Environmental sampling is a pathogen monitoring technique that has become important in the food industry. Many food processing companies have adopted environmental sampling as a way to verify good manufacturing practices and sanitation plans in their facilities. Environmental sampling is helpful because it gives better information on the source of product contamination than end product sampling. Two specific pathogens of concern to the food industry are Listeria monocytogenes and Salmonella enterica. Environmental samples are rarely analyzed immediately, but instead may be batched for later analysis or shipped to an off site testing facility. Multiple media on the market today is used for storage and transport of environmental samples. These various media types, differences in holding temperatures and time create variability in test sample conditions. Select time, temperature and media combinations were tested to determine their effect on Listeria monocytogenes and Salmonella enterica populations during transport and storage of samples. Cocktails of Listeria monocytogenes and Salmonella enterica were added separately to sample tubes containing D/E Neutralizing Broth, Neutralizing Buffer or Copan SRK Solution. Bacterial counts at 0, 12, 24 and 48 hours post inoculation were compared. Neutralizing Buffer and Copan SRK Solution maintained consistent bacterial populations at all temperatures. At 10° and 15°C, D/E Broth supported bacterial growth. This study helps validate the use of D/E Neutralizing Broth, Neutralizing Buffer and Copan SRK Solution for environmental sample transport and storage at proper holding temperatures. At temperatures >10°C Neutralizing Buffer or Copan SRK solution should be used if quantifying microbial recovery. / Master of Science

Campylobacter and Salmonella are foodborne pathogens commonly associated with raw poultry. Although there has been much research done on isolating these pathogens from poultry production environments using cloacal swabs, fecal samples, intestinal tract contents and dissection, research involving environmental sampling has been limited. New and/or improved environmental sampling methods may provide an easy, convenient, and less time-consuming way to collect samples. Coupling these sampling methods with PCR may provide a relatively simple, rapid, and robust means of testing for foodborne pathogens in a chicken house or flock prior to slaughter. Air, boot and sponge samples were collected from three commercial chicken grow-out houses located in southwestern Virginia when flocks were three, four, and five weeks old. Air samples were collected onto gelatin filters. Fecal/litter samples were collected from disposable booties worn over investigator's protective shoe coverings. Pre-moistened sponges were used to sample house feed pans and water dispensers on drink lines. A PCR method was used to qualitatively detect Campylobacter jejuni and Salmonella spp. Campylobacter jejuni was detected at each farm (house), across all three ages (3, 4, and 5 weeks), and from each sample type. Salmonella was not detected in any of the environmental samples. For all 270 samples, 41% (110/270) were positive for Campylobacter. Collectively, 28% (25/90) of air, 44% (40/90) of sponge, and 50% (45/90) of bootie samples were positive for Campylobacter. The methods used in this study are non-invasive to live animals, relatively rapid and specific, and could enable poultry processing facilities to coordinate scheduled processing of flocks with lower pathogen incidence, as a way to reduce post-slaughter pathogen transmission. / Master of Science

