Adherent-invasive Escherichia coli target the epithelial barrier

Involvement of intestinal microbes in the pathogenesis of chronic inflammatory bowel diseases (IBD, including Crohn disease and ulcerative colitis) is well established. However, the mechanisms by which bacteria lead to intestinal injury in IBD remain unclear and are the focus of current research. Using adherent-invasive Escherichia coli (AIEC) strain LF82, which is linked to Crohn disease, we recently demonstrated the ability of these intestinal microbes to disrupt the integrity of epithelial cells in an in vitro cell model. This disruption provides the bacteria a capacity to penetrate into and beyond the epithelial monolayer, replicate in cells, disseminate within the host, and induce a chronic immune response. These findings provide a link between microbes related to IBD, disruption of the intestinal epithelial cell barrier, and disease pathogenesis.

In this addendum, we provide a synopsis on current data concerning the role of AIEC in the pathogenesis of intestinal inflammation, summarise our recent findings, and highlight the central role of the epithelium in mucosal defence. We also discuss, in more detail, the potential implications of our findings and present ideas for future studies and targets for intervention.     

Adherent-invasive Escherichia coli in inflammatory bowel disease

Increased numbers of mucosa-associated Escherichia coli are observed in both major inflammatory bowel diseases, Crohn's disease (CD) and ulcerative colitis (UC). With the identification of mutations in the NOD2-encoding gene in patients with CD and given the intracellular location of NOD2, the presence of pathogenic invasive bacteria could be the link between innate immune response to invasive bacteria and the development of the inflammation. Adherent-invasive E. coli (AIEC) are isolated from ileal biopsies of 36.4% of patients with ileal involvement of CD. These pathogenic E. coli colonize the intestinal mucosa by adhering to intestinal epithelial cells and are also true invasive pathogens, able to invade intestinal epithelial cells and to replicate intracellularly. AIEC strains also survive and replicate extensively within macrophages without inducing host cell death, and their high replication rates induce the secretion of large amounts of tumor necrosis factor alpha (TNF-alpha). There is also evidence suggesting that AIEC is involved in the formation of granulomas. The presence of AIEC is restricted to CD patients. Mucosa-associated E. coli in patients with UC can adhere to intestinal epithelial cells and induce the secretion of IL-8, but there is no evidence that these E. coli strains are invasive.     

Strategy of Escherichia coli for crossing the blood-brain barrier

A major contributing factor to high mortality and morbidity associated with bacterial meningitis is the incomplete understanding of the pathogenesis of this disease: It is unclear how circulating bacteria cross the blood-brain barrier (BBB). Recent studies with Escherichia coli K1 show that successful traversal of the BBB requires a high degree of bacteremia, invasion of brain microvascular endothelial cells (BMEC), host cell actin cytoskeleton rearrangements and related signaling pathways, and traversal of the BBB as live bacteria. Several microbial determinants such as the K1 capsule, OmpA, Ibe proteins, AslA, TraJ, and CNF1 contribute to BMEC invasion. Of interest, E. coli K1 trafficking mechanisms differ from those of other meningitis-causing bacteria such as Listeria monocytogenes and group B streptococcus. Complete understanding of bacteria-BMEC interactions contributing to translocation of the BBB should assist in developing novel strategies to prevent bacterial meningitis.     

Interactions between enteropathogenic Escherichia coli and epithelial cells.

Enteropathogenic Escherichia coli (EPEC) may be considered a paradigm for a multistage interaction between pathogen and host cell. EPEC strains produce a type IV pilus that is associated with initial adherence to host cells, and these strains possess a type III secretion apparatus that is necessary for transducing signals to host cells. Secretion of three Esp proteins is required for activation of a phosphotyrosine-containing receptor that allows EPEC to bind intimately to host cells via the bacterial outer membrane protein intimin. Intimately attached bacteria rest upon a pedestal composed of host cytoskeletal proteins in an arrangement recognized as the attaching and effacing phenotype. The precise molecular interactions that lead to these dramatic alterations in the host cell cytoskeleton remain to be elucidated.     

Topoisomerase IV is a target of quinolones in Escherichia coli.

