A complex multi-vector processed beef value stream was biomapped and work-in-process (WIP) was sampled at various processing points that affected the product microbiome. Coliform Petrifilm™ was used to analyze samples qualitatively and quantitatively across the meat and glaze vectors. There was an impact (p≤0.05) of the glaze distribution system on coliforms. The pumps of the glaze system were fully disassembled and additional microbial data were obtained through swabbing. WIP and sponge samples were subjected to 16S V1/V2 gene amplification with primers designed for taxonomic identification. Sequencing was conducted on the Illumina MiSeq platform and bioinformatics were developed in QIIME 2 workflows. Bespoke sample groupings were created to compare process steps, with alpha and beta diversity measures analyzed using multivariate statistical models to assess microbial community dynamics. The pumps were found to harbor a diverse and distinct microbiota, acting as reservoirs for fermentative bacteria due to biofilm formation facilitated by carbohydrates in the glaze. The distinct microbial communities found in these pumps, especially one of the three systems, revealed the need for specialized sanitation. A novel weekly deep-clean process was developed and deployed to the glaze pumps utilizing guided disassembly and manual scrubbing, gaseous chlorine dioxide, and a quaternary ammonium hydrogen peroxide biofilm stripper. The implementation of this protocol led to a reduction in coliform presence (p≤0.05), with detection rates dropping from 1.14% to 0.08% in the product and from 32.82% to 0.63% in the glaze. To provide ongoing assessment of the value stream, a unique Environmental Monitoring Program (EMP) was developed to monitor WIP samples collected routinely. After enumeration on EC Petrifilm™, colonies were isolated and subjected to automated biochemical and MALDI-TOF identification. Results showed wide agreement (85.2%) between the two systems with discrepancies resolved by nanopore WGS. The EMP revealed a coliform microbiome with few similarities to the meat and glaze samples analyzed by 16S prior to the sanitation intervention. Isolates confirmed frequent identification of non-lactose fermenting organisms on EC Petrifilm™, illustrating the vulnerability of the method to break-through growth earlier than previously documented.

Environmental samples from food processing facilities and production (farms) were taken and analyzed for the presence of indicator organisms and Salmonella spp. on food contact (FC) and nonood contact (NFC) surfaces. Salmonella was isolated from both FC and NFC surfaces of a fruit and catfish-processing plant environment, but not from a dairy processing plant or from produce packaging facility environments. Scatter plots did not show a relationship between indicator organisms and the presence of Salmonella in processing environments, regardless of the facility/environment. Salmonella ser. Gaminara and Salmonella ser. Give were prevalent in the fruit processing environment. Persistence could not be determined, as Salmonella was not detected during subsequent samplings. Two modifications of the Rappaport Vassiliadis (RV) enrichment step were evaluated and compared to the standard enrichment methods for the recovery of Salmonella. Detection rates ranged from 10.2% to 21.2% and 22.3% to 31.9% for aquaculture and sweet potato farming environments, respectively. Salmonella ser. Newport and Salmonella ser. Javiana were prevalent in the sweet potato farming environment, whereas Salmonella ser. Newport and Salmonella ser. Hartford were prevalent in the aquaculture environment. PFGE analysis revealed clusters with a high degree of genetic similarity (greater than or equal to 90%) from the fruit-processing, aquaculture, and sweet potato environments, suggesting that they represented the same strain isolated from different sampling points. Molecular characterization of isolates revealed potential contamination paths to catfish and sweet potatoes.

Indicator organisms (IOs) such as Escherichia coli and Enterococcus spp. are used to predict the presence of pathogens in waters. Determining the relationships between environmental factors, IOs, and pathogens is a key to assessing water quality and ensuring public health, yet certain strains of E. coli and Enterococcus spp. survive for long periods in natural waters. Molecular subtyping, using repetitive extragenic palindromic DNA sequences (BOX-PCR), has been used to discriminate among environmental E. coli and Enterococcus spp. isolates. The reproducibility of BOX-PCR patterns varies with DNA purification methods; therefore, it is important to develop a standardized, rapid, high throughput DNA purification protocol for population biology studies. I have compared the effects of DNA purification methods on the reproducibility, cost, and speed of producing BOX-PCR patterns using three methods: a commercially available Qiagen kit (Qiagen DNeasy tissue), a whole cell method requiring no pre-treatment, and a method developed in-house using the MacConnell Mini-prep 96 (mini-prep) instrument. The whole cell method was the least expensive, but demonstrated the least precision (reproducibility). The Qiagen kit and the Mini-prep 96 showed high reproducibility (90-95%); however, the Mini-prep 96 is less expensive and very rapid, allowing processing of up to 192 isolates/day. Water and sediment from a Florida river were placed in an outdoor flume that maintained turbulent flow and oxic conditions in the water column (~11 mg/L). The flume was inoculated with seven E. coli strains and nine Enterococcus spp. of distinct BOX-PCR phylotypes. Putative "survivor" strains previously isolated from mesocosms and disinfected wastewater effluent and control laboratory strains were chosen to test the hypothesis of differential survival of strains under hydrodynamically active conditions. IO strains isolated each day were typed by BOX-PCR (n=100 isolates/day), revealing differential survival of certain E. coli and Enterococcus strains. Ultimately, a better understanding of the effect of hydrodynamic regime and phylotype distribution on IO survival in water will allow more accurate modeling of the fate of these organisms in aquatic environments. This will in turn lead to a better understanding of the organisms we use as indicators of pollution. This is necessary to ensure the health and safety of all recreational water users.

