Role of the U.S. Food and Drug Administration in the regulatory management of human listeriosis in the United States.

From 1986 to 2006, the incidence of listeriosis in the United States dropped from approximately seven to three cases per million population, a reduction that most likely reflects the joint efforts of industry, government, consumers, and academia. Herein, we describe the U.S. Food and Drug Administration (FDA) strategy over the past three decades to combat listeriosis. Specifically, we discuss early actions taken to address outbreaks during the 1980s, policy decisions regarding the presence of Listeria monocytogenes in FDA-regulated foods, FDA compliance programs with L. monocytogenes components, enforcement actions to remove L. monocytogenes-contaminated products from the market (i.e., recalls) or to prevent entry of such products into the market (i.e., import detentions and refusals), research milestones, outreach and education efforts, and selected special projects. Evolving demographic trends in the United States may pose a challenge to further reduction of the incidence of listeriosis.

Diarrhea in American infants and young children in the community setting: incidence, clinical presentation and microbiology.

OBJECTIVE: The characteristics and microbiology of the full spectrum of pediatric diarrhea occurring in the U.S. community setting are not well-understood. METHODS: Six-month prospective cohort study of 604 healthy 6- to 36-month-old children recruited by the Slone Center Office-based Research Network. RESULTS: The incidence of parent-defined diarrhea was 2.2 episodes per person-year. The median duration of diarrhea was 2 days with a median of 6 stools per episode. Outpatient visits and hospitalization were prompted by 9.7 and 0.3% of episodes, respectively. The most common microorganisms identified in healthy baseline stools were atypical enteropathogenic Escherichia coli (12.2%), enteroaggregative Escherichia coli (3.7%), Clostridium difficile (3.5%) and Clostridium perfringens (2.9%), and each of these was no more common in diarrhea stools. In contrast, all of the viruses analyzed were more prevalent in diarrhea specimens than in baseline specimens: enteric adenovirus (5.7% diarrhea versus 1.4% baseline), rotavirus (5.2% versus 1.4%), astrovirus (3.5% versus 1.4%), Sapporo-like virus (3.0% versus 0.8%) and norovirus (1.9% versus 0.8%). A likely pathogen was detected in 20.6% of diarrhea specimens. Vomiting and > or =16 stools in an episode were predictive of isolating a pathogen from the stool, each with a relative risk of approximately 2. CONCLUSIONS: Healthy young children in this study experienced more than 2 cases of diarrhea per person-year, but most were brief and do not require medical attention. Although most diarrhea-associated pathogens were viruses, no likely pathogen was found in almost 80% of cases; possible etiologies for these cases include currently unknown gastrointestinal infections, nongastrointestinal illnesses and dietary/environmental factors.

Prevalence, risk factors, O serogroups, and virulence profiles of Shiga toxin-producing bacteria from cattle production environments.

Shiga toxin (Stx)-producing bacteria are important human pathogens that have been linked with cattle and associated food products. We recovered Stx-producing bacteria from 27.5% of cattle, 6.8% of water, and 2.3% of wildlife samples from a cattle production area during an 11-month period. Positive samples were found during every month and on 98% of sampling days. We recovered isolates from all cattle operations sampled, and prevalence within operations ranged from approximately 5 to 33%. Cattle prevalence was associated with the presence of Stx-producing bacteria in water and the production group and environment of cattle, with an interaction between production group and environment. Odds of recovering isolates from cattle were highest for groups of adult cows and their unweaned calves in pasture environments. Overall, 49 O serogroups were identified from 527 isolates. Seventy of the isolates contained virulence genes that encoded intimin and enterohemorrhagic Escherichia coli hemolysin. These were serogroups O111, O157, O109, O103, O145, O172, O84, O26, O108, O117, O126, O159, O5, O69, O74, O98, and O-rough. Our results suggest that the prevalence of Stx-producing bacteria can be relatively high in cattle, and associated factors may not be entirely similar to those reported for serotype O157:H7. Although Stx-producing bacteria were frequently detected, the strains may not be equally pathogenic for humans given the wide variety of serogroups and virulence genes. However, focusing on O157:H7 in food safety and surveillance programs may allow other Stx-producing bacteria, which appear to be widespread in cattle, to go undetected.

