Human-Associated Methicillin-Resistant Staphylococcus aureus from a Subtropical Recreational Marine Beach

Reports of Staphylococcus aureus including methicillin-resistant S. aureus (MRSA) detected in marine environments have occurred since the early 1990s. This investigation sought to isolate and characterize S. aureus from marine waters and sand at a subtropical recreational beach, with and without bathers present, in order to investigate possible sources and to identify the risks to bathers of exposure to these organisms. During 40 days over 17 months, 1,001 water and 36 intertidal sand samples were collected by either bathers or investigators at a subtropical recreational beach. Methicillin-sensitive S. aureus (MSSA) and MRSA were isolated and identified using selective growth media and an organism-specific molecular marker. Antimicrobial susceptibility, staphylococcal cassette chromosome mec (SCCmec) type, pulsed-field gel electrophoresis (PFGE) pattern, multi-locus sequence type (MLST), and staphylococcal protein A (spa) type were characterized for all MRSA. S. aureus was isolated from 248 (37 %) bather nearby water samples at a concentration range of <2–780 colony forming units per ml, 102 (31 %) ambient water samples at a concentration range of <2–260 colony forming units per ml, and 9 (25 %) sand samples. Within the sand environment, S. aureus was isolated more often from above the intertidal zone than from intermittently wet or inundated sand. A total of 1334 MSSA were isolated from 37 sampling days and 22 MRSA were isolated from ten sampling days. Seventeen of the 22 MRSA were identified by PFGE as the community-associated MRSA USA300. MRSA isolates were all SCCmec type IVa, encompassed five spa types (t008, t064, t622, t688, and t723), two MLST types (ST8 and ST5), and 21 of 22 isolates carried the genes for Panton–Valentine leukocidin. There was a correlation (r?=?0.45; p?=?0.05) between the daily average number of bathers and S. aureus in the water; however, no association between exposure to S. aureus in these waters and reported illness was found. This report supports the concept that humans are a potential direct source for S. aureus in marine waters.     

Histone Deacetylase Inhibition Enhances Tissue Plasminogen Activator Release Capacity in Atherosclerotic Man

The expression of the tissue plasminogen activator (t-PA) gene appears to be under epigenetic control and can be affected by histone deacetylation inhibition. The study aimed to test if histone deacetalyase inhibitor treatment lead to increased t-PA release or reduced exhaustion in t-PA release in response to stimulation, as well as change in plasminogen activator inhibitor-1 (PAI-1) in subjects with coronary disease. In this clinical study, 16 post-myocardial infarction subjects, the perfused forearm model was used with isoprenaline provocation during 20 minutes, to stimulate local t-PA release. Each subject was measured twice on the same day (repeated stimuli sequences) as well as on two different occasions, without treatment and after four weeks of treatment with valproic acid (500 mg, twice daily). Net forearm release for t-PA in response to isoprenaline at minutes 1.5, 3, 6, 9, 12, 15 and 18 was measured, allowing assessment of cumulative t-PA release. There was a reduction in the exhaustion of cumulative t-PA release during repeated and prolonged stimulation with valproic acid treatment compared to non-treatment. Plasma PAI-1 antigen was decreased following treatment compared to non-treatment (18.4 ± 10.0 vs. 11.0 ± 7.1 nanograms/ml respectively, mean with 95% confidence interval). These findings demonstrate that histone deacetylation inhibition increases the capacity for endogenous t-PA release in subjects with vascular disease. Furthermore, the fibrinolytic balance is favored with suppressed PAI-1 levels. More studies are needed to establish the clinical relevance of these findings.

