The application of peptide nucleic acid probes for rapid detection and enumeration of eubacteria, Staphylococcus aureus and Pseudomonas aeruginosa in recreational beaches of S. Florida

A novel chemiluminescent in situ hybridization technique using peptide nucleic acids (PNA) was adapted for the detection of bacteria in beach sand and recreational waters in South Florida. The simultaneous detection and enumeration of eubacteria and the novel indicators, Staphylococcus aureus and Pseudomonas aeruginosa, was achieved within 6-8 h of processing. Following 5 h of incubation on TSA, soybean peroxidase-labeled peptide nucleic acid probes (Boston Probes, Boston, MA) targeting species-specific 16S rRNA sequences of P. aeruginosa and S. aureus were used to hybridize microcolonies of the target species in-situ. In addition, a universal probe for 16S rRNA sequences was used to target the eubacteria. Probes were detected after a light generating reaction with a chemiluminescent substrate and their presence recorded on Polaroid film. The probes showed limited cross-reactivity with mixed indigenous bacteria extracted from seawater and sand by shaking with phosphate-buffered saline (PBS). Specificity and cross-reactivity was tested on the reference bacterial genera Pseudomonas, Staphylococcus, Vibrio, Shigella, Salmonella, Acinetobacter, Enterobacter, Escherichia and Citrobacter. These tests confirmed that the probes were specific for the microorganisms of interest and were unaffected by high salt levels. The results of the PNA chemiluminescent in situ hybridization were compared with traditional plate count methods (PCM) for total 'freshwater' eubacteria, S. aureus and P. aeruginosa. Counts of eubacteria and S. aureus were comparable with numbers obtained from traditional plate counts but levels of P. aeruginosa were higher with PNA than with PCM. It is possible that PNA is more sensitive than PCM because it can detect microcolonies on the agar surface that never fully develop with the plate count method. We conclude that the in situ hybridization technique used here represents an important potential tool for the rapid monitoring of novel indicator organisms in beaches and recreational waters.     

Molecular and physiological evaluation of subtropical environmental isolates of Acanthamoeba spp., causal agent of Acanthamoeba keratitis

Previous molecular examination of Acanthamoeba spp. has resulted in the determination of distinct genotypes in this genus (designated T1-T12, T14). Genotype T4 has been responsible for the majority of cases of Acanthamoeba keratitis. Here we examine the relative abundance of environmental T4 isolates on beaches and ask whether they have temperature and salinity tolerances that could enhance pathogenicity. Twenty-four Acanthamoeba strains were isolated from beach sand (n = 20), soil (n = 3), and tap water (n = 1) in south Florida. Phylogenetic analysis identified 19 of 24 isolates as T4, the Acanthamoeba keratitis-associated genotype. The remaining isolates were genotype T5 (4) and T11 (1). Nearly all beach isolates were genotype T4, whereas the tap water and soil isolates were mostly T5. All amoebae grew at 0, 1.0, and 2.0% salt and 19 of 20 beach isolates also grew at 3.2%. No soil or tap-water acanthamoebae reproduced at 3.2%. All isolates grew at 37 degrees C and two (T5) at 42 degrees C. Little correlation existed between beach location, salt-tolerance, and genetic relatedness. Overall, the large majority of environmental isolates obtained were genotype T4, suggesting it may be the most common genotype in this environment and could be a potential source of Acanthamoeba keratitis infections.     

Induction of petite mutants in yeast Saccharomyces cerevisiae by the anticancer drug dequalinium

Dequalinium (DEQ), a drug with both antimicrobial and anticancer activity, induced the formation of petite (respiration-deficient) mutants in the yeast Saccharomyces cerevisiae. DEQ was found to be approximately 50-fold more potent than ethidium bromide (EB) at inducing petites. Analysis of the DEQ-induced petite mutants indicated a complete loss of mitochondrial DNA (<1 copy/cell). Prior to the loss of mtDNA, DEQ caused cleavage of the mtDNA into a population of fragments 30-40kbp in size suggesting that this drug causes petites by inducing a breakdown of mtDNA. The selective effect of DEQ on yeast mtDNA may underlie the antifungal activity of this chemotherapeutic agent.     

