Enteroaggregative Escherichia coli as a cause of acute diarrhea

Enteroaggregative Escherichia coli (EAEC) are considered an important emerging enteric and food-borne pathogen. The groups importantly affected by EAEC include international travelers, children in the developing world, and patients with HIV infection. EAEC does not commonly cause diarrheal illness in all hosts. ^ The reasons for the observed clinical variation in EAEC infection are multifactorial and are dependant on the pathogen, the inoculum ingested and the host susceptibility. A major obstacle in identifying the mechanism of pathogenesis for EAEC is the heterogeneity in virulence of strains. No EAEC virulence gene is consistently present in all diarrheagenic strains. However, a recent report suggests that a package of plasmid borne and chromosomal virulence factors are under the control of the described transcriptional activator aggR. Although the exact inoculum required for EAEC diarrheal illness is not known, a volunteer study has shown that oral ingestion of 10 10 cfu of virulent EAEC elicited diarrhea. Ongoing studies are being conducted to better define the exact infectious dose. There are also host factors associated with increased susceptibility of persons to diarrheal illness with EAEC. ^ The following three manuscripts: (1) review EAEC as an emerging enteric pathogen; (2) identify EAEC as a cause of acute diarrhea among different subpopulations worldwide; (3) identify virulence characteristics and the molecular epidemiology of EAEC isolates among travelers with diarrheal illness and describe the pathogenesis of EAEC infection. ^

Variation of genotype and prevalence of Noro virus in U.S. students in Mexico with Traveler's Diarrhea, summer of 2004 and 2005

Noro virus, a positive single stranded RNA virus has been identified as a major etiologic agent in food borne gastroenteritis and diarrheal diseases. The emergence of this organism as a major non-bacterial cause in such outbreaks is partly due to the improved diagnostic tools like Reverse Transcription Polymerase chain reaction (RTPCR) that enable its detection. Noro virus accounts for nearly 96% of non-bacterial gastroenteritis outbreaks in US (1). Travelers' Diarrhea (TD) has remained a constant public health risk in the developed nations for decades and bacteria like Entero toxigenic Escherichia coli, Entero aggregative Escherichia coli have been described as the main etiologic agents for TD (2-4). A possible viral contribution to TD has been discovered in two studies (5, 6). The current study was designed to determine the prevalence of Noro virus in a population of 107 US students with TD acquired in Mexico in 2005 and to compare the prevalence to the prevalence of Noro virus in a similar study done in 2004. This study involved the testing of clinical stool specimens from 107 subjects in 2005 for the presence of Noro virus using RTPCR. The prevalence of Noro virus in 2004 used for comparison to 2005 data was obtained from published data (5). All subjects were recruited as TD subjects in a randomized, double-blinded clinical trial comparing a standard three day dosing of Rifaximin with and without an anti motility drug Loperamide. The prevalence of Noro virus geno group I was similar in both years, but geno group II prevalence differed across the two years (p = 0.003). This study finding suggests that the prevalence of Noro virus geno groups varies with time even within a specific geographic location. This study emphasizes the need for further systematic epidemiologic studies to determine the molecular epidemiology and the prevalence patterns of different geno groups of this virus. These are essential to planning and implementation of public health measures to lessen the burden of TD due to Noro virus infection among US travelers. ^

Health education intervention to reduce risk of infectious disease transmission among community health workers and their clients

Indigent and congregate-living populations have high susceptibilities for disease and pose a higher risk for disease transmission to family, friends and to persons providing services to these populations. The adoption of basic infection control, personal hygiene, safe food handling and simple engineering practices will reduce the risk of infectious disease transmission to, from and among indigent and congregate-living populations. ^ The provision of social services, health promotion activities and other support services to indigent and congregate-living populations is an important aspect of many public health-related governmental, community-based and other medical care provider agencies. ^ In the interest of protecting the health of indigent and congregate-living populations, of personnel from organizations providing services to these populations and of the general community, an educational intervention is warranted to prevent the spread of blood-borne, air-borne, food-borne and close contact-borne infectious diseases. ^ An educational presentation was provided to staff from a community-based organization specializing in providing housing, health education, foodstuffs and meals and support services to disabled, low-income, homeless and HIV-infected individuals. The educational presentation delivered general best practices and standard guidelines. A pre and post test were administered to determine and measure knowledge pertinent to controlling the spread of infectious diseases between and among homeless shelter-living clients and between clients and the organization's staff. ^ Comparing pre-test and post-test results revealed areas of knowledge currently held by staff and other areas that staff would benefit from additional educational seminars and training. ^

