Invasive Enteric Pathogens Research Articles

A hierarchy of cell death pathways confers layered resistance to shigellosis in mice.

Bacteria of the genus Shigella cause shigellosis, a severe gastrointestinal disease driven by bacterial colonization of colonic intestinal epithelial cells. Vertebrates have evolved programmed cell death pathways that sense invasive enteric pathogens and eliminate their intracellular niche. Previously we reported that genetic removal of one such pathway, the NAIP-NLRC4 inflammasome, is sufficient to convert mice from resistant to susceptible to oral Shigella flexneri challenge (Mitchell et al., 2020). Here, we investigate the protective role of additional cell death pathways during oral mouse Shigella infection. We find that the Caspase-11 inflammasome, which senses Shigella LPS, restricts Shigella colonization of the intestinal epithelium in the absence of NAIP-NLRC4. However, this protection is limited when Shigella expresses OspC3, an effector that antagonizes Caspase-11 activity. TNFα, a cytokine that activates Caspase-8-dependent apoptosis, also provides potent protection from Shigella colonization of the intestinal epithelium when mice lack both NAIP-NLRC4 and Caspase-11. The combined genetic removal of Caspases-1, -11, and -8 renders mice hyper-susceptible to oral Shigella infection. Our findings uncover a layered hierarchy of cell death pathways that limit the ability of an invasive gastrointestinal pathogen to cause disease.

Nitrate Utilization Promotes Systemic Infection of Salmonella Typhimurium in Mice.

Salmonella Typhimurium is an invasive enteric pathogen that causes gastroenteritis in humans and life-threatening systemic infections in mice. During infection of the intestine, S. Typhimurium can exploit nitrate as an electron acceptor to enhance its growth. However, the roles of nitrate on S. Typhimurium systemic infection are unknown. In this study, nitrate levels were found to be significantly increased in the liver and spleen of mice systemically infected by S. Typhimurium. Mutations in genes encoding nitrate transmembrane transporter (narK) or nitrate-producing flavohemoprotein (hmpA) decreased the replication of S. Typhimurium in macrophages and reduced systemic infection in vivo, suggesting that nitrate utilization promotes S. Typhimurium systemic virulence. Moreover, nitrate utilization contributes to the acidification of the S. Typhimurium cytoplasm, which can sustain the virulence of S. Typhimurium by increasing the transcription of virulence genes encoding on Salmonella pathogenicity island 2 (SPI-2). Furthermore, the growth advantage of S. Typhimurium conferred by nitrate utilization occurred only under low-oxygen conditions, and the nitrate utilization was activated by both the global regulator Fnr and the nitrate-sensing two-component system NarX-NarL. Collectively, this study revealed a novel mechanism adopted by Salmonella to interact with its host and increase its virulence.

A tale of two habitats: Bacteroides fragilis, a lethal pathogen and resident in the human gastrointestinal microbiome.

Bacteroides fragilis is an obligately anaerobic Gram-negative bacterium and a major colonizer of the human large colon where Bacteroides is a predominant genus. During the growth of an individual clonal population, an astonishing number of reversible DNA inversion events occur, driving within-strain diversity. Additionally, the B. fragilis pan-genome contains a large pool of diverse polysaccharide biosynthesis loci, DNA restriction/modification systems and polysaccharide utilization loci, which generates remarkable between-strain diversity. Diversity clearly contributes to the success of B. fragilis within its normal habitat of the gastrointestinal (GI) tract and during infection in the extra-intestinal host environment. Within the GI tract, B. fragilis is usually symbiotic, for example providing localized nutrients for the gut epithelium, but B. fragilis within the GI tract may not always be benign. Metalloprotease toxin production is strongly associated with colorectal cancer. B. fragilis is unique amongst bacteria; some strains export a protein >99 % structurally similar to human ubiquitin and antigenically cross-reactive, which suggests a link to autoimmune diseases. B. fragilis is not a primary invasive enteric pathogen; however, if colonic contents contaminate the extra-intestinal host environment, it successfully adapts to this new habitat and causes infection; classically peritoneal infection arising from rupture of an inflamed appendix or GI surgery, which if untreated, can progress to bacteraemia and death. In this review selected aspects of B. fragilis adaptation to the different habitats of the GI tract and the extra-intestinal host environment are considered, along with the considerable challenges faced when studying this highly variable bacterium.