We have demonstrated that, in Escherichia coli, quinolone antimicrobial agents target topoisomerase IV (topo IV). The inhibition of topo IV becomes apparent only when gyrase is mutated to quinolone resistance. In such mutants, these antibiotics caused accumulation of replication catenanes, which is diagnostic of a loss of topo IV activity. Mutant forms of topo IV provided an additional 10-fold resistance to quinolones and prevented drug-induced catenane accumulation. Drug inhibition of topo IV differs from that of gyrase. (i) Wild-type topo IV is not dominant over the resistant allele. (ii) Inhibition of topo IV leads to only a slow stop in replication. (iii) Inhibition of topo IV is primarily bacteriostatic. These differences may result from topo IV acting behind the replication fork, allowing for repair of drug-induced lesions. We suggest that this and a slightly higher intrinsic resistance of topo IV make it secondary to gyrase as a quinolone target. Our results imply that the quinolone binding pockets of gyrase and topo IV are similar and that substantial levels of drug resistance require mutations in both enzymes.     

肠致病性大肠杆菌改变肠道上皮细胞极性的研究进展

上皮细胞构成了宿主阻止病原微生物入侵机体的生理屏障。细胞间连接,主要是紧密连接,将上皮细胞特殊的顶端和基底外侧膜位点分开,形成极化的上皮细胞,用于维持顶端-基底极性。肠致病性大肠杆菌通过破坏感染位点多种因素导致顶端-基底极性消失,包括上皮细胞屏障结构、黏附和极性蛋白的分布,以及极性复合物。本文主要阐述了紧密连接在维持上皮细胞极性中的作用、肠致病性大肠杆菌对紧密连接的破坏作用、肠致病性大肠杆菌对上皮细胞极性的影响及其作用机制,为肠致病性大肠杆菌作用机制的研究和防御措施提供参考。     

多重耐药大肠杆菌外膜屏障机制的研究

目的研究临床分离的多重耐药大肠杆菌耐药的外膜屏障机制。方法用尿素－ＳＤＳ－ＰＡＧＥ法对临床分离的大肠杆菌外膜孔蛋白进行分析，并用抽滤法测定大肠杆菌细胞内四环素浓度。结果临床分离的多重耐药大肠杆菌Ｒ３、Ｒ４、Ｒ２８、Ｒ３０外膜孔蛋白中均缺失ＯｍｐＦ，Ｒ３、Ｒ３０中同时缺失ＯｍｐＣ，Ｒ１２缺失ＯｍｐＡ。敏感菌株中均不缺失ＯｍｐＦ，但敏感株Ｓ２２缺失ＯｍｐＡ和ＯｍｐＣ。ＬＭ２１８（ＯｍｐＦ标准缺失株）、Ｒ２８细胞中稳态四环素浓度分别为敏感株Ｓ３６的８０％和４０％。结论外膜通透性的改变是大肠杆菌产生多重耐药的机制之一     

Lipopolysaccharide from Escherichia coli stimulates mucin secretion by cultured dog gallbladder epithelial cells

Biliary infection is associated with mucin hypersecretion by the biliary epithelium. Mucins have been identified as potent pronucleators of cholesterol in bile. The aim of the present study was to determine whether lipopolysaccharides (LPS) from different bacteria are capable of stimulating mucin secretion by cultured dog gallbladder epithelial (DGBE) cells, and to investigate the mechanism by which LPS stimulate mucin secretion. Mucin secretion by confluent monolayers of DGBE cells was quantified by measuring the secretion of [3H]-N-acetyl-D-glucosamine-labeled glycoproteins. Cell viability was evaluated by measuring the leakage of the enzyme, lactate dehydrogenase (LDH), into the culture medium. LPS, derived from Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa (200 microg/mL), all caused an increase in mucin secretion by the DGBE cells, without causing concomitant cell lysis. LPS from E. coli was found to be the most potent stimulator of mucin secretion, and increased mucin secretion by the DGBE cells to 252% +/- 14% of control. LPS from E. coli had no effect on intracellular cyclic adenosine monophosphate (cAMP) levels in the DGBE cells. Addition of the nitric oxide (NO)-releasing compound, NOR-4 (0.125-1 mmol/L), to the cells did not result in increased mucin secretion, and the NO synthase inhibitor, Nomega-nitro-L-arginine methyl ester (L-NAME) (4 or 10 mmol/L), did not inhibit the LPS-stimulated mucin secretion. Exogenous tumor necrosis factor alpha (TNF-alpha) (1-10 ng/mL) did cause a minor increase in mucin secretion by the DGBE cells, but the effect of LPS from E. coli on mucin secretion could not be inhibited by preincubation with a TNF-alpha antibody (10 microg/mL). We conclude that LPS stimulates mucin secretion by the gallbladder epithelium. Whether this stimulation is mediated by TNF-alpha remains to be determined.     

