In order to determine the source of bacteria in Tillamook Bay, Oregon, water samples were collected monthly for six months during the rainy season from October 1975 through March 1976 from the bay and its tributaries, the Kilchis, Trask, Tillamook, and Wilson Rivers. Fecal coliform levels of these samples were determined and the 1,917 bacteria isolated were tested for their resistance patterns to chloramphenicol (Cm), streptomycin (Sm), ampicillin (Am), tetracycline (Tc), chlortetracycline (Ct), oxytetracycline (Ot), neomycin (Nm), nitrofurazone (Ni), nalidixic acid (Na), sulfathiazole (Su), kanamycin (Km), and procaine penicillin G (Pe). The fecal coliform count per 100 ml of bay water ranged from 3.6 to 42.0. The counts for Tillamook River ranged from 13.5 to 112.0, Trask River from 0.0 to 132.0, Wilson River from 8.5 to 105.0, and Kilchis River from 0.5 to 13.9. The rise and fall of fecal coliform levels were characteristic of the sampling date and each sampling station showed its characteristic maximum and minimum levels. The 1,917 fecal coliform isolates showed 176 different resistance patterns to the 12 antibiotics tested. None of the patterns, however, was characteristic of any specific sampling site. The fecal coliform counts of the bay were statistically compared to 135 independent variables that included the fecal coliform counts of tributaries, temperature, river flow data, tide information, antibiotic use data, and the antibiotic resistance patterns. Bay fecal coliform levels were highly correlated with the fecal coliform counts of tributaries especially those of the Trask and Wilson Rivers, degree of resistance to antibiotics, recreational activities, and precipitation. Negative correlation existed between bay fecal coliform count and the ambient temperature. two potentially useful linear regression models to predict bay fecal coliform level were developed using a computerized stepwise multiple linear regression program. / Graduation date: 1977

The need to develop a renewable, environmentally friendly source of energy has become a primary focus in modern science, with bio gas showing considerable potential. Interest in the methanogenic Archaea has therefore grown in recent years and extensive studies have been carried out to investigate the population diversity in various habitats. Presently, there are only a few studies that have evaluated the microbial communities inhabiting the gastrointestinal tract of wildlife native to southern Africa. This study aimed to investigate the microbial diversity, in particular the bacterial and methanogen communities involved in fermentative digestion in the gastrointestinal tract of zebra. Assessment of the microbial diversity in zebra faeces included both culture-based techniques and nucleic acid targeting analysis via 16S rRNA gene sequencing. Quantitative analysis using selected solid media revealed high counts for aerobic and anaerobic Bacteria (7.51x108 and 2.45x109/gram of faecal sample respectively). The majority of aerobic colonies that were detected exhibited Bacillus-like morphology. Nucleic acid based analysis of the diversity of both Bacteria and methanogenic Archaea in zebra faecal material was performed. Both manual and kit based extractions were used for DNA isolation in order to compare the efficiency of the two methods. Results show that a vigorous mechanical treatment was best for the release of DNA from the faecal matter. Amplification of target gene regions was carried out using established primer pairs (ARCH69F/ARCH915R and EUB338F/EUB907R) for methanogen and bacterial DNA respectively. Amplified 16S rRNA gene regions were cloned into a high copy number vector and random clones were selected for evaluation. Clones containing the target gene were further analysed by ARDRA and were assigned to a specific phylotype. Two bacterial (105 clones in total) and three methanogen (178 clones in total) clone libraries were constructed, of which 24 phylotypes were established for Bacteria and 25 for methanogenic Archaea. A representative of each phylotype was analysed by sequencing and further phylogenetic analysis was conducted. Six bacterial phylotypes, which represented 56% of all bacterial clones, exhibited 99% sequence similarity to Bacillus species. Six methanogen phylotypes, which exhibited 99% sequence similarity to the hydrogenotrophic species Methanobrevibacter gottschalkii strain PG, were established to be predominant in zebra faeces. These phylotypes represented 71% of all archaeal clones selected for analysis in this study. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.

The glycerol enol ether, fecapentaene-12, is a direct acting fecal mutagen that is formed in the lower portion of the gastrointestional tract by anaerobic bacteria. The biological precursor to fecapentaene-12 is a natural product of mammalian origin whose role in the etiology of colon cancer is unknown. Preliminary evidence indicated that the precursor may be a plasmalogen with an intact pentaenol ether moiety. Further structural studies by means of degradative methods and chromatographic techniques enabled the structure of the precursor to be elucidated. Based on the structure of the precursor, the name plasmalopentaene-12 was coined. Synthetic methodology was developed for obtaining synthetic plasmalopentaene12. This was necessary in order to confirm the structure and to determine the precursor's biological role. The synthetic methodology proceeded through a novel "acyl migration" which enables the highly labile pentaenol ether to be generated late in the synthesis. Model studies indicated that this was a feasible pathway. It was also determined that this methodology may be highly adaptable to the synthesis of other plasmalogens and may also provide a new synthetic route to fecapentaene-12. / Ph. D.