Recombinant Human Secretory IgA Induces Salmonella Typhimurium Agglutination and Limits Bacterial Invasion into Gut-Associated Lymphoid Tissues.

As the predominant antibody type in mucosal secretions, human colostrum, and breast milk, secretory IgA (SIgA) plays a central role in safeguarding the intestinal epithelium of newborns from invasive enteric pathogens like the Gram-negative bacterium Salmonella enterica serovar Typhimurium (STm). SIgA is a complex molecule, consisting of an assemblage of two or more IgA monomers, joining (J)-chain, and secretory component (SC), whose exact functions in neutralizing pathogens are only beginning to be elucidated. In this study, we produced and characterized a recombinant human SIgA variant of Sal4, a well-characterized monoclonal antibody (mAb) specific for the O5-antigen of STm lipopolysaccharide (LPS). We demonstrate by flow cytometry, light microscopy, and fluorescence microscopy that Sal4 SIgA promotes the formation of large, densely packed bacterial aggregates in vitro. In a mouse model, passive oral administration of Sal4 SIgA was sufficient to entrap STm within the intestinal lumen and reduce bacterial invasion into gut-associated lymphoid tissues by several orders of magnitude. Bacterial aggregates induced by Sal4 SIgA treatment in the intestinal lumen were recalcitrant to immunohistochemical staining, suggesting the bacteria were encased in a protective capsule. Indeed, a crystal violet staining assay demonstrated that STm secretes an extracellular matrix enriched in cellulose following even short exposures to Sal4 SIgA. Collectively, these results demonstrate that recombinant human SIgA recapitulates key biological activities associated with mucosal immunity and raises the prospect of oral passive immunization to combat enteric diseases.

In vivo synthesis of bacterial amyloid curli contributes to joint inflammation during S. Typhimurium infection.

Reactive arthritis, an autoimmune disorder, occurs following gastrointestinal infection with invasive enteric pathogens, such as Salmonella enterica. Curli, an extracellular, bacterial amyloid with cross beta-sheet structure can trigger inflammatory responses by stimulating pattern recognition receptors. Here we show that S. Typhimurium produces curli amyloids in the cecum and colon of mice after natural oral infection, in both acute and chronic infection models. Production of curli was associated with an increase in anti-dsDNA autoantibodies and joint inflammation in infected mice. The negative impacts on the host appeared to be dependent on invasive systemic exposure of curli to immune cells. We hypothesize that in vivo synthesis of curli contributes to known complications of enteric infections and suggest that cross-seeding interactions can occur between pathogen-produced amyloids and amyloidogenic proteins of the host.

Efficacy of Rifaximin in Prevention of Travelers' Diarrhea: A Meta‐Analysis of Randomized, Double‐Blind, Placebo‐Controlled Trials

Rifaximin has been used successfully for the prevention of travelers' diarrhea (TD), the most general cause of disability among international travelers to developing tropical and semitropical regions. We sought to better evaluate the efficacy of rifaximin in the prevention of TD. Randomized controlled trials (RCTs) of rifaximin for the prevention of TD published in Pubmed, the Cochrane Central Register of Controlled Trials, Embase, and the Science Citation Index were searched. [Correction added on 3 October 2012, after first online publication: the phrase "protection of TD" was replaced with "prevention of TD".] The primary efficacy outcome was occurrence of TD over a 2-week treatment period. Secondary outcomes were requirement for antibiotic treatment, occurrence of mild diarrhea (MD), occurrence of TD in the third week after drug withdrawal, incidence of TD associated with isolation of diarrheagenic Escherichia coli (ie, ETEC, EAEC), and adverse events. Four RCTs with 502 participants were included in the systematic review. Rifaximin treatment showed a significant protection against TD (risk ratios, RR: 0.41, 95% CI: 0.30-0.56, p < 0.00001) and needed antibiotic-treated TD (relative risk [RR]: 0.30, 95% confidence interval [CI]: 0.18-0.49, p < 0.00001). There was no significant difference between rifaximin and placebo in the occurrence of MD (RR: 1.11, 95% CI: 0.78-1.59, p = 0.55) and the occurrence of TD in the third week after drug withdrawal (RR: 0.73, 95% CI: 0.30-1.73, p = 0.47). Enterotoxigenic E. coli was the major cause of TD, and all trials reported no differences in adverse events between rifaximin and placebo. Rifaximin can prevent TD caused by non-invasive enteric pathogens. Further research is needed for the treatment of invasive enteric pathogens. [Correction added on 3 October 2012, after first online publication: the phrase "Rifaximin can protect TD" was replaced with "Rifaximin can prevent TD".].