Shiga toxin-producing Escherichia coli as a possible etiological agent of chronic diarrhea.

We identified Shiga toxin-producing Escherichia coli (STEC) as the likely etiologic pathogen for chronic diarrhea in 2 patients, 1 of whom was immunocompromised with acquired immunodeficiency syndrome, and 1 of whom was immunocompetent. Both were treated with antibiotics, and neither developed systemic complications of the infection. These cases suggest that STEC infection should be considered in the differential diagnosis of chronic diarrhea.

Microbial-gut interactions in health and disease. Mucosal immune responses.

The host gastrointestinal tract is exposed to countless numbers of foreign antigens and has embedded a unique and complex network of immunological and non-immunological mechanisms, often termed the gastrointestinal 'mucosal barrier', to protect the host from potentially harmful pathogens while at the same time 'tolerating' other resident microbes to allow absorption and utilization of nutrients. Of the many important roles of this barrier, it is the distinct responsibility of the mucosal immune system to sample and discriminate between harmful and beneficial antigens and to prevent entry of food-borne pathogens through the gastrointestinal (GI) tract. This system comprises an immunological network termed the gut-associated lymphoid tissue (GALT) that consists of unique arrangements of B cells, T cells and phagocytes which sample luminal antigens through specialized epithelia termed the follicle associated epithelia (FAE) and orchestrate co-ordinated molecular responses between immune cells and other components of the mucosal barrier. Certain pathogens have developed ways to bypass and/or withstand defence by the mucosal immune system to establish disease in the host. Some 'opportunistic' pathogens (such as Clostridium difficile) take advantage of host or other factors (diet, stress, antibiotic use) which may alter or weaken the response of the immune system. Other pathogens have developed mechanisms for invading gastrointestinal epithelium and evading phagocytosis/destruction by immune system defences. Once cellular invasion occurs, host responses are activated to limit local mucosal damage and repel the foreign influence. Some pathogens (Shigella spp, parasites and viruses) primarily establish localized disease while others (Salmonella, Yersinia, Listeria) use the lymphatic system to enter organs or the bloodstream and cause more systemic illness. In some cases, pathogens (Helicobacter pylori and Salmonella typhi) colonize the GI tract or associated lymphoid structures for extended periods of time and these persistent pathogens may also be potential triggers for other chronic or inflammatory diseases, including inflammatory bowel disease and malignancies. The ability of certain pathogens to avoid or withstand the host's immune assault and/or utilize these host responses to their own advantage (i.e. enhance further colonization) will dictate the pathogen's success in promoting illness and furthering its own survival.

Effect of an oral Shiga toxin-binding agent on diarrhea-associated hemolytic uremic syndrome in children: a randomized controlled trial.

CONTEXT: Diarrhea-associated hemolytic uremic syndrome (HUS) is the most common cause of acute renal failure in children. Most cases are caused by an intestinal infection with Shiga toxin-producing strains of Escherichia coli. OBJECTIVE: To determine if administration of an oral agent that binds Shiga toxin could diminish the severity of diarrhea-associated HUS in pediatric patients. DESIGN, SETTING, AND PATIENTS: Multicenter, randomized, double-blind, placebo-controlled clinical trial of 145 children (96 experimental and 49 placebo) aged 6 months to 18 years with diarrhea-associated HUS conducted between July 27, 1997, and April 14, 2001, at 26 tertiary care pediatric nephrology centers in the United States and Canada. Trial included 2 phases, the hospital course for treatment of the acute illness and a 60-day outpatient follow-up period after discharge from the hospital. INTERVENTION: Patients were assigned to receive the binding agent, 500 mg/kg daily, or cornmeal placebo orally for 7 days in a 2:1 randomization scheme. MAIN OUTCOME MEASURES: Combined frequency of death or serious extrarenal events and need for dialysis in the experimental vs placebo group. RESULTS: A total of 62 patients (43%) were male and 123 (85%) were white. The median age of the patients was 4.2 years. Most patients (59%) were transferred from other hospitals to participating sites. The severity of disease at the time of randomization was comparable in the 2 groups. The prevalence of death or serious extrarenal events was 18% and 20% in the experimental and placebo groups, respectively (P =.82). Dialysis was required in 42% of experimental and 39% of placebo groups (P =.86). CONCLUSIONS: Oral therapy with a Shiga toxin-binding agent failed to diminish the severity of disease in pediatric patients with diarrhea-associated HUS.