Contamination of recreational and shellfish waters with fecal pollution is a major water quality issue with associated economic impacts and human health risks. Reliable fecal source identification and rapid, quantitative analyses are essential components of risk assessment. Enteric bacteria that are endemic to specific hosts have a potential role as public health indicators of fecal pollution. Building on previous work to discriminate ruminant and human fecal contamination, we cloned class Bacteroidetes 16S rRNA genes from pig, elk, dog, cat, and seagull fecal DNAs. Unique restriction patterns were identified among clones from each of the host species using Terminal Restriction Fragment Length Polymorphisms (T-RFLP). Clones exhibiting unique patterns were sequenced and analyzed phylogenetically, along with human, horse, and cattle sequences recovered from previous work. The analysis revealed both endemic and cosmopolitan (global) host distributions. The sequence data were used to identify host-specific genetic markers for pig and horse feces, and to design PCR primers that identify these sources of fecal pollution in water. There was a high degree of sequence overlap among the fecal Bacteroidetes of wild and domestic ruminants, and among human, domestic pet, and seagull Bacteroidetes. We compared fecal Bacteroidetes rRNA genes from these hosts using subtractive hybridization, a method that identifies differences between closely related genomes or gene sequences. A Bacteroidetes rDNA marker that distinguishes elk and cow feces was identified, as well as a host-specific marker for dog fecal Bacteroidetes. The four newly designed PCR primers were tested for specificity and sensitivity, and the dog primer was successfully used, along with the human and ruminant-specific primers, in a collaborative study comparing fecal source tracking methods. We also developed a real time Taq nuclease assay for quantification of fecal Bacteroidetes 16S rDNA, and compared it with an EPA-approved enumeration method for the current standard public health indicator, Escherichia coli, in serial dilutions of sewage primary influent. There was a strong, positive correlation between the methods, and the Taq nuclease assay was sensitive and much more rapid than the E. coli assay. PCR source identification and enumeration of fecal Bacteroidetes 16S rDNA show promise for application in a health risk-based analysis of fecal pollution. / Graduation date: 2005

No description available.

The ultimate objective of this work was to develop a real-time method for detecting and monitoring β-D-galactosidase as a suitable indicator of the potential presence of total coliform bacteria in water environments. Preliminary comparison of the chromogenic substrate, chlorophenol red β-D-galactopyranoside and the fluorogenic substrate, MuGAL, revealed unreliable results with the fluorogenic technique due to interference from compounds commonly found in environmental water samples. Thus, the chromogenic assay was further explored. Hydrolysis of the chromogenic substrate chlorophenol red β-D-galactopyranoside by β-D-galactosidase to yield chlorophenol red was the basis of this assay. Fundamental studies with chlorophenol red β-Dgalactopyranoside showed that β-D-galactosidase occurs extracellularly and in low concentrations in the polluted water environment. A direct correlation between enzyme activity and an increase in environmental water sample volume, as well as enzyme activity with total coliform colony forming unit counts were observed. Spectrophotometric detection was achieved within a maximum period of 24 h with a limit of detection level of 1 colony forming unit 100 ml[superscript -1]. This enzyme also exhibited physical and kinetic properties different from those of the pure commercially available β-D-galactosidase. Cell permeabilisation was not required for releasing enzymes into the extracellular environment. PEG 20 000 offered the best option for concentrating β-D-galactosidase. The source of β-D-galactosidase in the polluted environmental water samples was confirmed as Escherichia coli through SDS-PAGE, tryptic mapping and MALDI-TOF, thus justifying the further use of this method for detecting and/or monitoring total coliforms. Several compounds and metal ions commonly found in environmental water samples (as well as those used in water treatment processes) did have an effect on β-D-galactosidase. All the divalent cations except Mg [superscript 2+], at the concentrations studied, inhibited the relative activity of β-D-galactosidase in both commercial β-D-galactosidase and environmental samples. Immobilisation of chlorophenol red β-D-galactopyranoside onto a solid support material for the development of a strip bioprobe was unsuccessful, even though the nylon support material yielded some positive results. A monthly (seasonal) variation in β-Dgalactosidase activity from the environmental water samples was observed, with the highest activity coinciding with the highest monthly temperatures. Electro-oxidative detection and/or monitoring of chlorophenol red was possible. Chlorophenol red detection was linear over a wide range of concentrations (0.001-0.01 μg ml[superscript -1]). Interference by chlorophenol red β-D-galactopyranoside in the reduction window affected analysis. A range of phthalocyanine metal complexes were studied in an attempt to reduce fouling and/or increase the sensitivity of the biosensor. The selected phthalocyanine metal complexes were generally sensitive to changes in pH with a reduction in sensitivity from acidic pH to alkaline pH. The tetrasulphonated phthalocyanine metal complex of copper was, however, more stable with a minimum change of sensitivity. The phthalocyanine metal complexes were generally stable to changes in temperature. While only two consecutive scans were possible with the unmodified glassy carbon electrode, 77 consecutive scans were performed successfully with the CuPc-modified glassy carbon electrode. Among the phthalocyanine metal complexes studied, the CuPc-modified glassy carbon electrode therefore provided excellent results for the development of a biosensor. The CuPc modified-glassy carbon electrode detected 1 colony forming unit 100 ml[superscript -1] in 15 minutes, while the plain unmodified glassy carbon electrode required 6 hours to detect the equivalent number of colony forming units. CoPc, ZnPc and CuTSPc required 2, 2.25 and 1.75 h, respectively, to detect the same numbers of colony forming units. The CuPcmodified glassy carbon electrode detected 40 colony forming units 100 ml[superscript -1] instantly. In general, a direct correlation between colony forming units and current generated in the sensor was observed (R2=0.92). A higher correlation coefficient of 0.99 for 0-30 coliform colony forming units 100 ml[superscript -1] was determined. Current was detected in some water samples which did not show any colony forming units on the media, probably due to the phenomenon of viable but non-culturable bacteria, which is the major disadvantage encountered in the use of media for detecting indicator microorganisms. This novel biosensor therefore presents a very robust and sensitive technique for the detection and/or monitoring of coliform bacterial activity in water.

