Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a food-borne pathogen that causes hemorrhagic colitis and the hemolytic uremic syndrome. Colonization of the human gut mucosa and production of potent Shiga toxins are critical virulence traits of EHEC. Although EHEC O157:H7 contains numerous putative pili operons, their role in the colonization of the natural bovine or accidental human hosts remains largely unknown. We have identified in EHEC an adherence factor, herein called E. coli common pilus (ECP), composed of a 21-kDa pilin subunit whose amino acid sequence corresponds to the product of the yagZ (renamed ecpA ) gene present in all E. coli genomes sequenced to date. ECP production was demonstrated in 121 (71.6%) of a total of 169 ecpA ⁺ strains representing intestinal and extraintestinal pathogenic as well as normal flora E. coli . High-resolution ultrastructural and immunofluorescence studies demonstrated the presence of abundant peritrichous fibrillar structures emanating from the bacterial surface forming physical bridges between bacteria adhering to cultured epithelial cells. Isogenic ecpA mutants of EHEC O157:H7 or fecal commensal E. coli showed significant reduction in adherence to cultured epithelial cells. Our data suggest that ECP production is a common feature of E. coli colonizing the human gut or other host tissues. ECP is a pilus of EHEC O157:H7 with a potential role in host epithelial cell colonization and may represent a mechanism of adherence of both pathogenic and commensal E. coli .

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes hemorrhagic colitis and hemolytic uremic syndrome (HUS) by colonizing the gut mucosa and producing Shiga toxins (Stx). The only factor clearly demonstrated to play a role in EHEC adherence to intestinal epithelial cells is intimin, which binds host cell integrins and nucleolin, as well as a receptor (Tir) that it injects into the host cell. Here we report that EHEC O157:H7 produces adhesive type IV pili, which we term hemorrhagic coli pilus (HCP), composed of a 19-kDa pilin subunit (HcpA) that is encoded by the hcpA chromosomal gene. HCP were observed as bundles of fibers greater than 10 μm in length that formed physical bridges between bacteria adhering to human and bovine host cells. Sera of HUS patients, but not healthy individuals, recognized HcpA, suggesting that the pili are produced in vivo during EHEC infections. Inactivation of the hcpA gene in EHEC EDL933 resulted in significantly reduced adherence to cultured human intestinal and bovine renal epithelial cells and to porcine and bovine gut explants. An escN mutant, which is unable to translocate Tir, adhered less than the hcpA mutant, suggesting that adherence mediated by intimin-Tir interactions is a prelude to HCP-mediated adherence. An hcpA and stx1,2 triple mutant and an hcpA mutant had similar levels of adherence to bovine and human epithelial cells while a stx1,2 double mutant had only a minor defect in adherence, indicating that HCP-mediated adherence and cytotoxicity are independent events. Our data establish that EHEC O157:H7 HCP are intestinal colonization factors that are likely to contribute to the pathogenic potential of this food-borne pathogen.

Background: Native bees of the tribe Meliponini produce a distinct kind of propolis called geopropolis. Although many pharmacological activities of propolis have already been demonstrated, little is known about geopropolis, particularly regarding its antimicrobial activity against oral pathogens. The present study aimed at investigating the antimicrobial activity ofM. fasciculatageopropolis against oral pathogens, its effects onS. mutansbiofilms, and the chemical contents of the extracts. A gel prepared with a geopropolis extract was also analyzed for its activity onS. mutansand its immunotoxicological potential.Methods: Antimicrobial activities of three hydroalcoholic extracts (HAEs) of geopropolis, and hexane and chloroform fractions of one extract, were evaluated using the agar diffusion method and the broth dilution technique. Ethanol (70%, v/v) and chlorhexidine (0.12%, w/w) were used as negative and positive controls, respectively. Total phenol and flavonoid concentrations were assayed by spectrophotometry. Immunotoxicity was evaluated in mice by topical application in the oral cavity followed by quantification of biochemical and immunological parameters, and macro-microscopic analysis of animal organs.Results: Two extracts, HAE-2 and HAE-3, showed inhibition zones ranging from 9 to 13 mm in diameter forS. mutansandC. albicans, but presented no activity againstL.acidophilus. The MBCs for HAE-2 and HAE-3 againstS. mutanswere 6.25 mg/mL and 12.5 mg/mL, respectively. HAE-2 was fractionated, and its chloroform fraction had an MBC of 14.57 mg/mL. HAE-2 also exhibited bactericidal effects onS. mutansbiofilms after 3 h of treatment. Significant differences (p < 0.05) in total phenol and flavonoid concentrations were observed among the samples. Signs toxic effects were not observed after application of the geopropolis-based gel, but an increase in the production of IL-4 and IL-10, anti-inflammatory cytokines, was detected.Conclusions: In summary, geopropolis produced byM. fasciculatacan exert antimicrobial action againstS. mutansandC. albicans, with significant inhibitory activity againstS. mutansbiofilms. The extract with the highest flavonoid concentration, HAE-2, presented the highest antimicrobial activity. In addition, a geopropolis-based gel is not toxic in an animal model and displays anti-inflammatory effect.

EnterohemorrhagicEscherichia coli(EHEC) O157:H7 produces long bundles of polar type 4 pili (T4P) called HCP (forhemorrhagiccolipili) that form physical bridges between bacteria associating with human and animal epithelial cells. Here, we sought to further investigate whether HCP possessed other pathogenicity attributes associated with T4P production. Comparative studies performed with wild-type EHEC EDL933 and an isogenichcpAmutant revealed that HCP play different roles in the biology of this organism. We found that in addition to promoting bacterial attachment to host cells, HCP mediate (i) invasion of epithelial cells, (ii) hemagglutination of rabbit erythrocytes, (iii) interbacterial connections conducive to biofilm formation, (iv) specific binding to host extracellular matrix proteins laminin and fibronectin but not collagen, and (v) twitching motility. Nonadherent laboratoryE. colistrain HB101 complemented withhcpABCgenes on plasmid pJX22, which specifies for HCP overproduction in EDL933, became hyperadherent and invasive and produced a thick biofilm, suggesting that the presence of HCP confers HB101(pJX22) new attributes otherwise not exhibited by HB101. Analogous to other bacteria in which T4P are involved in the pathogenesis of several infectious diseases, our data strongly suggest that HCP display multiple functions that may contribute to EHEC colonization of different hosts and to virulence, survival, and transmission of this food-borne pathogen.

Bacterial resistance to the available marketed drugs has prompted the search of novel therapies; especially in regards of anti-virulence strategies that aim to make bacteria less pathogenic and/or decrease their probability to become resistant to therapy. Cinnamaldehyde is widely known for its antibacterial properties through mechanisms that include the interaction of this compound with bacterial cell walls. However, only a handful of studies have addressed its effects on bacterial virulence, especially when tested at sub-inhibitory concentrations. Herein, we show for the first time that cinnamaldehyde is bactericidal against Staphylococcus aureus and Enterococcus faecalis multidrug resistant strains and does not promote bacterial tolerance. Cinnamaldehyde actions were stronger on S. aureus as it was able to inhibit its hemolytic activity on human erythrocytes and reduce its adherence to latex. Furthermore, cinnamaldehyde enhanced the serum-dependent lysis of S. aureus. In vivo testing of cinnamaldehyde in Galleria mellonella larvae infected with S. aureus, showed this compound improves larvae survival whilst diminishing bacterial load in their hemolymph. We suggest that cinnamaldehyde may represent an alternative therapy to control S. aureus-induced bacterial infections as it presents the ability to reduce bacterial virulence/survival without promoting an adaptive phenotype.