Enteroinvasive Bacteria Research Articles

Enteroinvasive bacteria. Local study in specialized clinical care

Enteroinvasive bacteria. Local study in specialized clinical care

Virulence and antimicrobial resistance of Escherichia coli isolated from chicken meat, beef, and raw milk

. Two hundred and thirty-five strains of E. coli were recovered from beef, chicken meat, and raw milk sold in butcher shops and markets in the town of Jijel, East Algeria. A PCR investigation revealed the predominance of bacterial strains with extraintestinal pathogenic E. coli (ExPEC) genes (19.91%). Enteroaggregative (EAEC) and enteroinvasive (EIEC) bacteria were also detected. Strains from phylogenetic groups A and B1 were the most common; they accounted for 62.35% and 21.17% in chicken meat, 67.53% and 16.88% in raw milk and 64.38% and 25.97% in beef, respectively, followed by the strains from phylogenetic groups B2 and D. Moreover, E. coli from phylogenetic group D was more abundant in chicken than in beef or raw milk samples (P<0.05). Antibiogram results revealed the presence of three major antibiotic-susceptibility groups and more than a hundred antibiotypes. Between 50% and 70% of strains were resistant to antibiotics of the first group (ampicillin, amoxicillin, trimethoprim, sulfonamide and tetracycline), 20% to 50% to antibiotics of the second group (amoxicillin plus clavulanic acid, kanamycin, streptomycin, ciprofloxacin, flumequine and neomycin) and less than 20% to those of the third group (cephalothin, gentamicin and colistin). Additionally, the resistance to flumequine, trimethoprim, sulfamethoxazole-trimethoprim and tetracycline was more frequent in chicken meat strains than in those from beef and milk. It is important to point out that the strains carrying more than two virulence factors belonged to the B2 or D phylogenetic groups and had weaker resistance to antibiotics. The strains from the A and B1 groups had fewer virulence factors and showed high resistance to antibiotics.

Associations of bacterial enteropathogens with systemic inflammation, iron deficiency, and anemia in preschool-age children in southern Ghana.

Anemia remains a pervasive public health problem among preschool-age children in Ghana. Recent analyses have found that anemia in Ghanaian children, particularly in Southern regions, is largely attributable to infectious causes, rather than nutritional factors. Infections with enteropathogens can reduce iron absorption and increase systemic inflammation, but few studies have examined direct links between enteropathogens and anemia. This study investigated associations between detection of individual bacterial enteropathogens and systemic inflammation, iron deficiency, and anemia among 6- to 59-month-old children in Greater Accra, Ghana. Serum samples were analyzed from a cross-sectional sample of 262 children for concentrations of hemoglobin (Hb), biomarkers of systemic inflammation [C-reactive protein (CRP) and α-1-acid glycoprotein (AGP)], and biomarkers of iron status [serum ferritin (SF) and serum transferrin receptor (sTfR)]. Stool samples were analyzed for ten bacterial enteropathogens using qPCR. We estimated associations between presence of each enteropathogen and elevated systemic inflammation (CRP > 5 mg/L and AGP > 1 g/L), iron deficiency (SF < 12 μg/L and sTfR > 8.3 mg/L) and anemia (Hb < 110 g/L). Enteropathogens were detected in 87% of children’s stool despite a low prevalence of diarrhea (6.5%). Almost half (46%) of children had anemia while one-quarter (24%) had iron deficiency (low SF). Despite finding no associations with illness symptoms, Campylobacter jejuni/coli detection was strongly associated with elevated CRP [Odds Ratio (95% CI): 3.49 (1.45, 8.41)] and elevated AGP [4.27 (1.85, 9.84)]. Of the pathogens examined, only enteroinvasive Escherichia coli/Shigella spp. (EIEC/Shigella) was associated with iron deficiency, and enteroaggregative Escherichia coli (EAEC) [1.69 (1.01, 2.84)] and EIEC/Shigella [2.34 (1.15, 4.76)] were associated with anemia. These results suggest that certain enteroinvasive pathogenic bacteria may contribute to child anemia. Reducing exposure to enteropathogens through improved water, sanitation, and hygiene practices may help reduce the burden of anemia in young Ghanaian children.