A bench study was conducted to compare two membrane filter techniques for their efficiency in recovering fecal coliforms exposed to selected heavy metals. The effects on recovery by increasing time and metal concentration were also examined. The recovery methods employed included the standard membrane filter technique (S-MF) and a modified membrane filter technique (M-MF)~ which consisted of a lactose agar overlay and a five-hour preincubation at 35 C. The heavy metals Cd, Cr, Pb, and Zn were examined, each at two concentrations, to evaluate their effect on the recovery of the I. coli test organism, after exposure for 6, 24, and 48 hours. A statistical analysis of the data found the recoveries obtained by the M-MF to be significantly greater (.0001 level) than those of the S-MF, in all cases. Time was also found to significantly effect recovery, with recoveries decreasing as time increased. A significant difference was also found between the effects of the heavy metals tested and the concentrations of metals was likewise found to significantly effect recovery, with decreased recoveries being obtained at the high concentration of each metal. / Master of Science

Wastewater samples were collected from both the secondary settling and the chlorine contact tanks at a secondary sewage treatment plant (trickling filter) in Blacksburg, Virginia and analyzed for fecal coliforms using three procedures. Physical parameters including total suspended solids, DO, pH, turbidity, temperature and total chlorine residual were measured in effort to ascertain their effect on fecal coliform recoveries. The three procedures employed included the multiple-tube fermentation technique that yields the most probable number (MPN), the standard MF technique (SF-MF), and a modified MF technique (IF-MF) which consisted of a lactose overlay and a 5-hour incubation period at 44.5°C. A statistical analysis of the data showed that the means of the recoveries by the IF-MF technique were significantly greater (0.01 level) than those by the SF-MF technique in both the secondary settling tank and the chlorine contact tank samples. Recoveries by the IF-MF technique were comparable to those by the MPN technique when samples from the secondary settling basin were analyzed, but not in samples from the chlorine contact tank. However, the means of the IF-MF recovery procedure were within the 95 percent confidence interval associated with the MPN. No relationships could be established between the observed variations in the physical and chemical characteristics of the treated sewage samples and the fecal coliform densities. / Master of Science

Metabolic were used to evaluate Enterococcus as an indicator of faecal pollution. Enterococci were isolated using m-Enterococcus agar and speciated using conventional biochemical tests. Forty percent of the isolates were identified and metabolically characterized by the automated Biolog system. The biochemical test scheme recognized 16 enterococcal species, while Biolog recognized nine. Both methods identified E. faecalis at the greatest frequency. Overall species frequencies varied between the two methods. Biolog was unable to identify 31% of the isolates; 7% of the isolates were unidentified by the biochemical test scheme. Of the identified isolates, metabolic profiling with Biolog achieved speciation with 60 substrates. Unique profiles were obtained for 89% of the isolates. Isolates also demonstrated inter-trial differntial metabolism of substrates. This and the large number of unidentified isolates suggest great diversity among enterococci. Diversity and inter-trial metabolic inconsistencies will complicate use of enterococcal metabolic profiles as a source-tracking tool. / xxiii, 264 leaves ; 29 cm.