the association between biofilm producer ability and antimicrobial resistance. We isolated a total of 197 Staph. aureus strains (SCM = 111, CM = 86) and 119 Strep. uberis strains (SCM = 15, CM = 104) from milk samples obtained from 316 cows distributed in 24 dairy herds. Biofilm-forming ability was assessed using the microplate method, while antimicrobial susceptibility was determined using the disk diffusion method against 13 antimicrobials. Among the isolates examined, 57.3% of Staph. aureus and 53.8% of Strep. uberis exhibited the ability to produce biofilm, which was categorized as strong, moderate, or weak. In terms of antimicrobial susceptibility, Staph. aureus isolates displayed resistance to penicillin (92.9%), ampicillin (50.8%), and tetracycline (52.7%). Conversely, Strep. uberis isolates exhibited resistance to penicillin (80.6%), oxacillin (80.6%), and tetracycline (37.8%). However, no significant correlation was found between antimicrobial resistance patterns and biofilm formation ability among the isolates.

Suppression of Gαs synthesis by simvastatin treatment of vascular endothelial cells.

These studies explore the effects of statins on cyclic AMP-modulated signaling pathways in vascular endothelial cells. We previously observed (Kou, R., Sartoretto, J., and Michel, T. (2009) J. Biol. Chem. 284, 14734-14743) that simvastatin treatment of endothelial cells leads to a marked decrease in PKA-modulated phosphorylation of the protein VASP. Here we show that long-term treatment of mice with simvastatin attenuates the vasorelaxation response to the ß-adrenergic agonist isoproterenol, without affecting endothelin-induced vasoconstriction or carbachol-induced vasorelaxation. We found that statin treatment of endothelial cells dose-dependently inhibits PKA activation as assessed by analyses of serine 157 VASP phosphorylation as well as Epac-mediated Rap1 activation. These effects of simvastatin are completely reversed by mevalonate and by geranylgeranyl pyrophosphate, implicating geranylgeranylation as a critical determinant of the stain response. We used biochemical approaches as well as fluorescence resonance energy transfer (FRET) methods with a cAMP biosensor to show that simvastatin treatment of endothelial cells markedly inhibits cAMP accumulation in response to epinephrine. Importantly, simvastatin treatment significantly decreases Gα(s) abundance, without affecting other Gα subunits. Simvastatin treatment does not influence Gα(s) protein stability, and paradoxically increases the abundance of Gα(s) mRNA. Finally, we found that simvastatin treatment inhibits Gα(s) translation mediated by Akt/mTOR/eIF4/4EBP. Taken together, these findings establish a novel mechanism by which simvastatin modulates ß-adrenergic signaling in vascular wall, and may have implications for cardiovascular therapeutics.

Angiotensin-II and MARCKS: a hydrogen peroxide- and RAC1-dependent signaling pathway in vascular endothelium.

MARCKS is an actin-binding protein that modulates vascular endothelial cell migration and cytoskeleton signaling (Kalwa, H., and Michel, T. (2011) J. Biol. Chem. 286, 2320-2330). Angiotensin-II is a vasoactive peptide implicated in vascular physiology as well as pathophysiology; the pathways connecting angiotensin-II and cytoskeletal remodeling are incompletely understood. Here we show that MARCKS is expressed in intact arterial preparations, with prominent staining of the endothelium. In endothelial cells, angiotensin-II-promoted MARCKS phosphorylation is abrogated by PEG-catalase, implicating endogenous H(2)O(2) in the angiotensin-II response. Studies using the H(2)O(2) biosensor HyPer2 reveal that angiotensin-II promotes increases in intracellular H(2)O(2). We used a Rac1 FRET biosensor to show that angiotensin-II promotes Rac1 activation that is attenuated by PEG-catalase. siRNA-mediated Rac1 knockdown blocks angiotensin-II-stimulated MARCKS phosphorylation. Cell imaging studies using a phosphoinositide 4,5-bisphosphate (PIP(2)) biosensor revealed that angiotensin-II PIP(2) regulation depends on MARCKS and H(2)O(2). siRNA-mediated knockdown of MARCKS or Rac1 attenuates receptor-mediated activation of the tyrosine kinase c-Abl and disrupts actin fiber formation. These studies establish a critical role for H(2)O(2) in angiotensin-II signaling to the endothelial cytoskeleton in a novel pathway that is critically dependent on MARCKS, Rac1, and c-Abl.