In vivo transduction of an Stx-encoding phage in ruminants.

We assessed the ability of a kanamycin-marked Stx phage to move into a commensal, ovine Escherichia coli strain in the ruminant gastrointestinal tract. Transduction was detected in 19/24 sheep tested, resulting in the recovery of 47 transductants. Subtherapeutic doses of the quinolone antibiotic enrofloxacin did not increase the rate of transduction.

Characteristics of persistent diarrhea in a community-based cohort of young US children.

OBJECTIVE: The objective of the study was to define the characteristics and microbiology of persistent diarrhea (PD) in US children. METHODS: Six-month prospective cohort study of a convenience sample of 604 healthy 6- to 36-month-old children recruited by the Slone Center Office-based Research Network. RESULTS: Of 611 diarrhea episodes, 50 (8.2%) lasted < or = 14 days. The incidence of PD was 0.18 episodes per person-year, and the median duration of episodes was 22.0 days (range, 14-64 days). PD episodes were more likely than acute episodes to result in a medical visit (28.0% vs 8.2%; P = 0.0001). The most commonly used treatments were oral rehydration solution (12.0% of episodes) and antibiotics (6.0%). No bacterial or parasitic pathogens were associated with PD; but norovirus, rotavirus and sapovirus were each significantly more prevalent in PD stools compared with baseline stools, with relative risks of 12.4, 6.9 and 6.2, respectively. Fifty-nine per cent of the PD specimens tested were negative for all studied pathogens. CONCLUSIONS: PD occurs with a frequency of approximately 1 case per 5 person-years in US infants and young children. It seems to be a generally benign illness, with only 28% of cases presenting to medical care. Although viral pathogens seem to cause a minority of PD episodes in this population, most are not due to currently known infectious agents.

Food Safety: Bovine Spongiform Encephalopathy (Mad Cow Disease).

Bovine spongiform encephalopathy is just one of a group of diseases known as transmissible spongiform encephalopathies. Only recently has it become recognized that transmissible spongiform encephalopathies are likely due to proteins known as prions. Although it has been recognized that transmissible spongiform encephalopathies may readily spread within species, the recent observations that bovine spongiform encephalopathy in cattle may have originated from another transmissible spongiform encephalopathy in sheep, known as scrapie, is cause for concern. Further, bovine spongiform encephalopathy has now been strongly linked with a universally fatal human neurologic disease known as new variant Creutzfeldt-Jakob disease. Currently the only approach to preventing bovine spongiform encephalopathy, and subsequent new variant Creutzfeldt-Jakob disease in humans, from ingestion of bovine spongiform encephalopathy-infected material is to avoid consumption of contaminated food. Little can be done to treat food that will destroy prions and leave a palatable product. At this stage we are continuing to learn about transmissible spongiform encephalopathies and their implications on human health. This is an ever-changing situation and has an unpredictable element in terms of the extent of the current outbreaks in England and other parts of Europe.

Identification of motility and autoagglutination Campylobacter jejuni mutants by random transposon mutagenesis.