Flt3-ligand, IL-4, GM-CSF, and adherence-mediated isolation of murine lung dendritic cells: assessment of isolation technique on phenotype and function

Lung dendritic cells (DCs) are difficult to study due to their limited quantities and the complexities required for isolation. Although many procedures have been used to overcome this challenge, the effects of isolation techniques on lung DCs have not been reported. The current study shows that freshly isolated DCs (CD11c+) have limited ability to induce proliferation in allogeneic T cells, and are immature as indicated by low cell surface expression of costimulatory molecules compared with liver or splenic DCs. DCs isolated after overnight culture or from mice treated with Flt3L are phenotypically mature and potent stimulators of allogeneic T cells. DCs could not be propagated from lung mononuclear cells in response to IL-4 and GM-CSF. Contrary to data reported for nonpulmonary DCs, expression of CCR6 was decreased on mature lung DCs, and only a subset of mature DCs expressed higher levels of CCR7. Absence of CD8alpha expression indicates that freshly isolated DCs are myeloid-type, whereas mature DCs induced by overnight culture are both "lymphoid" (CD8alpha+) and "myeloid" (CD8alpha-). DCs from mice genetically deficient in CD8alpha expression were strong simulators of allogeneic T cells which was consistent with data showing that CD8alpha- DCs from CD8alpha-sufficient mice are better APCs compared with CD8alpha+ DCs from the same mice. These data show that freshly isolated lung DCs are phenotypically and functionally distinct, and that the isolation technique alters the biology of these cells. Therefore, lung DC phenotype and function must be interpreted relative to the technique used for isolation.     

Allosteric regulation of the biotin-dependent enzyme pyruvate carboxylase by acetyl-CoA

The activity of the biotin-dependent enzyme pyruvate carboxylase from many organisms is highly regulated by the allosteric activator acetyl-CoA. A number of X-ray crystallographic structures of the native pyruvate carboxylase tetramer are now available for the enzyme from Rhizobium etli and Staphylococcus aureus. Although all of these structures show that intersubunit catalysis occurs, in the case of the R. etli enzyme, only two of the four subunits have the allosteric activator bound to them and are optimally configured for catalysis of the overall reaction. However, it is apparent that acetyl-CoA binding does not induce the observed asymmetrical tetramer conformation and it is likely that, under normal reaction conditions, all of the subunits have acetyl-CoA bound to them. Thus the activation of the enzyme by acetyl-CoA involves more subtle structural effects, one of which may be to facilitate the correct positioning of Arg353 and biotin in the biotin carboxylase domain active site, thereby promoting biotin carboxylation and, at the same time, preventing abortive decarboxylation of carboxybiotin. It is also apparent from the crystal structures that there are allosteric interactions induced by acetyl-CoA binding in the pair of subunits not optimally configured for catalysis of the overall reaction.     

Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth. This system incorporates mechanical, topographical, adhesive and chemical signals. Mechanical properties are controlled by the type of material used to fabricate the electrospun fibers. In this protocol we use 30% methacrylated Hyaluronic Acid (HA), which has a tensile modulus of ~500 Pa, to produce a soft fibrous scaffold. Electrospinning on to a rotating mandrel produces aligned fibers to create a topographical cue. Adhesion is achieved by coating the scaffold with fibronectin. The primary challenge addressed herein is providing a chemical signal throughout the depth of the scaffold for extended periods. This procedure describes fabricating poly(lactic-co-glycolic acid) (PLGA) microspheres that contain Nerve Growth Factor (NGF) and directly impregnating the scaffold with these microspheres during the electrospinning process. Due to the harsh production environment, including high sheer forces and electrical charges, protein viability is measured after production. The system provides protein release for over 60 days and has been shown to promote primary nerve cell growth.     