Diffusely adherent Escherichia coli (DAEC) as a cause of acute diarrhea

Acute diarrhea is the most common medical problem in the developing countries. Infectious agents are responsible for a majority of cases of acute diarrhea. Knowing the cause of acute diarrhea is important to developing plans for disease prevention, control and therapy. Acute diarrhea is caused by many viruses, bacteria and parasites. ^ Travelers to developing countries of the world commonly develop diarrhea as a result of eating contaminated food or drinking contaminated water. About 30-50% of travelers who travel from industrialized countries like United States to the developing countries are at risk of developing diarrhea. High risk areas for travelers' diarrhea are Mexico, Latin America and Southeast Asia. Public restaurants are the common sites for exposure to this type of food-borne infectious disease in travelers. Food becomes contaminated when they are handled by people with fecal content on their hands. ^ The importance of Diffusely Adherent Escherichia Coli (DAEC) in travelers to these areas has not been well studied. Some of the studies looking at DAEC have shown the organism to be present in children without symptoms. Other studies have shown a relationship between DAEC infection and presence of symptoms. I have selected this topic because the patho-physiological processes in DAEC infection that allow intestinal and extra-intestinal infections to develop are not fully understood. DAEC related acute diarrhea is a relatively new topic of public health significance. There is a limited number of studies regarding the virulence and pathogenic mechanisms of DAEC. The presumed virulence factor of the organism is diffuse attachment to the intestinal lining of the infected host. However more research needs to be done to identify the pathogenic mechanisms and virulence factors associated with DAEC infection for better treatment planning and diarrhea prevention. ^

Toxoplasma gondii seroprevalence in ringed seals (Pusa hispida) from the Canadian Arctic

Toxoplasmosis is a significant public health threat for Inuit in the Canadian Arctic. This study aimed to investigate arctic seals as a possible food-borne source of infection. Blood samples collected from 828 seals in 7 Canadian Arctic communities from 1999 to 2006 were tested for Toxoplasma gondii antibodies using a direct agglutination test. Polymerase chain reaction (PCR) was used to detect T. gondii DNA in tissues of a subsample of seals. Associations between seal age, sex, species, diet, community and year of capture, and serological test results were investigated by logistic regression. Overall seroprevalence was 10.4% (86/828). All tissues tested were negative by PCR. In ringed seals, seroprevalence was significantly higher in juveniles than in adults (odds ratio = 2.44). Overall, seroprevalence varied amongst communities (P = 0.0119) and by capture year (P = 0.0001). Our study supports the hypothesis that consumption of raw seal meat is a significant source of infection for Inuit. This work raises many questions about the mechanism of transfer of this terrestrial parasite to the marine environment, the preponderance of infection in younger animals and the natural course of infection in seals. Further studies to address these questions are essential to fully understand the health risks for Inuit communities.

Restaurant Health and Hygiene: A Model Program with Theory-Based Guidelines for Implementation

Poor hygienic practices and illness of restaurant employees are major contributors to the contamination of food and the occurrence of food-borne illness in the United States, costing the food industry and society billions of dollars each year. Risk factors associated with this problem include lack of proper handwashing; food handlers reporting to work sick; poor personal hygiene; and bare hand contact with ready-to-eat foods. However, traditional efforts to control these causes of food-borne illness by public health authorities have had limited impact, and have revealed the need for comprehensive and innovative programs that provide active managerial control over employee health and hygiene in restaurant establishments. Further, the introduction and eventual adoption by the food industry of such programs can be facilitated through the use of behavior-change theory. This Capstone Project develops a model program to assist restaurant owners and operators in exerting active control over health and hygiene in their establishments and provides theory-based recommendations for the introduction of the program to the food industry.

Epidemiology of Opisthorchis viverrini

Opisthorchis viverrini is a food home trematode, important because of the sheer numbers of people infected and its serious morbidities such as hepatobiliary diseases and cholangiocarcinoma (CHCA). Although infections are identified throughout Southeast Asia, the epi-center is northeast Thailand, where high prevalence coexists with a high incidence of CHCA. In this review, we present the basic population features and the factors influencing transmission between the different hosts. (C) 2003 Elsevier B.V. All rights reserved.

Economic Impact of E.coli 0111 Outbreak in 1995 in Australia

Increased incidence of food-borne illnesses is a matter of significant concern for the community and the government alike. An outbreak of E.coli O111 that occurred in Australia in 1995 affected 200 people of whom 22 developed HUS while one person died. This study analyses the economic costs of the outbreak. The total cost of the outbreak is estimated to be A$5.61 million. Productivity loss represented the highest percentage of outbreak costs (66%) due to death, disability and chronic illness. The direct medical costs contributed 33%. The estimated loss could be even higher if all costs could be quantified. Nevertheless, the findings provide an idea to the policy maker regarding the extent and nature of the damage that could result from an outbreak. The severity of the damage warrants allocation of necessary resources to prevent such occurrences.