Enteric pathogens in German police officers after predominantly tropical deployments - A retrospective assessment over 5 years.

IntroductionThe study was performed to assess the infection risk of German police officers on predominantly tropical deployments, mostly United Nations missions, with gastrointestinal pathogens.MethodsPolice officers were offered PCR-based screening for gastrointestinal pathogens before and after deployment. The screening panel comprised enteroinvasive bacteria (Salmonella spp., Shigella spp./enteroinvasive Escherichia coli, Campylobacter jejuni, and Yersinia spp.), enteropathogenic protozoa (Entamoeba histolytica, Giardia duodenalis, Cryptosporidium spp., and Cyclospora cayetanensis), as well as enteric helminths (Ancyclostoma spp., Ascaris lumbricoides, Enterobius vermicularis, Hyme-nolepis nana, Necator americanus, African Schistosoma spp., Strongyloides stercoralis, Taenia saginata, Taenia solium, and Trichuris trichiura).ResultsG. duodenalis (n = 3), C. jejuni (n = 2), Salmonella spp. (n = 1), Shigella spp./enteroinvasive E. coli (n = 3), and S. stercoralis (n = 3) were detect in 12 out of 133 (9.0%) police officers. The majority had shown gastrointestinal symptoms on deployment and all were asymptomatic at the time of medical assessment. The major infection sites were Sub-Saharan Africa followed by Northern Africa and the Middle East.ConclusionsDeployment of police officers to tropical deployment sites on United Nations missions is associated with a considerable acquisition risk of gastrointestinal pathogens in a quantitatively relevant minority. Post-deployment screening is advisable to facilitate therapeutic and hygiene-related consequences.

Alternative folding to a monomer or homopolymer is a common feature of the type 1 pilus subunit FimA from enteroinvasive bacteria

Adhesive type 1 pili from enteroinvasive, Gram-negative bacteria mediate attachment to host cells. Up to 3000 copies of the main pilus subunit, FimA, assemble into the filamentous, helical quaternary structure of the pilus rod via a mechanism termed donor-strand complementation, in which the N-terminal extension of each subunit, the donor strand, is inserted into the incomplete immunoglobulin-like fold of the preceding FimA subunit. For FimA from Escherichia coli, it has been previously shown that the protein can also adopt a monomeric, self-complemented conformation in which the donor strand is inserted intramolecularly in the opposite orientation relative to that observed for FimA polymers. Notably, soluble FimA monomers can act as apoptosis inhibitors in epithelial cells after uptake of type 1-piliated pathogens. Here, we show that the FimA orthologues from Escherichia coli, Shigella flexneri, and Salmonella enterica can all fold to form self-complemented monomers. We solved X-ray structures of all three FimA monomers at 0.89–1.69 Å resolutions, revealing identical, intramolecular donor-strand complementation mechanisms. Our results also showed that the pseudo-palindromic sequences of the donor strands in all FimA proteins permit their alternative folding possibilities. All FimA monomers proved to be 50–60 kJ/mol less stable against unfolding than their pilus rod-like counterparts (which exhibited very high energy barriers of unfolding and refolding). We conclude that the ability of FimA to adopt an alternative, monomeric state with anti-apoptotic activity is a general feature of FimA proteins of type 1-piliated bacteria.

A meta-analysis of the use of rifaximin to prevent travellers' diarrhoea.