Salmonella Interaction with and Passage through the Intestinal Mucosa: Through the Lens of the Organism

Salmonella enterica serotypes are invasive enteric pathogens spread through fecal contamination of food and water sources, and represent a constant public health threat around the world. The symptoms associated with salmonellosis and typhoid disease are largely due to the host response to invading Salmonella, and to the mechanisms these bacteria employ to survive in the presence of, and invade through the intestinal mucosal epithelia. Surmounting this barrier is required for survival within the host, as well as for further dissemination throughout the body, and subsequent systemic disease. In this review, we highlight some of the major hurdles Salmonella must overcome upon encountering the intestinal mucosal epithelial barrier, and examine how these bacteria surmount and exploit host defense mechanisms.

Green banana-supplemented diet in the home management of acute and prolonged diarrhoea in children: a community-based trial in rural Bangladesh

To determine the effectiveness of green banana in the home management of acute (<7 days) or prolonged (≥ 7 days) diarrhoea at the community level. A cluster randomized field trial was conducted among 2968 Bangladeshi rural children 6-36 months old. Wards (villages) were randomly assigned to either a standard care group or a standard care plus green banana group where mothers were instructed to add cooked green banana to the diets of diarrhoeal children. Through a village-based surveillance system, diarrhoeal morbidity data (severity, duration, compliance) were collected for 14 days. Treatment effects were determined by analysing cumulative probability of cure by testing Cox proportional hazards models and relative risk (RR). The cumulative probability of cure was significantly (P < 0.001) different in children receiving GB for both acute [hazard ratio (HR) = 0.63 (95% CI: 0.56-0.67)] and prolonged diarrhoea [HR = 0.38 (95% CI: 0.26-0.59)]. The recovery rates of children with acute diarrhoea receiving GB (vs. control) were significantly more by day 3: 79.9%vs. 53.3% [(RR) = 0.47, 95% CI: 0.41-0.55], (P < 0.001) and day 7: 96.6%vs. 89.1% (RR = 0.32; 0.22-0.46), (P < 0.001). Children with prolonged diarrhoea receiving green banana had significantly higher recovery rates by day 10: 79.8%vs. 51.9% (RR = 0.42; 0.23-0.73), (P < 0.001) and day 14: 93.6%vs. 67.2% (RR = 0.22; 0.08-0.54), (P < 0.001). A green banana-supplemented diet hastened recovery of acute and prolonged childhood diarrhoea managed at home in rural Bangladesh.

MICROBIOLOGICAL ASPECTS ON IRRIGATION WATER QUALITY TO FIELD GROWN VEGETABLES

Food safety is an increasing problem in relation to vegetables, regarding both ready-to-eat produces, such as leafy salad, spinach and fruit as well as frozen produces that have not been exposed to pasteurization prior to freezing. Contamination may occur during the entire production chain. However, in connection with invasive enteric pathogens, slurry, farmyard manure, sewage sludge and irrigation water have been highlighted as potential sources for pathogen dispersal. Especially, the dispersal of Escherichia coli O157:H7 and Salmonella spp. is a considerable threat. An increasing number of outbreaks have been reported. To prevent the spread of invasive enteric bacteria by irrigation water, water sources, such as well water as well as community potable water have been proposed. The present paper describes pathogens of significance that may be spread by irrigation water supplied to field vegetables and presents some recent results on survival of human pathogens in water, soil and on vegetables. The current status of standards for microbiological quality of irrigation water to fresh or minimally processed fruit and vegetables is discussed.