Master of Science / Food Science Institute / Daniel Y.C. Fung / There are many different environmental sampling methods that are currently used in the industry. They include swab, sponge, flocked swab, direct agar contact, and M-Vac. Several studies have been conducted to determine the benefits and drawbacks of each method. Sampling methods utilized in this study were the swab, flocked swab, and M-Vac. Three surfaces were utilized in this study: ultra high density polypropylene, 304 stainless steel with a 2B finish, and 304 stainless steel with a 2B finish and a buffed surface. Surfaces sampled were 100 cm2. Prior to inoculation, surfaces were autoclaved for 15 min at 121 °C for sterilization. Surfaces were inoculated by either Listeria monocytogenes or Escherichia coli O157:H7 at a concentration of 9 log10 CFU/ml by painting the inoculum onto the surface with a sterilized paintbrush. Brushes were dipped in inoculum for 2 sec before painting from left to right once and then from up to down once. Brushes were redipped for 2 sec and the painting step was repeated. The same brush was used for all E. coli O157:H7 samples and a different brush was used for all L. monocytogenes samples. Then, the surfaces were allowed to dry for 30 min before sampling took place. Listeria monocytogenes samples were appropriately diluted and plated in duplicate onto Tryptic Soy Agar (TSA) and Modified Oxford Media (MOX). Escherichia coli O157:H7 samples were properly diluted and plated in duplicate onto TSA and MacConkey Sorbital Agar (MSA). After plating, dry surfaces were stained using LIVE/DEAD® BacLight™ Bacterial Viability Kit. The Zeiss LSM 5 Pascal confocal laser scanning electron microscope was used for microscopy images and photographs. Six 1 mm by 1 mm random and representative images were taken of each surface. Viable cell count results show that the sponge sampling method, in general, recovered a higher number of microorganisms. The swab was normally shown to recover the least number of microorganisms. When examining the microscopy images it can be concluded that biofilms are more easily formed with L. monocytogenes than E. coli O157:H7. Imaging also allowed for a visual representation of the remaining organisms that made it appear as if there was actually more bacteria recovery when the M-Vac sampling method was employed than when the sponge method was utilized.

Abstract Proof of concept for a continuous environmental sampling methodology that employs common terrestrial arthropods as environmental samplers was tested by analyzing pitfall, malaise and black light captures over a six month period over a replicated urban-suburban-rural gradient in Central Virginia. All arthropods captured at the nine sites were identified and assigned to aquatic, vegetation, or soil groups based on their association with these microhabitats. To offset variability in arthropod life history patterns and species abundance within habitat types, arthropod categories based on presence/absence data over the six month period were constructed to provide for sampling reliability within each microhabitat type. Arthropod categories ranged from single abundant species and families to synthetic groupings based on microhabitat associations (e.g., “soil beetles”), all of which could be easily identified. Mean weekly captures of individuals in each resulting category were compared within and among the nine sites using GLM or ranks analyses. Overall and weekly mean capture rates in the aquatic (two categories), soil (seven categories) and vegetation (11 categories) microhabitats were similar within each habitat type. With the exception of the two aquatic category members (midges and caddisflies), overall, monthly and weekly mean capture rates of all arthropod categories were highest in suburban and lowest in urban habitats. Results demonstrate reliability of the arthropod categories constructed and provide ground truthing for a continuously deployable and user-friendly arthropod-based system for monitoring environmental agents.

Stable isotopes of water are used as tracers for characterizing surface water/groundwater interactions. Gaps in sampling protocol for these tracers in low gradient canals limits their use in studies of canal-groundwater exchanges. Several sampling methods were developed to determine the temporal and spatial isotopic variation in a canal. The influence of a flow control gate on isotopic composition and the sensitivity of isotope mixing calculations to choice of sampling method were also evaluated. There was little variability in the isotopic composition of the canal along a cross section perpendicular flow. Some variation occurred monthly and seasonally. The greatest variability occurred between the upstream and downstream side of the flow control gates when the gates were closed. Mixing calculations were not sensitive to the choice of sampling method. This study shed light on isotope sampling methods in canals for canal-groundwater interactions studies. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2019. / FAU Electronic Theses and Dissertations Collection