Travellers' diarrhoea affects tens of millions of people travelling to less developed countries or regions annually. There are positive reports of the use of rifaximin, a non-absorbed, gut-selective antibiotic to prevent travellers' diarrhoea. This study will critically review and analyse clinical trials on the subject. Using the keywords [diarrhoea OR diarrhoea OR travel*] AND [rifaximin OR xifaxan OR xifaxanta OR normix OR rifagut], a preliminary search on the PubMed and Ovid databases yielded 411 papers published in English between 1 January 1988 and 1 July 2016. Of these, there were only five relevant clinical trials. The clinical trials were double-blind, placebo-controlled, randomized trials with a total of 879 subjects. The meta-analysis found significant reduction in risk of travellers' diarrhoea with rifaximin use compared to placebo (pooled RR 0.478, 95% CI: 0.375-0.610, and P < 0.001). For the entire travel and follow-up period, the risk of developing travellers' diarrhoea was significantly greater in individuals receiving the placebo than those receiving rifaximin (daily doses of 400-600 mg). Overall, rifaximin offered significant protection rates of 48-72%, with lower protection rates for Asian than Latin American countries. In terms of tolerability, similar rates of adverse events were reported for the rifaximin and placebo group ( P > 0.05), with no clinically significant or serious adverse events related to rifaximin use. There is good evidence supporting the use of rifaximin as a chemoprophylactic agent against travellers' diarrhoea, especially in individuals who are at high risk of severe complications from acute infectious diarrhoea. Rifaximin has an excellent tolerability/safety profile and demonstrated efficacy against diarrhoeagenic Escherichia coli and even enteroinvasive bacteria such Campylobacter species. Future studies should study the most effective dosing regimen for rifaximin chemoprophylaxis, as well as profile local antimicrobial resistance/susceptibility data in less developed regions to further guide rifaximin use.

Infection of human intestinal epithelial cells by invasive bacteria activates NF-κB and increases ICAM-1 expression through NOD1.

Background/AimsNucleotide-binding oligomerization domain 1 (NOD1) is required for primary intestinal epithelial cells (IECs) to respond to natural mucopeptides secreted by gram-negative bacteria. Infection of human IECs with invasive bacteria up-regulates intercellular adhesion molecule-1 (ICAM-1) expression. However, the role of NOD family members in host defense has been largely unknown. The aim of this study was to determine whether there is a functional role for NOD1 in the up-regulation of ICAM-1 expression in invasive bacteria-infected IECs.MethodsICAM-1 mRNA expression was compared between controls, Caco-2 or HT29 cells transfected with an empty vector, and IECs stably transfected with a dominant-negative (DN) NOD1. Expression was compared using qualitative reverse transcription polymerase chain reaction (RT-PCR), real-time RT-PCR, and flow cytometry after infection with enteroinvasive Escherichia coli O29:NM or Shigella flexneri. Nuclear factor kB (NF-κB) activation was determined by electrophoretic mobility shift assays.ResultsDN NOD1 significantly inhibited the up-regulation of ICAM-1 expression in response to an enteroinvasive bacterial infection. The Caco-2 cells transfected with DN NOD1 manifested marked inhibition of NF-kB activation in response to E. coli O29:NM infection.ConclusionsSignaling through NOD1 may play an essential role in neutrophil trafficking following infection with enteroinvasive bacteria.

Evaluation of immunogenicity and protective efficacy of combination heat-killed immunogens from three entero-invasive bacteria in rabbit model

Diarrhea is a very common health problem in both developing and developed countries. Among the major entero-invasive bacteria, Shigella, Salmonella and Campylobacter cause serious problems in different geographic regions. Recently we have shown immunogenicity and protective efficacy of heat killed multi-serotype Shigella immunogen in different animal models. In our present study, we have advanced our research by preparing a combination heat-killed immunogen of three different entero-invasive bacteria, Shigella, Salmonella and Campylobacter. After three doses on 0th, 14th and 28th day of oral immunization with tri-valent heat-killed (TVHK) immunogen in rabbit model, the immunogenicity was determined by differential count of white blood cells and immunoglobulin assay at various time points. During oral immunization differential count of lymphocytes increased where as polymorphonuclear leucocytes (PMN) count decreased. Serum IgG and IgA showed significant elevation during oral immunization and remained at a detectable value upto 120 days. Protection study was performed in both, in vitro and in vivo conditions, using bacteriocidal assay and rabbit ligated ileal loop model, respectively, which conferred protection against homologous bacteria. Moreover, immunoblot assay against whole cell lysate and lipopolysaccharide exhibited significant amount of antigen-specific immunoglobulins raised against three different bacteria which proved that proteins along with lipopolysaccharides played a pivotal role in immunogenicity and protective efficacy. This trivalent heat-killed immunogen could be a low-cost, simple, oral, non-living vaccine candidate for future use against invasive diarrhea.