Antisense Inhibition of Entamoeba histolytica Cysteine Proteases Inhibits Colonic Mucus Degradation

The exact role Entamoeba histolytica cysteine proteases play in overcoming the colonic mucus barrier, as a prerequisite to epithelial cell disruption, is not known. Herein, we determined whether E histolytica trophozoites expressing the antisense transcript to cysteine protease 5 (EhCP5) could degrade colonic mucin and destroy epithelial cells. Cysteine protease-deficient amoebae were generated by antisense inhibition of EhCP5, and assayed for proteolytic activity against [(35)S]cysteine-labeled mucin from LS 174T, and HT-29F Cl.16E cells. Recombinant EhCP5 mucinase activity was also assessed. Disruption of an intact mucus barrier and epithelial cell invasion by amoebae were measured using high mucin producing LS 174T and HT-29 Cl.16E monolayers or Chinese hamster ovary (CHO) cells devoid of a mucus barrier. Trophozoites with reduced cysteine protease activity were ineffective at degrading [(35)S]cysteine-labeled colonic mucin compared to wild-type amoebae by >60%. However, bioactive recombinant EhCP5 degraded >45% of purified native mucin, which was specifically inhibited by the cysteine proteinase (CP) inhibitor, E-64. Cysteine protease-deficient trophozoites could not overcome a protective intact mucus barrier and disrupt LS 174T or HT-29F Cl.16 cell monolayers; however, they readily adhere to and disrupt CHO monolayers devoid of a mucus barrier. These findings unravel a central role for E histolytica CPs as key virulence factors in disrupting an intact mucus barrier in the pathogenesis of intestinal amoebiasis.

Effects of Salmonella enterica serovars Typhimurium (ST) and Choleraesuis (SC) on chemokine and cytokine expression in swine ileum and jejunal epithelial cells

The gastrointestinal epithelium represents a barrier to potentially invasive enteric pathogens, maintains a role in innate immune surveillance, and is a source of both chemokine and cytokine chemotactic mediators in response to bacterial invasion. In the current study, we evaluated cytokine and chemokine mediators known to regulate movement of macrophages (macrophage migration inhibitory factor; MIF), neutrophils (IL8), dendritic cells (CCL20), and epithelial remodeling (osteopontin; OPN) in response to invasive swine enteropathogens Salmonella enterica serovar Typhimurium (ST) or Choleraesuis (SC). For the in vivo experiment, weaned pigs served as uninfected controls (0 h) or were given 3 × 10 9 CFU ST orally. Pigs were sacrificed at 8, 24, 48, and 144 h after inoculation and total RNA was extracted from defined segments of proximal (PI) and distal (DI) ileum. Relative expression of MIF and OPN were not affected by ST. IL8 expression was increased numerically ( P = 0.17 for the interaction term) at 24 and 144 h in the PI and these increases accounted for greater expression in the PI relative to the DI ( P < 0.05). Relative expression of CCL20 was increased at 24 h after ST ( P < 0.05). Next, we evaluated the time course of MIF, IL8, CCL20, and OPN mRNA expression induced by application of lipopolysaccharide (LPS), ST or SC in vitro using pig jejunal epithelial cells (IPEC-J2). Cells were grown to confluency on permeable membranes, and treated apically with LPS (10 ng/mL), ST or SC (10 8/well). After 1 h, cells were washed to remove LPS or extracellular bacteria, and media containing gentamicin was added to kill remaining extracellular bacteria. Media and RNA were collected at 1.5, 3, and 6 h after treatment. MIF mRNA was not affected by LPS or bacterial treatment. Similarly, IL8 expression was not affected by LPS, but was increased by ST and SC relative to controls at 1.5 and 3 h post exposure ( P < 0.05 for all comparisons). Treatment with SC increased CCL20 mRNA relative to controls at 3 h ( P < 0.05), while ST increased CCL20 at 1.5, 3, and 6 h with maximal expression at 6 h ( P < 0.05 for all comparisons). ST and SC increased polarized IL8 secretion. Our data demonstrate that invasive bacterial pathogens in the pig gastrointestinal tract trigger upregulation of selected cytokine and chemokine mediators, but serovars of Salmonella elicited differing patterns of activation in vitro.

