Human microbiota-secreted factors inhibit shiga toxin synthesis by enterohemorrhagic Escherichia coli O157:H7

Escherichia coli O157:H7 is a food-borne pathogen causing hemorrhagic colitis and hemolytic-uremic syndrome, especially in children. The main virulence factor responsible for the more serious disease is the Shiga toxin 2 (Stx2), which is released in the gut after oral ingestion of the organism. Although it is accepted that the amount of Stx2 produced by E. coli O157:H7 in the gut is critical for the development of disease, the eukaryotic or prokaryotic gut factors that modulate Stx2 synthesis are largely unknown. In this study, we examined the influence of prokaryotic molecules released by a complex human microbiota on Stx2 synthesis by E. coli O157:H7. Stx2 synthesis was assessed after growth of E. coli O157:H7 in cecal contents of gnotobiotic rats colonized with human microbiota or in conditioned medium having supported the growth of complex human microbiota. Extracellular prokaryotic molecules produced by the commensal microbiota repress stx(2) mRNA expression and Stx2 production by inhibiting the spontaneous and induced lytic cycle mediated by RecA. These molecules, with a molecular mass of below 3 kDa, are produced in part by Bacteroides thetaiotaomicron, a predominant species of the normal human intestinal microbiota. The microbiota-induced stx(2) repression is independent of the known quorum-sensing pathways described in E. coli O157:H7 involving SdiA, QseA, QseC, or autoinducer 3. Our findings demonstrate for the first time the regulatory activity of a soluble factor produced by the complex human digestive microbiota on a bacterial virulence factor in a physiologically relevant context.

Genotypes and antibiotic resistance of Campylobacter coli in fattening pigs

Campylobacter coli is a food-borne zoonotic pathogen causing human gastroenteritis worldwide. The organism is a commensal in the intestine of many food production animals including fattening pigs. The role of the pig as a potential reservoir for C. coli affecting human either directly or via poultry has hardly been investigated and genetic characterization of porcine strains is needed to address this question. For this aim multilocus sequence typing (MLST) and flaB typing was applied to 256 C. coli isolates from faeces of fattening pig collected during 2009 at different slaughterhouses in Switzerland. In addition genotypic resistances towards macrolides and quinolones based on point mutations in the 23S rRNA and gyrA genes, respectively, were determined. Of the 67 sequence types (STs) obtained by MLST, 37 were found for the first time. flaB typing revealed 46 different types with 14 of them being novel and was useful to further differentiate strains with an identical ST. Quinolone resistance was detected in 33.6% and macrolide resistance was found in 10.6% of isolates. Comparison with 99 C. coli pig isolates from 2001 revealed a significant decrease in antibiotic resistance towards both groups of antibiotics and there was high overlap between genotypes of 2001 and 2009. Little overlap of porcine genotypes was found with 97 C. coli isolates from poultry collected 2008, however, macrolide resistance was significantly higher in pig isolates. In conclusion, C. coli from Swiss pig are heterogeneous containing many novel STs, findings that could reflect the partitioned Swiss pig production with almost no international breed exchange. The antibiotic resistance echoes the use of corresponding drugs in the Swiss livestock production and indicates the efficacy of restrictive application of antibiotics in order to reduce resistances.

Role of milk and meat products as vehicles for antibiotic-resistant bacteria

This subject is reviewed under the following headings: Microbial contamination of raw meat and raw milk; Antibiotic resistance of food-borne pathogens; Antibiotic resistance of commensal and potentially pathogenic bacteria as a new threat in food microbiology; Antibiotic-resistant staphylococci in fermented meat and [in] milk products; Antibiotic-resistant Enterococcus sp. in fermented meat and [in] milk products; Enterococci in farm animals and meat; Enterococci in fermented food; Molecular characterization of resistance of food-borne enterococci; and Further ecological and epidemiological considerations of resistant live bacteria in food. It is concluded that further research is needed, particularly into the possible transfer of the resistance of bacteria consumed in meat or milk products to the indigenous bacteria of the human consumer.

Genotypes and antibiotic resistances of Campylobacter jejuni isolates from cattle and pigeons in dairy farms.

Campylobacter jejuni is the most common food-borne zoonotic pathogen causing human gastroenteritis worldwide and has assumed more importance in Italy following the increased consumption of raw milk. Our objectives were to get an overview of genotypes and antibiotic resistances in C. jejuni isolated from milk, cattle feces, and pigeons in dairy herds of Northern Italy. flaB-typing was applied to 78 C. jejuni isolates, previously characterized by Multi-Locus Sequence Typing, and genotypic resistances towards macrolides and quinolones based on point mutations in the 23S rRNA and gyrA genes, respectively, were determined. flaB-typing revealed 22 different types with one of them being novel and was useful to further differentiate strains with an identical Sequence Type (ST) and to identify a pigeon-specific clone. Macrolide resistance was not found, while quinolone resistance was detected in 23.3% of isolates. A relationship between specific genotypes and antibiotic resistance was observed, but was only significant for the Clonal Complex 206. Our data confirm that pigeons do not play a role in the spread of C. jejuni among cattle and they are not responsible for milk contamination. A relevant number of bulk milk samples were contaminated by C. jejuni resistant to quinolones, representing a possible source of human resistant strains.

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.

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.