Evaluation of the Impact of Stool Bacterial Cultures on Clinical Management on Patients Admitted With a Primary Diagnosis of Diarrhea

Introduction: Most cases of acute diarrhea are self-limited and due to viral or baterial infections. Stool cultures only test for the specific enteroinvasive bacteria, including Campylobacter jejuni, Salmonella species, Shigella species, Escherichia coli, which represent a small fraction of the infectious agents that can cause acute diarrhea. Previous studies have shown these organisms are positive in approximately 0.2 to 1.8% of cultures. We evaluated the effect of stool culture results on the management of inpatients with acute diarrhea. Methods: The database from the inpatient electronic medical records system at a university hospital was queried for stool bacterial cultures performed on inpatients from June 2010 through June 2014. Chart review was performed to assess the impact of stool culture results on clinical management and was limited to patients with an admitting diagnosis of acute diarrhea.Table 1: Stool Culture DistributionTable 2: Stool Culture DistributionFigure 1Figure 2Results: A total of 4576 stool cultures were evaluated within the study period. Of these stool cultures, 527 (11.5%) were positive, the most common organisms being: Candida sp. (n=282, 6.16%), Staphylococcus aureus (n=88, 1.92%), Campylobacter jejuni (n=25, 0.55%), Salmonella sp. (n=22, 0.48%), Shigella sp. (n=13, 0.28%). Escherchia coli 0157:H7 was not isolated in any cultures. Candida and S. aureus were considered contaminants and did not impact clinical management. This left 54 positive stool cultures for analysis, excluding duplicate cultures and those taken from patients without diarrhea. Seven cultures (15.5%) resulted in a change of the patient's management. Management changes included initiating antibiotics (n=4, 7.4%) and changing to narrower spectrum antibiotics (n=3, 5.6%). Results from 33 of the positive cultures (61%) were obtained after the patient was discharged with improved symptoms (with or without empiric antibiotic treatment). Results from the remaining 14 stool cultures (26%) were obtained during the patient's admission, but did not result in a change in management. Conclusion: In this large single center retrospective analysis, we demonstrate that bacterial stool cultures have a low rate of positivity, and positive culture results are of limited utility in guiding clinical management in patients admitted with acute diarrhea. Clinical judgment as to the benefits of culture results in guiding management should be considered prior to collecting them, and may enhance high value care and cost effectiveness.

PCR for enteric pathogens in high-prevalence settings. What does a positive signal tell us?

Background: Molecular methods, in particular PCR, are increasingly used for the diagnosis of enteric pathogens in stool samples. In high-endemicity settings, however, asymptomatic carriage or residual DNA from previous infections will hamper the interpretation of positive test results. We assessed the quantitative dimension of this problem in schoolchildren in the rural highlands of Madagascar. Methods: Stool samples were collected from 410 apparently healthy Madagascan schoolchildren and analysed by multiplex real-time PCR for enteroinvasive bacteria (Salmonella spp., Shigella spp./enteroinvasive Escherichia coli (EIEC), Campylobacter jejuni, Yersinia spp.), enteric protozoa (Entamoebea histolytica, Giardia duodenalis, Cryptosporidium spp., Cyclospora spp.), and helminths (Ascaris lumbricoides, Ancylostoma spp., Necator americanus, Strongyloides stercoralis). Symptoms of gastrointestinal disease were assessed. Results: Among the 410 samples, we detected Giardia duodenalis in 195, Campylobacter jejuni in 91, Ascaris lumbricoides in 72, Cyclospora cayetanenesis in 68, Shigella spp./EIEC in 56, and Strongyloides stercoralis and Cryptosporum spp. in 1 case each. Salmonella spp., Yersinia spp. and hookworms were not observed. Relative risk assessment suggested few and incoherent associations with pathogen detections, indicating asymptomatic carriage or DNA residuals. Only 26.1% of the schoolchildren were tested negative for all analysed pathogens. Conclusions: The very high risk of detecting traces of asymptomatic carriage or residual DNA from previous infections limits the value of highly sensitive PCR for the causal attribution of detected enteric pathogens from stool samples to an infectious gastrointestinal disease in the high-endemicity setting. Evaluated standards for the interpretation of such results are needed both for the diagnostic routine and for epidemiological assessments.

