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

High Numbers of Staphylococcus aureus at Three Bathing Beaches in South Florida

While the value of Staphylococcus aureus as an indicator for non-enteric diseases is unclear, understanding its prevalence in recreational beaches would prove useful, given its pathogenic potential. Staphylococcus aureus levels were evaluated in sand and seawater at three beaches during one year. To elucidate possible S. aureussources or colonization trends, distribution in sand was analyzed at Hollywood Beach. Staphylococcus aureus levels fluctuated throughout the study with highest average densities detected in dry sand (3.46 × 105 CFU/g, Hobie Beach), particularly at beaches with high human density. Patchy distribution marked hotspots of human use and/or possible bacterial re-growth. Data from a brief epidemiological survey indicated a very slight association between beach usage and skin conditions; suggesting high S. aureus levels in sand may not necessarily constitute major health risks. Because the possibility of disease transmission exists, particularly to children and immuno-compromised beach-goers, periodic surveying of highly frequented beaches seems warranted

Cellular functions of DNA gyrase and conditional gene regulation in the malaria parasite Plasmodium falciparum

Malaria is among the greatest disease threats to global public health. As part of the effort to combat the disease, the development of new anti-malarials remains imperative. The causative agent of malaria is a parasitic pathogen of the genus Plasmodium which is transmitted to humans through the bite of an anopheline mosquito. The parasite contains a relic, non-photosynthetic plastid (called the apicoplast) of red algal origin that was acquired by an ancestral form through a secondary endosymbiotic event. The apicoplast supports critical biosynthetic processes in the parasite that are divergent from the equivalent host pathway, providing substantial opportunity for the identification of possible drug targets. The Plasmodium DNA gyrase is a eubacterial type II topoisomerase that is expected to function within the apicoplast, with a principal role in regulating the topological transitions of the 35 kb circular apicoplast genome. In this study, the anti-bacterial ciprofloxacin was used to probe the cellular functions of DNA gyrase. For further characterization of enzyme functions, transgenic parasites were generated to develop a system for conditional gene regulation. Results from this study indicated that targeting DNA gyrase with ciprofloxacin inhibited the replication of apicoplast DNA and the accumulation of apicoplast RNA transcripts, and blocked the production of apicoplast ribosomal RNA. The apicoplast nucleoid appeared reduced in size and did not segregate normally, and the apicoplast displayed abnormal morphology. In contrast, mitochondrial DNA replication, transcription, and segregation were unaffected. The DNA gyrase appeared to associate with the apicoplast DNA at non-specific loci, implicating numerous functional roles for the enzyme. Results from the conditional gene regulation studies indicated that the Tet-transactivator system has potential applicability for controlling endogenous gene expression in P. falciparum. Artificial promoters were successfully targeted to the pfpm4 genomic locus, and piggyBac insertion of transactivator (TaTi2) expression cassettes resulted in stable TaTi2 expression. In washout studies, the temporal pattern of pfpm4 RNA expression closely followed the expression of tati2. In the induced state of target transgene expression the steady-state TaTi2 levels decreased, a phenomenon that will be further investigated

Fecal Indicator Organisms and Pathogenic Protozoa in South Florida Beach Sand: Implications for Public Health

Traditionally, the hygienic quality of beaches has been determined by monitoring the water for microbial indicators of fecal pollution. Beach sand, which may also be an important medium for the transmission of fecal borne pathogens, has rarely been examined. The aims of this study where to examine the prevalence of fecal indicator organisms in tidally affected beach sand and in dryer upper beach sand, relative to water; identify the potential sources of indicator organisms in beach sand; examine the prevalence of selected eukaryotic microbes at sandy beaches; and investigate the potential health risks related to beach use. Three south Florida Beaches (Ft. Lauderdale Beach, Hollywood Beach, Hobe Beach) were sampled bimonthly for a I year period. Significantly, enterococci, fecal coliform, and E. coli levels were consistently present at higher concentrations in beach sand compared to the seawater at all 3 study beaches. Levels of somatic and F-specific coliphages were also present at higher concentrations in beach sand. Microbial- source tracking analysis by carbon utilization profiling suggested that the predominate sources of enterococci in beach sand were seagulls, and transiently replicating indigenous populations. Acanthamoeba spp. was the most commonly isolated free-living naked amoeba in this study and molecular analysis revealed that 19 of the 20 beach sand clones were genotype T4, the Acanthamoeba keratitis-associated genotype. With respect to salinity, the growth characteristics of beach sand Acanthamoeba isolates were similar to Acanthamoeba isolated from corneal scrapings. Results from the beach survey indicated that beach goers may have an increased risk for acquiring contact related ailments at Hobe Beach. Accordingly, bacterial and viral fecal indicator microbes were detected at the highest frequency and greatest average concentrations from Robe Beach. Reports of enteric and respiratory related symptoms were not higher in beach goers compared to the control cohort