CD1d1-dependent control of the magnitude of an acute antiviral immune response

CD1d1-restricted NK T (NKT) cells rapidly secrete both Th1 and Th2 cytokines upon activation and are therefore thought to play a regulatory role during an immune response. In this study we examined the role of CD1d1 molecules and NKT cells in regulating virus-induced cytokine production. CD1d1-deficient (CD1KO) mice, which lack NKT cells, were infected with lymphocytic choriomeningitis virus, and spontaneous cytokine release from splenocytes was measured. We found that CD1KO mice produce significantly higher amounts of IL-2, IL-4, and IFN-gamma compared with wild-type controls postinfection. Depletion studies of individual lymphocyte subpopulations suggested that CD4+ T cells are required; however, isolation of specific lymphocyte populations indicated that CD4+ T cells alone are not sufficient for the increase in cytokine production in CD1KO mice. Splenocytes from lymphocytic choriomeningitis virus-infected CD1KO mice continued to produce enhanced cytokine levels long after viral clearance and cleared viral RNA faster than wild-type mice. There was no difference in the number of splenocytes between uninfected wild-type and CD1KO mice, whereas the latter knockout mice had an increased number of splenocytes after infection. Collectively, these data provide clear evidence that the expression of CD1d1 molecules controls the magnitude of the cell-mediated immune response to an acute viral infection.     

IL-12 p40 homodimer-dependent macrophage chemotaxis and respiratory viral inflammation are mediated through IL-12 receptor beta 1

Leukocyte recruitment to the airway lumen is a central feature of inflammatory conditions such as asthma and respiratory viral infection. Characterization of mediators that regulate leukocyte recruitment in these conditions revealed increased IL-12 p40 homodimer (p80) levels were associated with enhanced airway macrophage accumulation. To examine this association, we used in vivo and in vitro assays to demonstrate p80, but not IL-12 or p40, provided a macrophage chemoattractant signal. Macrophages from genetically deficient mice indicated p80-dependent chemotaxis was independent of IL-12 and required IL-12Rbeta1 (Rbeta1) expression. Furthermore, analysis of murine cell lines and primary culture macrophages revealed Rbeta1 expression, with an intact cytoplasmic tail, was necessary and sufficient to mediate p80-dependent chemotaxis. To examine the role for Rbeta1 in mediating macrophage accumulation in vivo, we contrasted Sendai virus-driven airway inflammation in wild-type and Rbeta1-deficient mice. Despite similar viral burden and production of the macrophage chemoattractant p80, the Rbeta1-deficient mice displayed a selective decrease in airway macrophage accumulation and resistance to viral-dependent mortality. Thus, Rbeta1 mediates p80-dependent macrophage chemotaxis and inhibition of the p80-Rbeta1 interaction may provide a novel anti-inflammatory strategy to manipulate the inflammation associated with asthma and respiratory viral infection.     

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.     

Characterization of the Bacterial Communities of Life Stages of Free Living Lone Star Ticks (Amblyomma americanum)

The lone star tick (Amblyomma americanum) is an abundant and aggressive biter of humans, domestic animals, and wildlife in the southeastern-central USA and an important vector of several known and suspected zoonotic bacterial pathogens. However, the biological drivers of bacterial community variation in this tick are still poorly defined. Knowing the community context in which tick-borne bacterial pathogens exist and evolve is required to fully understand the ecology and immunobiology of the ticks and to design effective public health and veterinary interventions. We performed a metagenomic survey of the bacterial communities of questing A. americanum and tested 131 individuals (66 nymphs, 24 males, and 41 females) from five sites in three states. Pyrosequencing was performed with barcoded eubacterial primers targeting variable 16S rRNA gene regions 5–3. The bacterial communities were dominated by Rickettsia (likely R. amblyommii) and an obligate Coxiella symbiont, together accounting for 6.7–100% of sequences per tick. DNAs from Midichloria, Borrelia, Wolbachia, Ehrlichia, Pseudomonas, or unidentified Bacillales, Enterobacteriaceae, or Rhizobiales groups were also detected frequently. Wolbachia and Midichloria significantly co-occurred in Georgia (p<0.00001), but not in other states. The significance of the Midichloria-Wolbachia co-occurrence is unknown. Among ticks collected in Georgia, nymphs differed from adults in both the composition (p = 0.002) and structure (p = 0.002) of their bacterial communities. Adults differed only in their community structure (p = 0.002) with males containing more Rickettsia and females containing more Coxiella. Comparisons among adult ticks collected in New York and North Carolina supported the findings from the Georgia collection despite differences in geography, collection date, and sample handling, implying that the differences detected are consistent attributes. The data also suggest that some members of the bacterial community change during the tick life cycle and that some sex-specific attributes may be detectable in nymphs.