Surveillance of Food- and Smear-Transmitted Pathogens in European Soldiers with Diarrhea on Deployment in the Tropics: Experience from the European Union Training Mission (EUTM) Mali.

Introduction. Since 2013, European soldiers have been deployed on the European Union Training Mission (EUTM) in Mali. From the beginning, diarrhea has been among the most “urgent” concerns. Diarrhea surveillance based on deployable real-time PCR equipment was conducted between December 2013 and August 2014. Material and Methods. In total, 53 stool samples were obtained from 51 soldiers with acute diarrhea. Multiplex PCR panels comprised enteroinvasive bacteria, diarrhea-associated Escherichia coli (EPEC, ETEC, EAEC, and EIEC), enteropathogenic viruses, and protozoa. Noroviruses were characterized by sequencing. Cultural screening for Enterobacteriaceae with extended-spectrum beta-lactamases (ESBL) with subsequent repetitive sequence-based PCR (rep-PCR) typing was performed. Clinical information was assessed. Results. Positive PCR results for diarrhea-associated pathogens were detected in 43/53 samples, comprising EPEC (n = 21), ETEC (n = 19), EAEC (n = 15), Norovirus (n = 10), Shigella spp./EIEC (n = 6), Cryptosporidium parvum (n = 3), Giardia duodenalis (n = 2), Salmonella spp. (n = 1), Astrovirus (n = 1), Rotavirus (n = 1), and Sapovirus (n = 1). ESBL-positive Enterobacteriaceae were grown from 13 out of 48 samples. Simultaneous infections with several enteropathogenic agents were observed in 23 instances. Symptoms were mild to moderate. There were hints of autochthonous transmission. Conclusions. Multiplex real-time PCR proved to be suitable for diarrhea surveillance on deployment. Etiological attribution is challenging in cases of detection of multiple pathogens.

Liver-Resident Macrophage Necroptosis Orchestrates Type 1 Microbicidal Inflammation and Type-2-Mediated Tissue Repair during Bacterial Infection

Kupffer cells, the phagocytes of fetal origin that line the liver sinusoids, are key contributors of host defense against enteroinvasive bacteria. Here, we found that infection by Listeria monocytogenes induced the early necroptotic death of Kupffer cells, which was followed by monocyte recruitment and an anti-bacterial type 1 inflammatory response. Kupffer cell death also triggered a type 2 response that involved the hepatocyte-derived alarmin interleukin-33 (IL-33) and basophil-derived interleukin-4 (IL-4). This led to the alternative activation of the monocyte-derived macrophages recruited to the liver, which thereby replaced ablated Kupffer cells and restored liver homeostasis. Kupffer cell death is therefore a key signal orchestrating type 1 microbicidal inflammation and type-2-mediated liver repair upon infection. This indicates that beyond the classical dichotomy of type 1 and type 2 responses, these responses can develop sequentially in the context of a bacterial infection and act interdependently, orchestrating liver immune responses and return to homeostasis, respectively.

Linear synthesis of the branched pentasaccharide repeats of O-antigens from Shigella flexneri 1a and 1b demonstrating the major steric hindrance associated with type-specific glucosylation.