Enteroaggregative Escherichia coli

Enteroaggregative Escherichia coli (EAEC) are an increasingly important cause of diarrhoea. E. coli belonging to this category cause watery diarrhoea, which is often persistent and can be inflammatory. EAEC have been implicated in sporadic diarrhoea in children and adults, in both developing and developed countries, and have been identified as the cause of several outbreaks worldwide. EAEC are defined by their ability to adhere to epithelial cells in a characteristic "stacked-brick" pattern but are otherwise highly heterogeneous. Genes that could contribute to the pathogenicity of EAEC encode adhesins, toxins, and other factors, all of which are only partially conserved. Practicable tools are needed to improve diagnosis and identify risk factors. EAEC-infected individuals can be treated with fluoroquinolones but there is a need to examine alternative treatment protocols.     

Enterotoxigenic Escherichia coli

The enterotoxigenic Escherichia coli are a pervasive cause of serious diarrheal illness in developing countries. Presently, there is no vaccine to prevent these infections, and many features of the basic pathogenesis of these organisms remain poorly understood. Until very recently most pathogenesis studies had focused almost exclusively on a small subset of known “classical” virulence genes, namely fimbrial colonization factors and the heat-labile (LT) and heat stable (ST) enterotoxins. However, recent investigations of pathogen-host interactions reveal a surprisingly complex and intricately orchestrated engagement involving the interplay of classical and “novel” virulence genes, as well as participation of genes highly conserved in the E. coli species. These studies may inform further rational approaches to vaccine development for these important pathogens.     

Quinolone-resistant Escherichia coli

Quinolones (nalidixic acid - NAL, norfloxacin - NOR, ciprofloxacin - CIP and gatifloxacin - GAT) were tested against Escherichia coli isolated from urine (385 patient samples) by disk diffusion (DD) and agar dilution (AD) methods. Fifty-three samples (13.8%) were classified as resistant to at least one of the quinolones tested. CIP and NOR susceptibilities were the same (91.4%) and they were similar to GAT (92.7%). Susceptibility to NAL, detected by the disk diffusion method, was used to predict susceptibility to NOR, CIP and GAT by the agar dilution method. The sensitivity and specificity of NAL were 100% and 95%, respectively. Twelve samples were analyzed for mutations in the quinolone resistance-determining region (QRDR) of the gyrA and parC genes. Sequencing of these genes failed to find any mutations in the quinolone-sensitive isolates. However, three mutations were observed in the isolates resistant to all the quinolones tested - two in gyrA and one in parC. A single mutation in gyrA was found in the strains that were resistant to nalidixic acid but fluoroquinolone-sensitive. These findings support the suggestion that NAL could be used as a marker for susceptibility to fluoroquinolones in routine microbiology laboratories. The overall resistance rate to quinolones in the present study was 13.8%, which is higher than that observed in other studies carried out in developed countries. Our findings serve as a warning that resistance to this group of antimicrobial agents is increasing.     

Pathogenesis of bacteriuria in elderly women: the role of Escherichia coli adherence to vaginal epithelial cells

In vitro Escherichia coli adherence to exfoliated epithelial cells obtained from a group of elderly women was compared with adherence values in young healthy women in an attempt to define the role of adherence as an independent host risk factor for the acquisition of bacteriuria. Utilizing four uropathogenic strains of E. coli possessing differing fimbrial adherence mechanisms, no evidence was found of increased adherence receptivity of vaginal epithelial cells for bacteria in elderly women. In contrast, there was evidence of significantly reduced attachment of two of four strains in the elderly women (p less than .02). Based upon preliminary studies using four E. coli strains, susceptibility to bacterial adherence does not appear to be a mechanism responsible for the increased frequency of E. coli bacteriuria in elderly women.