Campylobacter jejuni has been identified as the leading cause of acute bacterial diarrhea in the United States, yet compared with other enteric pathogens, considerably less is understood concerning the virulence factors of this human pathogen. A random in vivo transposon mutagenesis system was recently developed for the purpose of creating a library of C. jejuni transformants. A total of 1,065 C. jejuni transposon mutants were screened for their ability to swarm on motility agar plates and autoagglutinate in liquid cultures; 28 mutants were subsequently identified. The transposon insertion sites were obtained by using random-primed PCR, and the putative genes responsible for these phenotypes were identified. Of these mutants, all 28 were found to have diminished motility (0 to 86% that of the control). Seventeen motility mutants had insertions in genes with strong homology to functionally known motility and chemotaxis genes; however, 11 insertions were in genes of unknown function. Twenty motility mutants were unable to autoagglutinate, suggesting that the expression of flagella is correlated with autoagglutination (AAG). However, four mutants expressed wild-type levels of surface FlaA, as indicated by Western blot analysis, yet were unable to autoagglutinate (Cj1318, Cj1333, Cj1340c, and Cj1062). These results suggest that FlaA is necessary but not sufficient to mediate the AAG phenotype. Furthermore, two of the four AAG mutants (Cj1333 and Cj1062) were unable to invade INT-407 intestinal epithelial cells, as determined by a gentamicin treatment assay. These data identify novel genes important for motility, chemotaxis, and AAG and demonstrate their potential role in virulence.

Comparison of Shiga toxin production by hemolytic-uremic syndrome-associated and bovine-associated Shiga toxin-producing Escherichia coli isolates.

There is considerable diversity among Shiga toxin (Stx)-producing Escherichia coli (STEC) bacteria, and only a subset of these organisms are thought to be human pathogens. The characteristics that distinguish STEC bacteria that give rise to human disease are not well understood. Stxs, the principal virulence determinants of STEC, are thought to account for hemolytic-uremic syndrome (HUS), a severe clinical consequence of STEC infection. Stxs are typically bacteriophage encoded, and their production has been shown to be enhanced by prophage-inducing agents such as mitomycin C in a limited number of clinical STEC isolates. Low iron concentrations also enhance Stx production by some clinical isolates; however, little is known regarding whether and to what extent these stimuli regulate Stx production by STEC associated with cattle, the principal environmental reservoir of STEC. In this study, we investigated whether toxin production differed between HUS- and bovine-associated STEC strains. Basal production of Stx by HUS-associated STEC exceeded that of bovine-associated STEC. In addition, following mitomycin C treatment, Stx2 production by HUS-associated STEC was significantly greater than that by bovine-associated STEC. Unexpectedly, mitomycin C treatment had a minimal effect on Stx1 production by both HUS- and bovine-associated STEC. However, Stx1 production was induced by growth in low-iron medium, and induction was more marked for HUS-associated STEC than for bovine-associated STEC. These observations reveal that disease-associated and bovine-associated STEC bacteria differ in their basal and inducible Stx production characteristics.

Detection of Shiga-Like Toxin-Producing Escherichia coli in Ground Beef and Milk by Commercial Enzyme Immunoassay.

Shiga-like toxin (SLT)-producing Escherichia coli (SLTEC) is the leading cause of acute renal failure among children. SLTEC are most commonly ingested from contaminated food, and because cattle are a major reservoir, ground beef and milk have been a significant source of contamination associated with multiperson outbreaks. While serotype O157:H7 has been principally identified in the United States there are many other SLTEC serotypes associated with human disease. We have therefore examined the utility of an enzyme immunoassay (EIA) for Shiga-like toxins as a means of detecting the presence of low levels of multiple SLTEC serotypes in ground beef and milk. In the present study we demonstrated that it is possible to detect low levels (approximately 1 SLTEC per g of ground beef) in both small-scale (2 g of beef per 5 ml) and standard large-scale (25 g of beef per 225 ml) food microbial cultures. The EIA was also capable of allowing detection of SLTEC in nonspiked retail ground beef samples: we were able to recover SLTEC isolates (O113:Hu; O22:H-; O82:H8) from 3 of 12 ground beef samples. The EIA detected SLTs produced in spiked milk samples when as few as 1 SLTEC per ml was added. Overall the EIA proved to be a highly sensitive way to detect the presence of SLTEC in either ground beef or milk samples after overnight enrichment culturing in an appropriate broth and should provide a rapid and convenient method for the detection of multiple pathogenic SLTEC serotypes.