Global changes in gene expression observed at the transition from growth to stationary phase in Listeria monocytogenes ScottA batch culture

Listeria monocytogenes is a food-borne Gram-positive bacterium that is responsible for a variety of infections (worldwide) annually. The organism is able to survive a variety of environmental conditions and stresses, however, the mechanisms by which L. monocytogenes adapts to environmental change are yet to be fully elucidated. An understanding of the mechanism(s) by which L. monocytogenes survives unfavourable environmental conditions will aid in developing new food processing methods to control the organism in foodstuffs. We have utilized a proteomic approach to investigate the response of L. monocytogenes batch cultures to the transition from exponential to stationary growth phase. Proteomic analysis showed that batch cultures of L. monocytogenes perceived stress and began preparations for stationary phase much earlier (approximately A(600) = 0.75, mid-exponential) than predicted by growth characteristics alone. Global analysis of the proteome revealed that the expression levels of more than 50% of all proteins observed changed significantly over a 7-9 h period during this transition phase. We have highlighted ten proteins in particular whose expression levels appear to be important in the early onset of the stationary phase. The significance of these findings in terms of functionality and the mechanistic picture are discussed.

Glove Use in Restaurants: Efficacy is Questionable

Apparently there seems to be a growing consensus on the part of both industry managers and consumers that the use of gloves is an effective barrier to the spread of food-borne illness. However, with more than 13 years’ experience as a food service manager and executive, the author has discovered otherwise.

Consumers’ Reactions to Sanitation in Casual Dining, Quick-Service, and Fine Dining Restaurants

Consumers’ concern about food safety, sanitation, and health has increased since food-borne illnesses still frequently occur in the US. This article explored consumers’ perceptions, emotions, and behavioral intention about the sanitation of the physical environment in three different restaurant settings, casual dining, quick-service, and fine dining restaurants. Disgust was the most strongly felt negative emotion, but no significant differences were found for negative emotional reactions to dirty conditions among the three types of restaurants. Positive emotional reactions were significantly different among the restaurant types. Behavioral intention was also significantly different among the three restaurant types as a reaction to dirty food. The findings help restaurant owners and managers understand how consumers feel and react to “dirty” food, service staff, or dining room tables in casual, quick-service and fine dining restaurant.

Bioengineering strategies to improve functional qualities of nisin

The abuse of antibiotics and the emergence of multi-drug resistant bacterial strains have created the need to explore alternative methods of controlling microbial pathogens. The bacteriocin family of antimicrobial peptides has been proposed as one such alternative to classic antibiotics. Nisin A belongs to the subgroup of bacteriocins called the lantibiotics, which contain several unusual amino acids as a consequence of enzyme-mediated post-translational modifications. As nisin is produced by generally regarded as safe (GRAS) microorganisms, it could potentially be applied in a clinical setting. However, as lantibiotics are naturally produced in such small quantities, this can hinder their industrial potential. In order to overcome this, several approaches can be utilised. For example, given the gene encoded nature of lantibiotics, genetic engineering approaches can be implemented in order to yield variants with enhanced properties. Here, the use of mutagenesis-based strategies was employed to obtain a derivative of nisin with enhanced bioactivity in vitro. Investigations with purified peptide highlighted the enhanced specific activity of this variant, nisin M21V, against food-borne Listeria monocytogenes strains. Furthermore, this specific enhanced bioactivity was evident in a mouse model of listeriosis. Reductions in bioluminescence and microbial counts in organs from infected mice were observed following treatment with nisin M21V compared to that of wild-type nisin A. Peptide bioengineering approaches were also implemented to obtain additional novel derivatives of nisin. The generation of “S5X” and “S33X” banks (representing a change of natural serines at positions 5 and 33 to all possible alternative residues) by a combination of site-saturation and site-directed mutagenesis led to the identification of several derivatives exhibiting improved stability. This allowed the rational design of variants with enhanced stability compared to that of wild type nisin. Another means of tackling issues associated with lantibiotic yield is to combine lantibiotics with other antimicrobials. This could circumvent the need for enhanced production while also reducing concentrations of the peptide antimicrobials. We observed that combinations of nisin variants and low levels of plant essential oils (thymol, carvacrol, trans-cinnamaldehyde) significantly controlled Gram negative foodborne pathogens in in vitro assays compared to nisin A-essential oil combinations. This enhanced control was also evident in model food systems. Nisin variants used in conjunction with carvacrol significantly reduced numbers of E. coli O157:H7 in apple juice while a commercial nisin preparation used in combination with citric acid significantly controlled C. sakazakii in infant milk formula. It is noteworthy that while nisin is generally associated with Gram positive targets, upon combination with plant essential oils the spectrum of inhibition was broadened to Gram negative targets.