This thesis examined the efficacy of various sampling and detection methods used for environmental monitoring of Listeria species on wooden surfaces used for cheese aging. Government agencies including the Food and Drug Administration (FDA) and United States Department of Agriculture (USDA) recommend enrichment methods coupled with use of environmental sponges and swabs. Our study compared efficacy of sponge swabs manufactured by 3M™ and World Bioproducts. There is a lack of research validating the best performing swab type and enrichment method combination that is sensitive when used on rough, porous surfaces. The sensitivity of these environmental sampling tools and methods are critical considerations to effectively monitor the presence of Listeria species on wooden boards used during aging of artisan cheese. Seasoned spruce wooden shelves, cut into 100cm2 replicates, were spot inoculated with varying concentrations of Listeria species inocula, the Listeria species strains consisted of two L. monocytogenes strains and a Green Florescence Protein (GFP) expressing strain of L. innocua. The inoculated wooden surface was swabbed with three environmental sampling sponge/swab formats (World Bioproducts© EZ ReachTM environmental swabs (WBEZ) with HiCap (WBHC) and Dey-Engley (WBDE) neutralizing broths; and 3MTM environmental swabs (3MTM) with Dey-Engley neutralizing broth). Enumeration methods were used to determine the low target limits of detection. Once the low target concentrations were identified, five enrichment methods consisting of 3MTM Listeria Environmental Plate, FDA, Dual Enrichment, modified USDA, and modified FDA were challenged against low concentrations of Listeria species inocula (0.01 cfu/cm2, 0.1 cfu/cm2, 1 cfu/cm2) and the three environmental sponge swab formats. Performance of the swab formats was assessed by collection of naturally contaminated environmental samples (n=405) from dairy farm environments, swabbing where wooden surfaces existed, and analyzed using the most effective enrichment methods found from previous experiments. Lastly, the wooden surfaces and sponge swabs were observed under a Florescent Microscope using GFP L. innocua to visually determine how each sponge material of the 3M™ and World Bioproducts recovered the inocula. When wood surfaces were inoculated at high concentration levels of Listeria spp., all swab formats performed equally for detecting Listeria. Success of positive recovery at low concentrations was variable, where enrichment methods and swabs were not dependent on each other. The swab format that worked best for detecting low levels of Listeria species was the WBDE sponge swab. The WBDE swab also performed the best in dairy farm environmental sampling. The m-USDA enrichment method was found to be most effective in recovery and repair of low and potentially injured Listeria spp. Wooden surfaces are rough and porous and should be taken into consideration when creating an environmental sampling plan for these food contact surfaces. All swabs and methods performed with only slight variation, but the variation could be significant when monitoring wooden shelves with low level contamination of Listeria species. Artisan cheesemakers who use wooden shelves during the aging of their cheese, should ensure use of the most sensitive detection methods.

Rapid bioassessment techniques used for aquatic macroinvertebrate and plants in wetlands were tested in New South Wales, Australia. Wetlands surveyed ranged from coastal wetlands in the North and Central parts of the State, to tableland wetlands west of Sydney. Wetlands varied in dominant vegetation, hydrology, substrate and level of human impairment. Different options for sampling (mesh size, live-picking times, quadrat size, number of samples or quadrats) and analysis (taxonomic resolution, transformations, biotic indices, multivariate and univariate analyses) are compared to determine optimal sampling effort and evaluate the effects of errors or variability. Results show that, for wetlands of New South Wales, sampling procedures developed and tested in streams and other regions of Australia may not be the most efficient. Using the data from 21 wetlands in New South Wales, a number of analytical techniques were evaluated for the effects of errors. Results show that species-level multivariate analysis is more sensitive in detecting less obvious differences between wetlands (i.e., small effect sizes), while family-level analyses are more appropriate for large effect sizes. A modified waterplant index was developed that is simpler and has a wider application than the other Australian options available. Inherent problems in each index tested were addressed. The results show that the process of summarising a large amount of information into a single value will result in the loss of both information and variability between samples and this cumulative effect of error may effect the assessment of wetland condition. The practical outcome of this thesis is a set of standardised steps to assess wetland quality using biological assemblages. The results show that protocols and indices for rivers are not directly transferable to palustrine, vegetation dominated wetlands. I present protocols that are more appropriate to wetlands and recognise that each protocol would need to be adapted for each wetland type. Despite the need for flexible protocols, I promote the need for a standard approach to wetland sampling and the need for consideration of the effects of errors in sampling designs. This study highlights the need for more research on the response of specific stressors to wetlands flora and fauna. The results from this study also show that wetland macroinvertebrates and plant communities can be used as surrogates in multivariate analyses for detecting large differences between wetlands (wetland types) but that impact assessment requires more detailed investigations including species identification and careful consideration of the choice of reference and control sites. In conclusion I emphasise the need for scientific rigour in the use of biological indicators and consideration of the effects of errors and implications to sampling designs. / Doctor of Philosophy (PhD)

