Emerging pathogens are organisms whose incidence has increased within the past two decades. In the last 40 years, several pathogens have emerged to cause infectious waterborne and foodborne diseases, thus causing a significant public health concern. Enterobacter sakazakii and Naegleria fowleri are emerging pathogens that have been documented to cause fatal infections. E. sakazakii is an emerging foodborne pathogen that represents a significant health risk by causing infections resulting in septicemia, meningitis and necrotizing enterocolitis in neonates, premature infants and also elderly immunocompromised individuals. Naegleria fowleri is a water-based protozoan flagellate that is the cause of primary amoebic meningoencephalitis; a fatal disease that mostly infects children and young adults through water-related recreational activities. The focus of this dissertation is to identify environmental reservoirs of Enterobacter sakazakii and to determine inactivation strategies to control Naegleria fowleri by chlorine and ultraviolet disinfection. In Appendix A, samples from various household kitchens were collected to determine the presence of E.sakazakii. The highest percentage of E.sakazakii was isolated from kitchen sponges (8%; n=50) and dishrags (10%; n=50). This study provided information on the presence of E.sakazakii on environmental surfaces in the kitchen. In Appendix B, our recent research has determined that N. fowleri is present in 8% (n=143) of municipal drinking water wells in central and southern Arizona. Therefore, guidelines need to be established for treatment of water with various disinfectants to control the growth and proliferation of N.fowleri. In Appendix C, the Ct values (concentration (mg/l) × exposure time) for chlorine inactivation of N. fowleri trophozoites and cysts were determined using the Efficiency Hom Kinetic Model (EHM). The Ct values for 99% inactivation of trophozoites and cysts were estimated to be 9 and 31, respectively. The ultraviolet light dose required for the 99% inactivation of N.fowleri trophozoites and cysts was determined to be 63 mW.sec/cm² and 13 mW.sec/cm², respectively.

Surrogate organisms have been used to study highly pathogenic organisms, or organisms that cannot be cultured in the laboratory. Surrogates are selected based on multiple similarities to the pathogen, such as morphology, genome size and structure, and environmental characteristics. This dissertation utilized MS2 coliphage as a surrogate for norovirus and Ebola virus in three environments. MS2 is an icosahedral, single-stranded RNA bacteriophage. It is a male-specific coliphage that infects the bacteria Escherichia coli. Its properties, such as morphology and survival in the environment, have been likened to those of many enteric viruses. Because of this, it has been used as a surrogate for pathogenic enteric viruses for disinfection testing on surfaces, in water and in food; modeling the movement and survival of pathogens in different environments; and transfer properties from surfaces. This dissertation utilized MS2 as a surrogate in three different studies. In the first, MS2 is used as a surrogate for human enteric viruses in irrigation canals to predict the re-suspension of pathogenic viruses from bed sediment into overlying irrigation water using a flume to re-create field conditions in the laboratory. MS2 re-suspension into the overlying water was characterized at varying flow rates and velocities using two sediment types. Its overall re-suspension was not statistically significantly different (p > 0.05) between flow rates. The additional studies in this dissertation used MS2 as a surrogate for Ebola virus in human waste. Ebola virus is a BSL-4 organism that is spread through direct contact with bodily fluids. It is found in bodily fluids in concentrations between 10^5.5 and 10⁸ genome copies per milliliter. In the first study using MS2 as a surrogate for Ebola virus, efficacies of four disinfectants were tested using 10¹² PFU of MS2 in one liter containing 2.25% (w/v) organic matter at three contact times (1, 15 and 30 minutes). The purpose of this study was to assess the disinfectants on reducing virus in waste before toilet flushing. Peracetic acid and quaternary ammonium formulation were found to reduce the concentration of MS2 in the toilet bowl the fastest (within one minute) with the greatest reduction (2.26 and 1.99 log₁₀), when compared with the other disinfectants. Reductions observed from hydrogen peroxide were significantly less than those from peracetic acid and quaternary ammonium (p < 0.05). The contamination of restroom surfaces by MS2 was also evaluated after toilet flushing with and without disinfectant treatment. All four disinfectants were found to significantly reduce the viral concentrations on fomites after 15 minutes of contact (p < 0.05). Despite disinfectant use, three sites were contaminated in 100% of trials (N = 18). These were the toilet bowl rim, the toilet seat top and underside. The final study evaluated the inactivation of MS2 and several other viruses by thermophilic and mesophilic anaerobic digestion. Little information is available on the influence of the wastewater treatment process, specifically anaerobic digestion, on emerging viruses, such as Ebola virus. It is important to evaluate this process due to the environmental disposal and discharge of wastewater and solids into the environment. All viruses were recoverable after mesophilic digestion (reductions from 1.8-6.6 log₁₀ per mL), except the lipid-containing bacteriophage Φ6. Thermophilic digestion inactivated all viruses significantly (p = 0.0011) more than mesophilic digestion. The reductions by thermophilic digestion ranged from 2.8-7.1 log₁₀ per mL. The inactivation between the initial concentration and both digestion types was statistically significant (p = 0.007).