Trial registration

EU Clinical Trials Register 2012-004950-27     

Decreased mortality in a rat model of acute postinfarction heart failure

INTRODUCTION: The rat model of postinfarction heart failure (HF) has been very valuable in experimental cardiology. One disadvantage of this model is the very high acute mortality (70-80%). The aim of this study was to evaluate whether measures of intensive cardiac care applied to rats with acute myocardial infarction (MI) would reduce mortality. METHODS: Male Sprague-Dawley rats weighing approximately 300 g were used. The animals were randomized into two groups. The intensive care group (IC) n=20 and conventional care group (CC) n=20. Experimental MI was induced by ligation of the left coronary artery producing large anterolateral MI. Animals in the IC group received isoflurane anesthesia and respiratory support postoperatively. The heart rhythm was monitored continuously and ventricular arrhythmias were treated with amiodarone and cardioversion. RESULTS: Mortality rate within 24 h was 4/20 (20%) in the IC group and 14/20 (70%) in the CC group (p<0.01). This represents a 3.5-fold reduction in acute mortality rate. CONCLUSIONS: The use of amiodarone, respiratory support, isoflurane gas anesthesia, and electrical cardioversion of malignant arrhythmias are simple and effective measures to reduce mortality in rats with acute MI and HF. Improving survival rates increases cost-efficiency and ethical acceptance of this important experimental HF model.     

Hyperinsulinemia: effect on cardiac mass/function, angiotensin II receptor expression, and insulin signaling pathways

To investigate the association between hyperinsulinemia and cardiac hypertrophy, we treated rats with insulin for 7 wk and assessed effects on myocardial growth, vascularization, and fibrosis in relation to the expression of angiotensin II receptors (AT-R). We also characterized insulin signaling pathways believed to promote myocyte growth and interact with proliferative responses mediated by G protein-coupled receptors, and we assessed myocardial insulin receptor substrate-1 (IRS-1) and p110 alpha catalytic and p85 regulatory subunits of phospatidylinositol 3 kinase (PI3K), Akt, MEK, ERK1/2, and S6 kinase-1 (S6K1). Left ventricular (LV) geometry and performance were evaluated echocardiographically. Insulin decreased AT1a-R mRNA expression but increased protein levels and increased AT2-R mRNA and protein levels and phosphorylation of IRS-1 (Ser374/Tyr989), MEK1/2 (Ser218/Ser222), ERK1/2 (Thr202/Tyr204), S6K1 (Thr421/Ser424/Thr389), Akt (Thr308/Thr308), and PI3K p110 alpha but not of p85 (Tyr508). Insulin increased LV mass and relative wall thickness and reduced stroke volume and cardiac output. Histochemical examination demonstrated myocyte hypertrophy and increases in interstitial fibrosis. Metoprolol plus insulin prevented the increase in relative wall thickness, decreased fibrosis, increased LV mass, and improved function seen with insulin alone. Thus our data demonstrate that chronic hyperinsulinemia decreases AT1a-to-AT2 ratio and increases MEK-ERK1/2 and S6K1 pathway activity related to hypertrophy. These changes might be crucial for increased cardiovascular growth and fibrosis and signs of impaired LV function.     

Presence of Pathogens and Indicator Microbes at a Non-Point Source Subtropical Recreational Marine Beach