Shigella flexneri serotypes 1b and 1a are Gram-negative enteroinvasive bacteria causing shigellosis in humans. The O-antigen from S. flexneri 1b is a { → 2)-[3Ac/4Ac]-α-L-RHAP-(1 → 2)-α-L-Rhap-(1 → 3)-[2Ac]-α-L-Rhap-(1 → 3)-[α-D-Glcp-(1 → 4)]-β-D-GlcpNAc-(1 → }n branched polysaccharide ({(Ac)AB(Ac)C(E)D}n). It is identical to that from S. flexneri 1a, except for the 2C-acetate. A concise synthesis of the disaccharide ED, trisaccharides (Ac)C(E)D and C(E)D, tetrasaccharides B(Ac)C(E)D and BC(E)D, and pentasaccharides AB(Ac)C(E)D and ABC(E)D is described starting from a 2-N-acetyl-D-glucosaminide acceptor and using the imidate glycosylation chemistry. The E residue was efficiently introduced via a potent stereoselective [E + D] coupling. In contrast, harsh conditions and appropriate tuning of the donor were required for a high yielding [C + ED] glycosylation. Irrespective of the level of steric bulk at residue C, glycosylation at O-3D of the ED acceptor generated a major change of conformation of the D residue within the obtained C(E)D trisaccharide, as attested by NMR data. Proper manipulation of the constrained C(E)D trisaccharide was necessary to proceed with the stepwise chain elongation at O-3C of an acceptor having the 2C-O-acetyl already in place. The protected intermediates went through a one- to three-step deprotection sequence to give the propyl glycoside targets, as portions of the O-antigens from both S. flexneri 1a and 1b. Protecting group removal was clearly associated with conformational relief, yielding oligosaccharides, for which NMR data were consistent with a (4)C1 conformation for the 3,4-di-O-glycosylated residue D, as in the native bacterial polymers.

Effectiveness of rifaximin in prevention of diarrhoea in individuals travelling to south and southeast Asia: a randomised, double-blind, placebo-controlled, phase 3 trial

Travellers' diarrhoea causes substantial acute and long-term morbidity. Chemoprophylaxis with fluoroquinolones or rifaximin is effective in prevention of diarrhoea in individuals travelling to Latin America and Africa. Little evidence is available to support the protective effect of antimicrobial drugs in south and southeast Asia, where enteroinvasive and antibiotic-resistant bacteria cause a substantial proportion of diarrhoeal episodes. We aimed to assess the effectiveness of rifaximin in prevention of diarrhoea in individuals travelling to south and southeast Asia. We did this double-blind, placebo-controlled, single-centre, parallel-group, clinical trial in Tübingen, Germany, between Nov 12, 2009, and Sept 3, 2012. Individuals aged 18-64 years who were planning a 6-28 day journey to south and southeast Asia were randomly assigned (1:1), according to a randomisation list (permuted block size of eight) generated by an independent statistician, to receive placebo or rifaximin 200 mg tablets twice daily. All members of the study team, including investigators, those assessing outcomes, and data analysts, were masked to treatment allocation. The primary endpoint was time to the first episode of classic travellers' diarrhoea, defined as three or more loose stools in 24 h, accompanied by one or more enteric symptoms. Analyses were by intention to treat and per protocol. We randomly assigned 258 participants to rifaximin (n=129) or placebo (n=129), of whom 239 (93%) returned a completed diary and were included in the primary effectiveness analysis. 48 (41%) of 117 participants in the placebo group and 30 (25%) of 122 in the rifaximin group reported classic episodes of travellers' diarrhoea. From departure to 7 days after return, rifaximin provided 48% protection (95% CI 16-68) by lowering the incidence of travellers' diarrhoea from 1·99 (1·50-2·64) per 100 person-days in the placebo group to 1·04 (0·72-1·48) in the intervention group (incidence rate ratio 0·52, 95% CI 0·32-0·84; p=0·005). The number needed to treat was 5·70 (95% CI 3·44-16·69) to prevent one case of classic travellers' diarrhoea during the first 3 weeks of follow-up. The per-protocol analysis essentially corroborated the findings from the intention-to-treat analysis. We recorded one serious adverse event in a participant in the rifaximin group who had grade 3 right lower quadrant abdominal pain 72 h after the last intake of study drug. The complaints were considered unlikely to be related to use of the drug. Rifaximin is moderately effective in prevention of diarrhoea in individuals travelling to south and southeast Asia. Similar studies are needed to inform travellers and practitioners about the effectiveness of this drug at other popular destinations.