Bacteriophage control of Shiga toxin 1 production and release by Escherichia coli.

The stx genes of many Shiga toxin-encoding Escherichia coli (STEC) strains are encoded by prophages of the lambda bacteriophage family. In the genome of the Stx1-encoding phage H-19B, the stx(1)AB genes are found approximately 1 kb downstream of the late phage promoter, p(R)', but are known to be regulated by the associated iron-regulated promoter, p(Stx1). Growth of H-19B lysogens in low iron concentrations or in conditions that induce the prophage results in increased Stx1 production. Although the mechanism by which low iron concentration induces Stx1 production is well understood, the mechanisms by which phage induction enhances toxin production have not been extensively characterized. The studies reported here identify the factors that contribute to Stx1 production after induction of the H-19B prophage. We found that replication of the phage genome, with the associated increase in stx(1)AB copy number, is the most quantitatively important mechanism by which H-19B induction increases Stx1 production. Three promoters are shown to be involved in stx(1)AB transcription after phage induction, the iron-regulated p(Stx1) and the phage-regulated p(R) and p(R)' promoters, the relative importance of which varies with environmental conditions. Late phage transcription initiating at the p(R)' promoter, contrary to previous findings in the related Stx2-encoding phage phi 361, was found to be unnecessary for high-level Stx1 production after phage induction. Finally, we present evidence that phage-mediated lysis regulates the quantity of Stx1 produced by determining the duration of Stx1 accumulation and provides a mechanism for Stx1 release. By amplifying stx(1)AB copy number, regulating stx(1)AB transcription and allowing for Stx1 release, the biology of the Stx-encoding phages contributes greatly to the production of Stx, the principal virulence factor of STEC.

Shiga toxin 1 triggers a ribotoxic stress response leading to p38 and JNK activation and induction of apoptosis in intestinal epithelial cells.

Shiga toxins made by Shiga toxin-producing Escherichia coli (STEC) are associated with hemolytic uremic syndrome. Shiga toxins (Stxs) may access the host systemic circulation by absorption across the intestinal epithelium. The effects of Stxs on this cell layer are not completely understood, although animal models of STEC infection suggest that, in the gut, Stxs may participate in both immune activation and apoptosis. Stxs have one enzymatically active A subunit associated with five identical B subunits. The A subunit inactivates ribosomes by cleaving a specific adenine from the 28S rRNA. We have previously shown that Stxs can induce multiple C-X-C chemokines in intestinal epithelial cells in vitro, including interleukin-8 (IL-8), and that Stx-induced IL-8 expression is linked to induction of c-Jun mRNA and p38 mitogen-activated protein (MAP) kinase pathway activity. We now report Stx1 induction of both primary response genes c-jun and c-fos and activation of the stress-activated protein kinases, JNK/SAPK and p38, in the intestinal epithelial cell line HCT-8. By 1 h of exposure to Stx1, mRNAs for c-jun and c-fos are induced, and both JNK and p38 are activated; activation of both kinases persisted up to 24 h. Stx1 enzymatic activity was required for kinase activation; a catalytically defective mutant toxin did not activate either. Stx1 treatment of HCT-8 cells resulted in cell death that was associated with caspase 3 cleavage and internucleosomal DNA fragmentation; this cytotoxicity also required Stx1 enzymatic activity. Blocking Stx1-induced p38 and JNK activation with the inhibitor SB202190 prevented cell death and diminished Stx1-associated caspase 3 cleavage. In summary, these data link the Stx1-induced ribotoxic stress response with both chemokine expression and apoptosis in the intestinal epithelial cell line HCT-8 and suggest that blocking host cell MAP kinases may prevent these Stx-associated events.