The application of next generation sequencing to profile microbe related cheese quality defects

High throughput next generation sequencing, together with advanced molecular methods, has considerably enhanced the field of food microbiology. By overcoming biases associated with culture dependant approaches, it has become possible to achieve novel insights into the nature of food-borne microbial communities. In this thesis, several different sequencing-based approaches were applied with a view to better understanding microbe associated quality defects in cheese. Initially, a literature review provides an overview of microbe-associated cheese quality defects as well as molecular methods for profiling complex microbial communities. Following this, 16S rRNA sequencing revealed temporal and spatial differences in microbial composition due to the time during the production day that specific commercial cheeses were manufactured. A novel Ion PGM sequencing approach, focusing on decarboxylase genes rather than 16S rRNA genes, was then successfully employed to profile the biogenic amine producing cohort of a series of artisanal cheeses. Investigations into the phenomenon of cheese pinking formed the basis of a joint 16S rRNA and whole genome shotgun sequencing approach, leading to the identification of Thermus species and, more specifically, the pathway involved in production of lycopene, a red coloured carotenoid. Finally, using a more traditional approach, the effect of addition of a facultatively heterofermentative Lactobacillus (Lactobacillus casei) to a Swiss-type cheese, in which starter activity was compromised, was investigated from the perspective of its ability to promote gas defects and irregular eye formation. X-ray computed tomography was used to visualise, using a non-destructive method, the consequences of the undesirable gas formation that resulted. Ultimately this thesis has demonstrated that the application of molecular techniques, such as next generation sequencing, can provide a detailed insight into defect-causing microbial populations present and thereby may underpin approaches to optimise the quality and consistency of a wide variety of cheeses.

A prospective view of animal and human Fasciolosis

Fasciolosis, a food-borne trematodiasis, results following infection with the parasites, Fasciola hepatica and Fasciola gigantica. These trematodes greatly affect the global agricultural community, infecting millions of ruminants worldwide and causing annual economic losses in excess of US $3 billion. Fasciolosis, an important zoonosis, is classified by WHO as a neglected tropical disease with an estimated 17 million people infected and a further 180 million people at risk of infection. The significant impact on agriculture and human health together with the increasing demand for animal-derived food products to support global population growth demonstrate that fasciolosis is a major One Health problem. This review details the problematic issues surrounding fasciolosis control, including drug resistance, lack of diagnosis and the threat that hybridization of the Fasciola species poses to future animal and human health. We discuss how these parasites may mediate their long-term survival through regulation and modulation of the host immune system, by altering the host immune homeostasis and/or by influencing the intestinal microbiome particularly in respect to concurrent infections with other pathogens. Large genome, transcriptome and proteomic data sets are now available to support an integrated One Health approach to develop novel diagnostic and control strategies for both animal and human disease.

Molecular Profiling of Shiga Toxin-Producing Escherichia coli and Enteropathogenic E. coli Strains Isolated from French Coastal Environments

Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) strains may be responsible for food-borne infections in humans. Twenty-eight STEC and 75 EPEC strains previously isolated from French shellfish-harvesting areas and their watersheds and belonging to 68 distinguishable serotypes were characterized in this study. High-throughput real-time PCR was used to search for the presence of 75 E. coli virulence-associated gene targets, and genes encoding Shiga toxin (stx) and intimin (eae) were subtyped using PCR tests and DNA sequencing, respectively. The results showed a high level of diversity between strains, with 17 unique virulence gene profiles for STEC and 56 for EPEC. Seven STEC and 15 EPEC strains were found to display a large number or a particular combination of genetic markers of virulence and the presence of stx and/or eae variants, suggesting their potential pathogenicity for humans. Among these, an O26:H11 stx1a eae-β1 strain was associated with a large number of virulence-associated genes (n = 47), including genes carried on the locus of enterocyte effacement (LEE) or other pathogenicity islands, such as OI-122, OI-71, OI-43/48, OI-50, OI-57, and the high-pathogenicity island (HPI). One O91:H21 STEC strain containing 4 stx variants (stx1a, stx2a, stx2c, and stx2d) was found to possess genes associated with pathogenicity islands OI-122, OI-43/48, and OI-15. Among EPEC strains harboring a large number of virulence genes (n, 34 to 50), eight belonged to serotype O26:H11, O103:H2, O103:H25, O145:H28, O157:H7, or O153:H2.