A Soluble Form of the Pilus Protein FimA Targets the VDAC-Hexokinase Complex at Mitochondria to Suppress Host Cell Apoptosis

Inhibition of apoptotic response of host cells during an early phase of infection is a strategy used by many enteroinvasive bacterial pathogens to enhance their survival. Here, we report the identification of a soluble form of the pilus protein FimA from the culture supernatants of E. coli K1, Salmonella, and Shigella that can potently inhibit Bax-mediated release of cytochrome c from isolated mitochondria. Similar to the infected cells, HCT116 cells stably expressing FimA display a delay in the integration of Bax into outer mitochondrial membrane induced by apoptotic stimuli. FimA targets to mitochondria through binding to VDAC1, which is a prerequisite step for E. coli K1 to render the short-term blockade of apoptotic death in the host cells. Interestingly, FimA strengthens the VDAC1-hexokinase interaction and prevents dissociation of hexokinase from VDAC1 triggered by apoptotic stimuli. Together, these data thus reveal a paradigm of antiapoptosis mechanism undertaken by the enteroinvasive bacteria.

Characterisation of Early Mucosal and Neuronal Lesions Following Shigella flexneri Infection in Human Colon

Background Shigella, an enteroinvasive bacteria induces a major inflammatory response responsible for acute rectocolitis in humans. However, early effect of Shigella flexneri (S. flexneri) infection upon the human mucosa and its microenvironement, in particular the enteric nervous system, remains currently unknown. Therefore, in this study, we sought to characterize ex vivo the early events of shigellosis in a model of human colonic explants. In particular, we aimed at identifying factors produced by S. flexneri and responsible for the lesions of the barrier. We also aimed at determining the putative lesions of the enteric nervous system induced by S. flexneri.Methodology/Principal FindingsWe first showed that, following 3 h of infection, the invasive but not the non-invasive strain of S. flexneri induced significant desquamation of the intestinal epithelial barrier and a reduction of epithelial height. These changes were significantly reduced following infection with SepA deficient S. flexneri strains. Secondly, S. flexneri induced rapid neuronal morphological alterations suggestive of cell death in enteric submucosal neurones. These alterations were associated with a significant increase in the proportion of vasoactive intestinal peptide (VIP) immunoreactive (IR) neurons but not in total VIP levels. The NMDA receptor antagonist MK-801 blocked neuronal morphological changes induced by S. flexneri, but not the increase in the proportion of VIP-IR.Conclusions/SignificanceThis human explant model can be used to gain better insight into the early pathogenic events following S. flexneri infection and the mechanisms involved.

Interaction of Phospholipase C-γ1 with Villin Regulates Epithelial Cell Migration

Interaction of Phospholipase C-γ1 with Villin Regulates Epithelial Cell Migration

Lactoferrin downregulates pro-inflammatory cytokines upexpressed in intestinal epithelial cells infected with invasive or noninvasive Escherichia coli strainsThis paper is one of a selection of papers published in this Special Issue, entitled 7th International Conference on Lactoferrin: Structure, Function, and Applications, and has undergone the Journal’s usual peer review process.

Intestinal epithelial cells are able to differentially interact with commensal or pathogenic microorganisms, triggering a physiological or destructive inflammation, respectively. To mimic commensal-enteroinvasive bacteria-host cell interaction, we infected Caco-2 cells with noninvasive Escherichia coli HB101 and with recombinant invasive E. coli HB101(pRI203). Using DNA microarray mRNA profiling and ELISA assays, we studied the expression of several cytokine and cytokine-related genes in infected Caco-2 cells in the absence or presence of bovine lactoferrin (bLf). Infection of Caco-2 cells with the noninvasive strain induced a slight increase in the expression of interleukin 8 (IL-8), whereas infection with invasive E. coli HB101(pRI203) induced a significant increase in the expression of IL-8 as well as other pro-inflammatory cytokines. The addition of bLf, in native- or holo-form, did not influence expression of cytokine genes by uninfected Caco-2 cells, but it decreased expression of IL-8 by cells infected with E.coli HB101. Moreover, except for IL-8, bLfs dramatically downregulated pro-inflammatory cytokines upexpressed by Caco-2 cells infected with the invasive strain. Although IL-8 was decreased by bLfs, it remained upregulated, suggesting that it could be a signal of persistence of intracellular bacteria. The bLf ability to reduce expression of some pro-inflammatory cytokines, which appears independent of its iron saturation, might represent an important natural mechanism in regulating epithelial cell responses to pathogenic bacteria and in limiting cell damage and the spread of infections.

