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.pili ͉ enterohemorrhagic Escherichia coli ͉ normal flora

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.

Propolis is a resinous substance made by bees. It possesses many biological activities, and many studies have reported its potential application in the control of dental caries. However, variability in the chemical composition of propolis is a potential problem in its quality control, especially since propolis has already been incorporated into products for oral use. Therefore, a critical analysis of the available data on propolis is warranted. The present review discusses the in vitro and in vivo studies published in the period between 1978 and 2008 regarding the effects of propolis on Streptococcus mutans growth, bacterial adherence, glucosyltransferase activity, and caries indicators. Several investigations carried out with crude propolis extracts, isolated fractions, and purified compounds showed reductions in Streptococcus mutans counts and interference with their adhesion capacity and glucosyltransferase activity, which are considered major properties in the establishment of the cariogenic process. Data from in vivo studies have demonstrated reductions in Streptococcus mutans counts in saliva, the plaque index, and insoluble polysaccharide formation. These findings indicate that propolis and/or its compounds are promising cariostatic agents. However, the variation in the chemical composition of propolis due to its geographical distribution is a significant drawback to its routine clinical use. Thus, further studies are needed to establish the quality and safety control criteria for propolis in order for it to be used in accordance with its proposed activity.

Since viable bacteria can persist in tooth cavities regardless of the technique used for caries removal, the objective of the present randomized clinical trial was to examine the microflora of primary teeth treated by complete or partial removal of carious dentin. Deciduous molars with acute carious lesions in the inner half of dentin and vital pulp were randomly divided into two groups of 16: complete removal, in which the carious dentin was completely removed with the help of a caries detector dye, and partial removal, in which the carious dentin was completely removed from the dentinoenamel junction and lateral walls, while the necrotic carious dentin from the cavity floor was only removed superficially. Dentin samples were obtained with a sterile No. 3 bur after caries removal and 3–6 months after protection with calcium hydroxide cement and restoration of the cavities with resin composite. The samples were stored in thioglycolate. Decimal dilutions were then prepared and seeded for the enumeration of Streptococcus spp., mutans streptococci, Lactobacillus spp. and total microorganisms. Before sealing, a larger number of microorganisms was detected in teeth submitted to partial caries removal compared to the complete removal group. However, after sealing the level of colonization was similar in the two groups for all microorganisms studied. The results suggest that persistence of bacteria does not seem to be a reason for reopening of cavities in deciduous teeth after partial caries removal.

BackgroundNative 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 of M. fasciculata geopropolis against oral pathogens, its effects on S. mutans biofilms, and the chemical contents of the extracts. A gel prepared with a geopropolis extract was also analyzed for its activity on S. mutans and its immunotoxicological potential.MethodsAntimicrobial 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.ResultsTwo extracts, HAE-2 and HAE-3, showed inhibition zones ranging from 9 to 13 mm in diameter for S. mutans and C. albicans, but presented no activity against L. acidophilus. The MBCs for HAE-2 and HAE-3 against S. mutans were 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 on S. mutans biofilms 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.ConclusionsIn summary, geopropolis produced by M. fasciculata can exert antimicrobial action against S. mutans and C. albicans, with significant inhibitory activity against S. mutans biofilms. 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.

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.

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Enterohemorrhagic Escherichia coli (EHEC) O157:H7 produces long bundles of polar type 4 pili (T4P) called HCP (for hemorrhagic coli pili) 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 isogenic hcpA mutant 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 laboratory E. coli strain HB101 complemented with hcpABC genes 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.Type 4 pili (T4P) represent a unique class of adhesive pili described as long bundles of flexible and filamentous polymers, whose pilin subunits are mounted in a helical fashion and exposed on the surface of several of gram-negative bacteria of clinical, industrial, and environmental importance. These pili are defined on the basis of their structural, biochemical, morphological, and antigenic characteristics. Some are considerably highly conserved in different bacteria and share several major features, including a short conserved signal peptide, a hydrophobic amino-terminal domain, and a carboxy-terminal disulfide bond (57).T4P have been described in Escherichia coli pathogroups such as enteropathogenic E. coli (17), enterotoxigenic E. coli (18), and enterohemorrhagic E. coli (EHEC) (66), as well as in other gram-negative pathogenic bacteria, including Moraxella catarrhalis (30), species of Neisseria (36), Pseudomonas aeruginosa (32), and Vibrio cholerae (60). A number of cellular functions associated with pathogenicity have been attributed to T4P, such as adhesion to host cells, microcolony and biofilm formation, bacterial aggregation, receptors for phages, immune evasion, twitching motility, DNA uptake, and cell signaling (9). EHEC O157:H7 is an emerging and significant food-borne pathogen that has been implicated in many outbreaks in the United States and other countries (21, 50). The clinical manifestations of EHEC infections range from self-limiting diarrhea to hemorrhagic colitis, which can evolve to severe complications known as hemolytic urem...