Swimming in ocean water, including ocean water at beaches not impacted by known point sources of pollution, is an increasing health concern. This study was an initial evaluation of the presence of indicator microbes and pathogens and the association among the indicator microbes, pathogens, and environmental conditions at a subtropical, recreational marine beach in south Florida impacted by non-point sources of pollution. Twelve water and eight sand samples were collected during four sampling events at high or low tide under elevated or reduced solar insolation conditions. The analyses performed included analyses of fecal indicator bacteria (FIB) (fecal coliforms, Escherichia coli, enterococci, and Clostridium perfringens), human-associated microbial source tracking (MST) markers (human polyomaviruses [HPyVs] and Enterococcus faecium esp gene), and pathogens (Vibrio vulnificus, Staphylococcus aureus, enterovirus, norovirus, hepatitis A virus, Cryptosporidium spp., and Giardia spp.). The enterococcus concentrations in water and sand determined by quantitative PCR were greater than the concentrations determined by membrane filtration measurement. The FIB concentrations in water were below the recreational water quality standards for three of the four sampling events, when pathogens and MST markers were also generally undetectable. The FIB levels exceeded regulatory guidelines during one event, and this was accompanied by detection of HPyVs and pathogens, including detection of the autochthonous bacterium V. vulnificus in sand and water, detection of the allochthonous protozoans Giardia spp. in water, and detection of Cryptosporidium spp. in sand samples. The elevated microbial levels were detected at high tide and under low-solar-insolation conditions. Additional sampling should be conducted to further explore the relationships between tidal and solar insolation conditions and between indicator microbes and pathogens in subtropical recreational marine waters impacted by non-point source pollution.Global estimates indicate that each year more than 120 million cases of gastrointestinal disease and 50 million cases of severe respiratory diseases are caused by swimming and bathing in wastewater-polluted coastal waters (42). Swimming-related illness is attributed predominantly to exposure to microbial pathogens, which enter the water through point sources, such as sewage outfalls. Water quality at beaches may also be impacted by non-point sources, such as storm water runoff, sand resuspension, animal fecal inputs, and human bather shedding (8, 12, 22, 47, 59).The concentration of indicator microorganisms in a body of recreational water is used to estimate the health risk to bathers. These microbes serve as surrogates for microbial pathogens. Studies show that the U.S. Environmental Protection Agency (EPA)-recommended indicator microbe for marine beaches, enterococci, shows a significant correlation with illness in marine beaches impacted by point source pollution (38, 54). However, a similar correlation has not been identified at beaches impacted by non-point source pollution or subtropical marine beaches (17, 29, 38, 54).The failure to consistently demonstrate an association between enterococci and illness at non-point source beaches calls into question the ability of indicator microbes to predict the presence of pathogens. Studies conducted on the west coast of the United States have shown that indicators are often not correlated with measured pathogens at non-point source beaches (31, 32, 33, 37). Additional studies conducted in a subtropical environment, such as that of South Florida, where this study was conducted, have repeatedly shown the limited accuracy of indicator microbe standards for determining the presence of pathogens (27, 35). This lack of correlation is understandable since an indicator microbe, such as enterococci, may come from relatively low-risk sources of fecal pollution and therefore may not be related to human or other high-risk sources of fecal pollution and pathogens (9). It has also been shown in both subtropical and temperate climates that indicator bacteria can multiply in the environment, resulting in a false impression of increased microbial pollution and pathogen presence (4, 7, 19, 24, 41, 45, 57, 58). Environmental factors, such as tide, rain, and solar insolation, can also have significant and varying effects on the levels of indicator and pathogenic microbes (21, 24, 33).The lack of correlation between pathogens and indicator microbes at non-point source beaches can result in two problematic scenarios. If indicator microbes are absent and pathogens are present (false-negative scenario), regulatory monitoring may fail to identify the potential adverse health effects on bathers due to the pathogens. This problem is likely to occur since indicator bacteria are less resistant to environmental stresses and disinfection at wastewater treatment plants than certain pathogens (6, 15). However, if indicator microbes are present and pathogens are absent (false-positive scenario), there can be unnecessary economic losses due to recreational beach advisories and/or closures. A 4-month closure of Huntington Beach in 1999 due to microbial standard violations resulted in the loss of millions of dollars in tourism income to the business community and almost 2 million dollars in beach closure investigation fees (55; for a review, see reference 27).Given these two possible scenarios, the relationship between indicator microbes and pathogens under different environmental conditions at non-point source beaches representing different geographic and climatic settings should be assessed further. Investigation of this relationship would require a large sample size in order to establish possible significant associations between the various factors and targets. The objective of this study was to conduct a preliminary evaluation of the presence of indicator microbes and pathogens and the possible association between indicator microbes, pathogen measurements, and environmental conditions at a subtropical recreational marine beach in South Florida. Because of cost limitations when multiple targets, including pathogens, are screened, this study was not intended to establish a conclusive relationship between the various factors and targets but was intended to provide insight into both the presence of organisms and possible associations which should be investigated further. Such information would be useful for understanding the potential health risks to bathers from non-point sources of microbes and would also contribute to determining the appropriateness of using indicator microbes to monitor the water quality at non-point source beaches. Although previous studies have assessed the presence of either viral, protozoan, or bacterial pathogens along with indicator microbes in point or non-point source recreational beach waters (27, 33, 37, 47), to our knowledge, this is the first study to assess the presence of all three classes of pathogens (viral, protozoan, and bacterial) as well as indicator microbes at a non-point source recreational beach. This study is also the first study to sample for all these microbes in both water and sand at a non-point source recreational beach. Through analysis of the various microbes under different targeted environmental conditions, this study also included a preliminary evaluation of the sources of microbial contaminants and pathogens and the effectiveness of various analytical methods for microbe detection. The latter part of the study included a comparison of three different methods for enterococcus enumeration, as well as an innovative method for simultaneously concentrating protozoans and viruses from water samples.     