TLR Signaling in the Gut in Health and Disease

The human intestine has evolved in the presence of diverse enteric microflora. TLRs convert the recognition of pathogen-associated molecules in the gut into signals for anti-microbial peptide expression, barrier fortification, and proliferation of epithelial cells. Healing of injured intestinal epithelium and clearance of intramucosal bacteria require the presence of intact TLR signaling. Nucleotide oligomerization domain (Nod)1 and Nod2 are additional pattern recognition receptors that are required for defense against invasive enteric pathogens. Through spatial and functional localization of TLR and Nod molecules, the normal gut maintains a state of controlled inflammation. By contrast, patients with inflammatory bowel disease demonstrate inflammation in response to the normal flora. A subset of these patients carry polymorphisms in TLR and CARD15/NOD2 genes. A better understanding of the delicate regulation of TLR and Nod molecules in the gut may lead to improved treatment for enteric infections and idiopathic inflammatory bowel diseases.

The rovA mutant of Yersinia enterocolitica displays differential degrees of virulence depending on the route of infection.

Yersinia enterocolitica is an invasive enteric pathogen that causes significant inflammatory disease. Recently, we identified and characterized a global regulator of virulence (rovA). When mice are infected orally with the rovA mutant they are attenuated by 50% lethal dose (LD(50)) analysis and have altered kinetics of infection. Most significantly, mice orally infected with the rovA mutant have greatly reduced inflammation in the Peyer's patches compared to those infected with wild-type Y. enterocolitica. However, we present data here indicating that when the rovA mutant bacteria are delivered intraperitoneally (i.p.), they are significantly more virulent than when delivered orally. The i.p. LD(50) for the rovA mutant is only 10-fold higher than that of the wild-type Y. enterocolitica, and there are significant inflammatory responses to the rovA mutant that are evident in the liver and spleen. Altogether, these data suggest that the RovA regulon may be required for the early events of the infection that occur in the Peyer's patches. Furthermore, these data suggest that the RovA regulon may be dispensable for Y. enterocolitica systemic disease and inflammatory responses if the Peyer's patches are bypassed.

Analysis by High Density cDNA Arrays of Altered Gene Expression in Human Intestinal Epithelial Cells in Response to Infection with the Invasive Enteric BacteriaSalmonella

Many clinically important enteric pathogens initiate disease by invading and passing through the intestinal epithelium, a process accompanied by increased epithelial expression of proinflammatory cytokines. To further define the role intestinal epithelial cells play in initiating and modulating the host response to infection with invasive bacteria, hybrid selection on high density cDNA arrays was used to characterize the mRNA expression profile of approximately 4,300 genes in human intestinal epithelial cells after infection with the prototypic invasive bacteria, Salmonella. Selected findings were further evaluated by reverse transcription-polymerase chain reaction, Northern blot analysis, and protein assays. Epithelial infection with Salmonella significantly up-regulated mRNA expression of a relatively small fraction of all genes tested. Of these, several cytokines (granulocyte colony-stimulating factor, inhibin A, Epstein-Barr virus-induced gene 3, interleukin-8, macrophage inflammatory protein-2alpha), kinases (TKT, Eck, HEK), transcription factors (interferon regulatory factor-1), and HLA class I were the most prominent. Furthermore, the transcription factor NF-kappaB is shown to be important for inducible mRNA expression for a broad group of genes tested. These findings expand the repertoire of known epithelial cell responses to infection with an invasive enteric pathogen. The results also show that evaluation of mRNA expression profiles by cDNA array analysis is a powerful approach to characterizing and understanding host-pathogen interactions.

Mucosal and systemic immune responses in humans after primary and booster immunizations with orally administered invasive and noninvasive live attenuated bacteria.