Battling enteroinvasive bacteria: Nod1 comes to the rescue

Recognition of pathogenic bacteria by mammalian hosts is largely mediated by membrane-bound Toll-like receptors (TLRs). Recently, a family of cytosolic proteins, termed NODs, with homology to plant disease-resistance gene products has been implicated in sensing microbes within the cytosol. The role of NOD family members in host defense is largely unknown. However, a recent report revealed that Nod1 is a crucial sensor for certain enteroinvasive bacteria that avoid TLR signaling. This finding suggests that Nod1 plays an important role in the initial recognition of pathogenic bacteria at epithelial surfaces, such as the gut, where innate immune responses to commensal bacteria must be avoided.

Dangerous--and Not So Dangerous--Liaisons

### Contents #### News [The Roots of Plant-Microbe Collaborations][1] #### Reviews How Viruses Enter Animal Cells A. E. Smith and A. Helenius [Bacterial Invasion: The Paradigms of Enteroinvasive Pathogens][2] P. Cossart and P. J. Sansonetti Intracellular Parasite Invasion Strategies L. D. Sibley Ancient Invasions: From Endosymbionts to Organelles S. D. Dyall et al. See related Science Express by [Horn et al.][3] I n many biological systems, one organism, often a virus, bacterium, or other microbe, invades another, generally at the cellular level. The ramifications can be profound. For example, a virus infecting a single animal cell can ultimately lead to the demise of the whole animal. Other microbial parasites may also have catastrophic consequences for their hosts, as seen in the terrible toll taken by malarial parasites in the develoing world. Cellular invasions can also have more benign outcomes, as when microbial symbioses are established in the roots of legumes, allowing them to gain essential nitrogen nutrients; or in our ancient past, when bacteria took up residence in prototype eukaryotic cells and evolved into mitochondria, the cell's energy powerhouses. In this special issue, we look at a variety of these cellular invasions and examine the intracellular niches established by invading microorganisms. Smith and Helenius (p. 237) describe invasions by animal viruses, which employ multiple strategies to allow the viral nucleic acids to gain access to the host cell replication machinery and generate progeny viruses. Some bacterial pathogens can also take up residence within eukaryotic host cells. Cossart and Sansonetti (p. [242][2]) look at infections by a group of enteroinvasive bacteria, including Salmonella , Shigella, Yersinia , and Listeria , that cause certain types of food poisoning. When these microbes invade cells, they establish a privileged intracellular niche within which they can grow and divide, protected from host defense mechanisms. In Science Express, Horn et al. ([www.sciencemag.org/cgi/content/abstract/1096330][3]) present a comparative and phylogenetic genome analysis of a chlamydia-related symbiont of free-living amoebas. They suggest that ancient chlamydiae were major inventors of mechanisms used by intracellular bacterial pathogens for survival in, and exploitation of, eukaryotic host cells. Another group of invasive organisms, the apicomplexan parasites, includes Plasmodium , which causes malaria, and the organism responsible for toxoplasmosis, which can damage multiple organs in immune-compromised individuals. Sibley (p. 248) reviews how apicomplexan parasites gain entry to susceptible host cells. Dyall et al. (p. 253) describe how, way back in evolutionary history, endosymbionts developed into organelles such as mitochondria and chloroplasts, thus producing early eukaryotes. In an accompanying News story (p. [234][1]), Jean Marx looks at the molecular interactions required for the establishment of two types of intracellular symbioses that are critical for plant growth and thus for modern agriculture: In one, nitrogen-fixing rhizobial bacteria set up housekeeping in legume roots, and in the other, mycorrhizal fungi invade the roots of many plants and help them to acquire phosphate. Keeping yourself to yourself may be the done thing in polite society, but in the cut and thrust of the real life of invasive organisms, it's simply not an option. [1]: /lookup/doi/10.1126/science.304.5668.234 [2]: /lookup/doi/10.1126/science.1090124 [3]: http://www.sciencemag.org/cgi/content/abstract/1096330