Relation of beta-casomorphin to apnea in sudden infant death syndrome

Sudden infant death syndrome (SIDS) is the most common cause of death in infants and its pathogenesis is complex and multifactorial. The aim of this review is to summarize recent novel findings regarding the possible association of beta-casomorphin (beta-CM) to apnea in SIDS, which has not been widely appreciated by pediatricians and scientists. beta-CM is an exogenous bioactive peptide derived from casein, a major protein in milk and milk products, which has opioid activity. Mechanistically, circulation of this peptide into the infant's immature central nervous system might inhibit the respiratory center in the brainstem leading to apnea and death. This paper will review the possible relationship between beta-CM and SIDS in the context of passage of beta-CM through the gastrointestinal tract and the blood-brain barrier (BBB), permeability of the BBB to peptides in infants, and characterization of the casomorphin system in the brain.     

Mutational analyses of Epstein-Barr virus glycoprotein 42 reveal functional domains not involved in receptor binding but required for membrane fusion

Epstein-Barr virus (EBV) is a human gammaherpesvirus associated with malignancies of both epithelial and lymphoid origin. Efficient infection of the latent host reservoir B lymphocytes involves the binding of glycoproteins gp350/220 for initial attachment, followed by the concerted action of gH, gL, gB, and gp42 for membrane fusion. The type II membrane protein gp42 is required for infection of B cells and assembles into a complex with gH and gL. The cellular host receptor for gp42, class II human leukocyte antigen (HLA), has been structurally verified by crystallization analyses of gp42 bound to HLA-DR1. Interestingly, the crystal structure revealed a hydrophobic pocket consisting of many aromatic and aliphatic residues from the predicted C-type lectin domain of gp42 that in other members of the C-type lectin family binds major histocompatibility complex class I or other diverse ligands. Although the hydrophobic pocket does not bind HLA class II, mutational analyses presented here indicate that this domain is essential for EBV-induced membrane fusion. In addition, mutational analysis of the region of gp42 contacting HLA class II in the gp42-HLA-DR1 cocrystal confirms that this region interacts with HLA class II and that this interaction is also important for EBV-induced membrane fusion.     

In vivo metabolic imaging of cardiac bioenergetics in transgenic mice

Recent advances in transgenic technology have made the mouse a particularly interesting small animal in cardiovascular research. Increasingly sophisticated experimental methods and tools are needed for detailed characterization of cardiovascular physiology and biochemistry in the mice. The objective of this study was to develop a method for noninvasive evaluation of cardiac energy metabolism in the mouse. Cardiac gated (31)P magnetic resonance spectroscopy using Image Selected in Vivo Spectroscopy (ISIS) method was applied in old mice overexpressing bovine growth hormone (bGH) (n = 5) and control mice (n = 5). The localized volumes of interest were 128 and 112 microL, respectively. Phosphocreatine-to-ATP ratio was 1.5 +/- 0.13 in the bGH mice and 2.1 +/- 0.04 in the control group (P < 0.01). The study demonstrates the feasibility of application of volume-selective (31)P MRS for evaluation of cardiac energy metabolism in the mouse under maintained physiological conditions.     

Tissue-specific tropomyosin isoform composition.

Four distinct genes encode tropomyosin (Tm) proteins, integral components of the actin microfilament system. In non-muscle cells, over 40 Tm isoforms are derived using alternative splicing. Distinct populations of actin filaments characterized by the composition of these Tm isoforms are found differentially sorted within cells (Gunning et al. 1998b). We hypothesized that these distinct intracellular compartments defined by the association of Tm isoforms may allow for independent regulation of microfilament function. Consequently, to understand the molecular mechanisms that give rise to these different microfilaments and their regulation, a cohort of fully characterized isoform-specific Tm antibodies was required. The characterization protocol initially involved testing the specificity of the antibodies on bacterially produced Tm proteins. We then confirmed that these Tm antibodies can be used to probe the expression and subcellular localization of different Tm isoforms by Western blot analysis, immunofluorescence staining of cells in culture, and immunohistochemistry of paraffin wax-embedded mouse tissues. These Tm antibodies, therefore, have the capacity to monitor specific actin filament populations in a range of experimental systems.