The mucosal and systemic immune responses after primary and booster immunizations with two attenuated live oral vaccine strains derived from a noninvasive (Vibrio cholerae) and an invasive (Salmonella typhi) enteric pathogen were comparatively evaluated. Vaccination with S. typhi Ty21a elicited antibody-secreting cell (ASC) responses specific for S. typhi O9, 12 lipopolysaccharide (LPS), as well as significant increases in levels of immunoglobulin G (IgG) and IgA antibodies to the same antigen in serum. A strong systemic CD4(+) T-helper type 1 cell-mediated immune (CMI) response was also induced. In contrast to results with Ty21a, no evidence of a CMI response was obtained after primary immunization with V. cholerae CVD 103-HgR in spite of the good immunogenicity of the vaccine. Volunteers who received a single dose of CVD 103-HgR primarily developed an IgM ASC response against whole vaccine cells and purified V. cholerae Inaba LPS, and seroconversion of serum vibriocidal antibodies occurred in four of five subjects. Serum IgG anti-cholera toxin antibody titers were of lower magnitude. For both live vaccines, the volunteers still presented significant local immunity 14 months after primary immunization, as revealed by the elevated baseline antibody titers at the time of the booster immunization and the lower ASC, serum IgG, and vibriocidal antibody responses after the booster immunization. These results suggest that local immunity may interfere with colonization of the gut by both vaccine strains at least up to 14 months after basis immunization. Interestingly, despite a low secondary ASC response, Ty21a was able to boost both humoral (anti-LPS systemic IgG and IgA) and CMI responses. Evidence of a CMI response was also observed for one of three volunteers given a cholera vaccine booster dose. The direct comparison of results with two attenuated live oral vaccine strains in human volunteers clearly showed that the capacity of the vaccine strain to colonize specific body compartments conditions the pattern of vaccine-induced immune responses.

Apoptosis of human intestinal epithelial cells after bacterial invasion.

Epithelial cells that line the human intestinal mucosa are the initial site of host invasion by bacterial pathogens. The studies herein define apoptosis as a new category of intestinal epithelial cell response to bacterial infection. Human colon epithelial cells are shown to undergo apoptosis following infection with invasive enteric pathogens, such as Salmonella or enteroinvasive Escherichia coli. In contrast to the rapid onset of apoptosis seen after bacterial infection of mouse monocyte-macrophage cell lines, the commitment of human intestinal epithelial cell lines to undergo apoptosis is delayed for at least 6 h after bacterial infection, requires bacterial entry and replication, and the ensuing phenotypic expression of apoptosis is delayed for 12-18 h after bacterial entry. TNF-alpha and nitric oxide, which are produced as components of the intestinal epithelial cell proinflammatory program in the early period after bacterial invasion, play an important role in the later induction and regulation of the epithelial cell apoptotic program. Apoptosis in response to bacterial infection may function to delete infected and damaged epithelial cells and restore epithelial cell growth regulation and epithelial integrity that are altered during the course of enteric infection. The delay in onset of epithelial cell apoptosis after bacterial infection may be important both to the host and the invading pathogen since it provides sufficient time for epithelial cells to generate signals important for the activation of mucosal inflammation and concurrently allows invading bacteria time to adapt to the intracellular environment before invading deeper mucosal layers.

Prevalence of invasive ability and other virulence-associated characteristics in Providencia alcalifaciens strains isolated in São Paulo, Brazil.

Providencia alcalifaciens is an invasive enteric pathogen. The present study determined the prevalence of invasive ability in P. alcalifaciens strains isolated in São Paulo, Brazil, mainly from patients with diarrhoea. Invasion of HeLa cells was found in 17 (42%) of 41 strains studied. Most (88%) of the invasive strains were isolated from diarrhoeal stools. The invasive property was identified in 50% of P. alcalifaciens strains isolated as pure cultures or from stool samples where no other enteropathogen was identified. All the invasive strains caused actin condensation in infected cells. Plasmid profile analysis showed the presence of plasmids of 35.8-180 kb in 70% of the strains regardless of their invasive ability, suggesting that invasiveness in P. alcalifaciens is not plasmid related. No homology with a probe for gene sequences for invasion of enteroinvasive Escherichia coli and Shigella strains was identified in colony hybridisation assays. The invasive property of P. alcalifaciens was confirmed in the present study, but this characteristic did not predominate among strains isolated from patients with diarrhoea in São Paulo City. The presence of other virulence mechanisms and the role of non-invasive P. alcalifaciens strains as a cause of diarrhoea remain to be established.