Bacterial source tracking (BST) methods often involve the use of phenotypic or genotypic fingerprinting techniques to compare indicator bacteria such as Escherichia coli isolated from unknown sources against a library of fingerprints from indicator bacteria found in the feces of various known source animals. The predictive capability of a library is based in part on how well the library isolates reflect the true population diversity of indicator bacteria that can potentially impact a water body. The purpose of this study was to compare the behavior of E. coli population structures in the feces of humans, beef cattle and horses across different parameters. Ribotyping and antibiotic resistance analysis were used to "fingerprint", or subtype E. coli isolates. Significantly greater diversity was observed in the E. coli population of horses compared to the human or beef cattle sampled. Subtype sharing between individuals from all host categories was infrequent, therefore the majority of E. coli subtypes were sampled from a single individual. The dominant E. coli populations of nine individuals (three per host source category) were monitored over time, which demonstrated that E. coli subtypes within a host individual vary on a monthly time frame, and an increase in the frequency of subtype sharing was noted between individuals within the same source group over time. The E. coli population of a single human that had just finished antibiotic treatment was studied on a daily basis for one month. The loss of an E. coli subtype with high antibiotic resistance was observed over time, however there was a single dominant E. coli subtype that was present at every sampling event during the entire month. Geographic distinctiveness of E. coli populations was investigated by sampling four herds located in different geographical regions. We observed that E. coli populations are not geographically distinct, but are somewhat individual-specific, as most E. coli isolates had a subtype that was found in a single individual. This study defines factors that should be considered when constructing a successful BST library, and suggests that E. coli may not be the appropriate indicator organism for BST.

Title from PDF of title page. / Document formatted into pages; contains 117 pages. / Thesis (M.S.)--University of South Florida, 2003. / Includes bibliographical references. / Text (Electronic thesis) in PDF format. / ABSTRACT: Bacterial source tracking (BST) methods often involve the use of phenotypic or genotypic fingerprinting techniques to compare indicator bacteria such as Escherichia coli isolated from unknown sources against a library of fingerprints from indicator bacteria found in the feces of various known source animals. The predictive capability of a library is based in part on how well the library isolates reflect the true population diversity of indicator bacteria that can potentially impact a water body. The purpose of this study was to compare the behavior of E. coli population structures in the feces of humans, beef cattle and horses across different parameters. Ribotyping and antibiotic resistance analysis were used to "fingerprint", or subtype E. coli isolates. Significantly greater diversity was observed in the E. coli population of horses compared to the human or beef cattle sampled. / ABSTRACT: Subtype sharing between individuals from all host categories was infrequent, therefore the majority of E. coli subtypes were sampled from a single individual. The dominant E. coli populations of nine individuals (three per host source category) were monitored over time, which demonstrated that E. coli subtypes within a host individual vary on a monthly time frame, and an increase in the frequency of subtype sharing was noted between individuals within the same source group over time. The E. coli population of a single human that had just finished antibiotic treatment was studied on a daily basis for one month. The loss of an E. coli subtype with high antibiotic resistance was observed over time, however there was a single dominant E. coli subtype that was present at every sampling event during the entire month. Geographic distinctiveness of E. coli populations was investigated by sampling four herds located in different geographical regions. We observed that E. / ABSTRACT: coli populations are not geographically distinct, but are somewhat individual-specific, as most E. coli isolates had a subtype that was found in a single individual. This study defines factors that should be considered when constructing a successful BST library, and suggests that E. coli may not be the appropriate indicator organism for BST. / System requirements: World Wide Web browser and PDF reader. / Mode of access: World Wide Web.