Cystic fibrosis (CF) is an autosomal recessive disorder affecting 70,000 individuals worldwide. This disease is characterized by the buildup of mucus in the airways leading to chronic lung infections resulting in pulmonary failure and death in 95% of CF patients. Routine surveillance of CF pathogens using traditional microbiology culture guides management and treatment of CF patients. Molecular profiling studies have revealed emerging pathogens that may play a role in CF lung disease by either directly causing infection or upregulating the virulence factors of classic CF pathogens, such as P. aeruginosa; however, routine CF culture protocols have not been modified to detect these organisms. The goal of this study was to expand the data relevant to the use of microbiology cultures for the management and treatment of CF patients at Virginia Commonwealth University Medical Center (VCUMC) by directly selecting for emerging CF pathogens in culture. This was accomplished by developing,optimizing, and implementing an agar to select for colistin-resistant non-fermenting Gram- negative rods (NF GNRS). In addition, McKay agar and anaerobic media were utilized to recover members of the Streptococcus anginosus group (SAG) and anaerobes in CF respiratory samples. The prevalences of SAG, anaerobes, and colistin-resistant NF GNRs recovered on study media from 75 adult and pediatric CF patients at VCUMC were 17.33%, 41.33%, and 4% respectively. Approximately 62% of patients culture-positive for SAG were also infected with P. aeruginosa and 53.8% of SAG recovered in culture were from CF patients experiencing PE. These findings further support the claim that interspecies interactions among emerging and classic CF pathogens may result in periods of clinical instability or PE. Twenty-eight of the 75 patients were culture-positive for Veillonella species, with the majority of samples collected during a period of surveillance. Four colistin-resistant NF GNRs were isolated on the study media alone. The selective nature of the study media prevented the mixed respiratory flora and classic CF pathogens from overgrowing and obscuring the growth of these colistin-resistant NF GNRs. The presence and role of emerging pathogens in the CF patient population at VCUMC warrants further investigation; therefore, the routine culture protocol needs to be revised to recover and select for those organisms thought to play a role in PE and lung function decline.

Campylobacter enteritis in humans is primarily associated with C. jejuni/coli infection. Other species cause campylobacteriosis relatively infrequently; while this could be attributed to bias in diagnostic methods, the pathogenicity of non-jejuni/coli Campylobacter spp. such as C. upsaliensis and C. helveticus (isolated from dogs and cats) is uncertain. Galleria mellonella larvae are suitable models of the mammalian innate immune system and have been applied to C. jejuni studies. This study compared the pathogenicity of C. jejuni, C. upsaliensis, and C. helveticus isolates. Larvae inoculated with either C. upsaliensis or C. helveticus showed significantly higher survival than those inoculated with C. jejuni. All three Campylobacter species induced indistinguishable histopathological changes in the larvae. C. jejuni could be isolated from inoculated larvae up to eight days post-inoculation whereas C. upsaliensis and C. helveticus could only be isolated in the first two days. There was a significant variation in the hazard rate between batches of larvae, in Campylobacter strains, and in biological replicates as random effects, and in species and bacterial dose as fixed effects. The Galleria model is applicable to other Campylobacter spp. as well as C. jejuni, but may be subject to significant variation with all Campylobacter species. While C. upsaliensis and C. helveticus cannot be considered non-pathogenic, they are significantly less pathogenic than C. jejuni.

This study focuses on emerging E. coil serotypes and has developed methods for the isolation and identification of non-0157 STEC and EPEC. A basal medium for the isolation of these pathogens was developed as well as a serogroup specific PCR assay for the detection of the 02 serogroup. These culture and molecular based techniques have proven to be valuable in the detection, identification, and epidemiological investigation of these groups of emerging pathogens. These methods were applied to 1) a farm study, whereby samples (faecal and soil) and 2) an abattoir study, whereby samples (hide and carcass) were analysed for the presence of non-0157 STEC and EPEC. Isolates were subsequently characterised in terms of serotype/serogroup and virulence markers. The data generated by this work has illustrated the extent of non-0157 STEC and EPEC contamination in the farm and abattoir environments, thus providing scientific background upon which control strategies may be based.