HIV-1 Tat modulates invasion by a bacterial enteric pathogen into a human intestinal cell line.

To explore the possibility that an HIV-1 gene product may modulate entry of an invasive enteric pathogen into a terminally differentiated human intestinal cell line. HIV-1 Tat was selected for investigation because of its unique ability to cross cell membranes. After transient transfection of HT29-C1 cells with plasmids containing HIV-1 long terminal repeat (LTR)-lacZ plus a Tat expression cassette, or with a pSR-lacZ control plasmid, bacterial invasion assays were performed on both groups of cells utilizing a clinical Salmonella isolate. Assays were performed concurrently on a control group of non-transfected cells. A second series of experiments compared bacterial invasion into cells transfected with the Tat expression vector alone versus cells transfected with either an isogenic expression vector that did not make Tat, or with pSR-lacZ. Finally, the ability of exogenous Tat protein to transactivate an HIV-1 LTR-chloramphenicol acetyltransferase (CAT) plasmid which had been transfected into HT29-C1 cells and to modulate Salmonella invasion was also assessed. HT29-C1 cells transfected with a Tat expression vector, either alone or in combination with another plasmid, were significantly less susceptible to bacterial invasion than cells that either did not undergo transfection, were transfected with an otherwise isogenic expression vector without Tat, or transfected with an unrelated plasmid. Duplicate experiments also demonstrated that exogenous purified Tat protein transactivated an HIV-1 LTR-CAT plasmid which had been transfected into HT29-C1 cells and inhibited Salmonella invasion compared with unexposed cells. HIV-1 Tat inhibits Salmonella invasion of a human enterocyte cell line whether the protein is expressed intracellularly or provided exogenously.

Characteristics of invasion of HEp-2 cells by Providencia alcalifaciens.

Previous studies with three isolates from diarrhoeal stools suggested that Providencia alcalifaciens is an invasive enteric pathogen that also causes actin condensation in infected cells. These findings were extended in the present study with a further 14 diarrhoeal stool isolates of P. alcalifaciens and HEp-2 cell monolayers for invasion assays. Studies on invasion characteristics with two selected isolates suggested that P. alcalifaciens required prior growth at 37 degrees C for better invasion. Invasion and actin condensation were inhibited by an agent that inhibits microfilament formation, but not by agents that inhibit receptor-mediated endocytosis, microtubule formation, endosome acidification or receptor recycling. In time-course assays with HEp-2 cell monolayers maintained in medium containing gentamicin, P. alcalifaciens showed a small degree of multiplication after invasion of the cells, but viable bacteria could not be recovered over a 24-h period although the integrity of the cell monolayer was preserved during this period.

FOOD POISONING SYNDROMES

Despite our society's advances in sanitation, food preservation, and hygiene, the prevalence of foodborne disease remains high (12.6 million cases per year in the United States). Although there is a constant need for education of food handlers and consumers, there is also a need for continued vigilant monitoring of coastal waters, meat packing facilities, and imported foods. As long as antibiotics are used in poultry and cattle feeds, one can expect the incidence of antibiotic-resistant foodborne pathogens to rise. There are several promising areas of research in the field of foodborne illnesses. Molecular biologists are actively characterizing the genes that enable invasive enteric pathogens such as Salmonella and Yersinia to enter tissues, and the bacterial toxins associated with secretory diarrheas continue to be the subject of intense scrutiny. Epidemiologists are implementing new techniques such as DNA fingerprinting and multilocus enzyme electrophoresis for tracing pathogens in disease outbreaks. Similarly, the use of computers in the food industry facilitates the tracing of contaminated foods. Although the rates of foodborne illness may not decrease significantly during the next decade, we can expect more rapid identification and tracing of outbreaks as well as an improved understanding of the pathogenesis of the foodborne diseases.