Water treatment facilities have been relying on indicator bacteria to assess the quality of water for decades. The purpose of this group of studies is to investigate the predictive capabilities of conventional and alternative indicators for pathogenic microorganisms in disinfection processes and treated wastewater effluents. In addition, the possibility that diversity of indicator bacteria, as well as overall bacterial diversity, correlate with fecal contamination in water bodies has been investigated. Indicator organisms (total coliforms, fecal coliforms, enterococci, C. perfringens, and coliphages) as well as pathogens (enteroviruses, Giardia, and Cryptosporidium) were enumerated from six wastewater treatment facilities at various stages of treatment. Statistical analyses were conducted to determine if the indicator organisms (individually or as a set) could predict the presence or absence of pathogens. Single indicator organism analysis failed to correlate with the occu rrence pathogens, thus monitoring a suite of indicator organisms may be a better measure to predict the presence of pathogens. The product of chlorine residual concentration and contact time (CT) was identified as a factor for determining the log10 reduction of enteric viruses in wastewater treatment facilities that used chloramines for disinfection.Samples were also collected from river waters and sediments in watersheds with different human population densities to identify the impact of anthropogenic activities on bacterial diversity. 16S rRNA restriction fragment length polymorphism (RFLP), ribotyping, and denaturing gradient gel electrophoresis (DGGE) were used to determine total coliform, Escherichia coli, and bacterial community population structures, respectively. The concentrations of indicator organisms were significantly different among the river sites in sediments, but not in water column. The population diversity measurements were not significantly different among the river sites; while the indicator population and bacterial community structures were dissimilar in water column vs. associated sediment samples. Accumulation curves demonstrated that greater than 20 isolates must be sampled at most of the sites to represent the dominant populations. A better understanding of the relationship between the indicator organisms and pathogens as well as knowledge of the ecology of indicator organisms in pristine and anthropogenically impacted waters may contribute to water quality restoration and public health protection.

Current research is revealing that stormwater can carry pathogens and that this stormwater is entering wastewater treatment facilities. During periods of intense rainfall, not only can stormwater carry higher amounts of pathogens, but it also increases the flow rate to the wastewater treatment facility. In many instances, the flow rate exceeds the facilities' treatment capacity and can impact treatment performance. The purpose of this study was to identify whether wastewater treatment is impaired during periods of increased rainfall, and to compare current policies that address this issue. The study was conducted using a case study approach to analyze historical precipitation and wastewater treatment data from facilities located in Clearwater and St. Petersburg, Florida. The effluent from the biological nutrient removal system operated at the facilities located in Clearwater was compared to the effluent from the activated sludge treatment system operated by the facility located in St. Petersburg. Statistical analyses were conducted to identify significant differences in either the loading or performance of wastewater treatment facilities under wet and dry flow conditions. In this case, the Clearwater facilities operating below their treatment capacity were better equipped to handle peak wet weather flows and efficiently treat wastewater than the St. Petersburg facility which has a less advanced treatment system and was operating at and above its treatment capacity.

Waterborne pathogen presence caused by fecal pollution is a leading cause of morbidity and mortality worldwide. In developed countries, this problem can result in waterborne outbreaks. Research suggests that there is a need for better fecal indicators because current methods (total coliforms and E. coli) are insufficient. This study investigated Bacteroidales 16S rRNA markers as a microbial source tracking tool in an agricultural watershed. Correlations between pathogens and markers were also investigated. Water quality monitoring was conducted following assay validation of ruminant-, bovine-, human-specific, and universal Bacteroidales markers. Results revealed a positive relationship between E. coli and the universal marker. Ruminant- and bovine-specific marker detection was associated with increased runoff due to precipitation; however, the human associated marker was not detected. Furthermore, no correlations between Campylobacter, Salmonella, or E. coli O157:H7 could be made. Consequently, these techniques have potential to become a powerful tool; however, further research is needed

Floc-pathogen interactions are important determinants of the fate of pathogens in aquatic systems. The dissociation of bacteria from particles due to shear stress can significantly increase the presence of free-floating pathogenic bacteria in the aqueous phase. This has implications for pathogen transport and water quality. This study evaluated the interactions of water-borne pathogens with particles in selected aquatic ecosystems. Three experimental chapters and one concluding chapter is presented. Chapter 3 assesses the strength of the floc-microorganism association under different energy levels in relation to the physico-chemical properties and the bioorganic content of flocs from six different aquatic environments (SB, CSO, RF, AG, ML, MN); Chapter 4 evaluates how energy dissociates bacteria and affects microbial diversity in free-floating and particle-associated fractions in cohesive bed sediments (BedS) and suspended flocs (SusF) of three sites (SB, CSO, RF). Chapter 5 studies the diversity and succession among free-floating and particle-associated bacteria at different energy levels and the abundance of antibiotic resistance genes and Class 1 integrons (intI1) as a result of ecosystem perturbation in the six initial sites. Different strategies, such as standard laboratory analytical methods, as well as techniques based on analytical chemistry, biochemistry and molecular biology were used to accomplish these objectives. The bioorganic and physico-chemical properties of flocs and sediments, and the energy effects these structures are exposed to, play a role in the assessment of pathogen risk in water systems. Molecular approaches showed a significant difference in the composition of free-floating and particle-associated assemblages after simulated flow conditions and detected earlier differences in the dissociation of bacteria, compared to plating techniques. The analysis of integrons provided evidence for horizontal gene transfer events. Free-floating and particle-associated bacterial assemblages are potential genetic reservoirs for antibiotic resistance genes. This research shows that particles act as reservoirs for microorganisms, providing an early warning for potential indicators of human health risk in water systems and could determine the presence of future clinically relevant antibiotic resistance mechanisms and/or pathogenic microbial gene transfer in sediments, demonstrating the need to improve the existing protocols and methodologies that assess water quality. / Natural Sciences and Engineering Research Council of Canada (NSERC)

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2006. / Härtill 4 uppsatser.