Graduation date: 2002

The primary step in the microbiological assessment of highly dynamic and complex food processing conditions is environmental sampling. The objectives of this study were to: (1) compare the efficacy of four sampling devices including Microbial-Vac system (MV), cellulose sponge (SP), polyester swab (SW) and composite tissue (CT), for the recovery of Listeria monocytogenes and Brochothrix thermosphacta on five surfaces and (2) to determine if there was a significant difference between the recovery of low (10 CFU/900cm2) and high (100 CFU/900cm2) L. monocytogenes inoculum levels using the sampling devices in a simulated food processing environment. Surfaces used for this study were stainless steel (SS), polyethylene cutting board (CB), polyurethane conveyor belt (PB), open hinge flat top conveyor belt (FT) and mesh conveyor belt (MB). Food contact surfaces were inoculated with L. monocytogenes to obtain a final cell population of 10 (low) or 100 (high) CFU/900 cm2. An average cell density of 10,000 CFU/25 cm2 was used for inoculating B. thermosphacta on each of the surfaces. Inoculated surfaces were dried and held for two hours at 4˚C then sampled and processed for detection. Because L. monocytogenes is a "zero tolerance" pathogen in ready-to-eat foods, the qualitative analysis included an enrichment step to detect presence/absence in the sample. In comparison, B. thermosphacta was directly plated in order to quantify the recovery capability of each device. Results indicated for recovery of 100 CFU/900 cm2 L. monocytogenes, there was no difference among devices on SS, CB or PB surfaces (p>0.05). However, a significant difference was detected at 10 CFU/900 cm2 on SS between MV and CT, 62.97 and 17.34%, respectively (p=0.0086). Results for FT indicated MV was superior over SP and SW (p=0.0004) for detection of high and low L. monocytogenes. There was no difference for the quantitative recovery of B. thermosphacta on PB and SS; however, there was a difference (p=0.0371) among devices on CB indicating MV was superior over SP and CT. The swab recovered 3.25 log CFU/25cm2 from flat top belts and was significantly lower (p=0.0259) than MV and SP devices, 4.29 and 4.12 log CFU/25cm2, respectively.

Clostridium difficile is a common and potentially fatal cause of antibiotic-associated diarrhoea and pseudomembranous colitis worldwide. It has been isolated from patients and their surroundings, in healthcare facilities and from the community. C. difficile is able to survive for many months on inanimate surfaces in the form of spores. PCR ribotyping is used in the UK to characterise and identify strain diversity. Investigating how the most problematic strains respond to cleaning regimes may influence the control of disease. This work used the University Hospitals of Leicester Trust as a case study for this purpose of understanding the epidemiology of this pathogen within healthcare facilities. Five individual agar media were compared based on their abilities to recover and resuscitate damaged ribotype 027 spores, a strain associated with disease outbreaks and increased severity. Controlled laboratory experiments with a sub-lethal dose of a germicide were conducted before C. difficile recovery from hospital wards. An additional two sampling campaigns acquired environmental strains. C. difficile isolation after routine cleaning demonstrated the inefficiency of the current recovery regime as C. difficile spores were recovered using direct contact plates, enrichment broths, and resuscitation media. This study used layering of non-selective agar over selective agar, identifying a potential link in the proportions of media following the use of sponges in environmental sampling. All strains were characterised by ribotyping; ribotype 027 was isolated from all sampling cohorts. A four-month epidemiological study was conducted into the ribotype prevalence and distribution from C. difficile-positive faecal specimens. A second survey investigated these effects with a modification of C. difficile detection from faecal samples. Hydrogen peroxide vapour is currently being explored as a means of decontamination of healthcare-associated infections. Inactivation kinetics of ribotype 027 spores were analysed in response to vapour and liquid exposure of hydrogen peroxide. No reports thus far have explored such kinetics and controlled decontamination with both clinical and non-clinical strains. Evidence strongly suggests spores can be inactivated with its application. Furthermore, this study revealed there appears to be significant differences in susceptibility and inactivation of different C. difficile ribotypes.