Adherence Inhibition of Cronobacter sakazakii to Intestinal Epithelial Cells by Lactoferrin

Cronobacter sakazakii is now recognized as an opportunistic pathogen and has been implicated in rare but severe cases of necrotizing enterocolitis, meningitis, and sepsis in neonates. The first step in bacterial pathogenesis requires that the organism adheres to host cells surfaces; therefore, agents that inhibit adherence might be useful for preventing infections. Lactoferrin, an iron binding protein found in milk, has been shown to inhibit bacterial adherence by direct interaction and disruption of bacterial surfaces. Therefore, the goal of this research was to assess the ability of two different types of bovine lactoferrin, alone and in combination with a 1:1 blend of galactooligosaccharides and polydextrose, to inhibit adherence of C. sakazakii to a HEp-2 human cell line. Results showed that the adherence of C. sakazakii was significantly reduced at a minimum lactoferrin concentration of 10 mg/ml. However, in combination with the oligosaccharide blend, no synergistic effect was observed in adherence inhibition. These results suggest that lactoferrin might interact with C. sakazakii and directly inhibit adhesion to tissue culture cells.     

Surface-layer protein extracts from Lactobacillus helveticus inhibit enterohaemorrhagic Escherichia coli O157:H7 adhesion to epithelial cells

Adherence of intestinal pathogens, including Escherichia coli O157:H7, to human intestinal epithelial cells is a key step in pathogenesis. Probiotic bacteria, including Lactobacillus helveticus R0052 inhibit the adhesion of E. coli O157:H7 to epithelial cells, a process which may be related to specific components of the bacterial surface. Surface-layer proteins (Slps) are located in a paracrystalline layer outside the bacterial cell wall and are thought to play a role in tissue adherence. However, the ability of S-layer protein extract derived from probiotic bacteria to block adherence of enteric pathogens has not been investigated. Human epithelial (HEp-2 and T84) cells were treated with S-layer protein extract alone, infected with E. coli O157:H7, or pretreated with S-layer protein extract prior to infection to determine their importance in the inhibition of pathogen adherence. The effects of S-layer protein extracts were characterized by phase-contrast and immunofluorescence microscopy and measurement of the transepithelial electrical resistance of polarized monolayers. Pre-treatment of host epithelial cells with S-layer protein extracts prior to E. coli O157:H7 infection decreased pathogen adherence and attaching-effacing lesions in addition to preserving the barrier function of monolayers. These in vitro studies indicate that a non-viable constituent derived from a probiotic strain may prove effective in interrupting the infectious process of an intestinal pathogen.     

Antisense RNA decreases AP33 gene expression and cytoadherence by <Emphasis Type="Italic">T. vaginalis</Emphasis>

Background  

Host parasitism by Trichomonas vaginalis is complex. Adherence to vaginal epithelial cells (VECs) is mediated by surface proteins. We showed before that antisense down-regulation of expression of adhesin AP65 decreased amounts of protein, which lowered levels of T. vaginalis adherence to VECs. We now perform antisense down-regulation of expression of the ap33 gene to evaluate and confirm a role for AP33 in adherence by T. vaginalis. We also used an established transfection system for heterologous expression of AP33 in T. foetus as an additional confirmatory approach.     

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal colonization. The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.     

Adherence ability and serum resistance of different hospital clusters of Acinetobacter baumannii

The role of mechanical ventilation and catheters in favouring Acinetobacter baumannii infections needs to be better understood. This study evaluated the adherence of 19 isolates of different hospital clusters of A. baumannii to abiotic surfaces and epithelial cells (HEp-2). Of the hydrophobic isolates, 80% adhered to polystyrene, indicating a close relationship between hydrophobicity and adherence. All isolates adhered to epithelial cells to different degrees, and 73·7% showed an aggregated pattern. Analysis of the serum resistance of catheter-tip isolates showed that all were resistant. These worrisome results showed that the high capacity of A. baumannii to adhere to surfaces and survive in human serum could hinder treatment and control of this pathogen.     

The role of DNA base excision repair in filamentation in Escherichia coli K-12 adhered to epithelial HEp-2 cells

Base excision repair (BER) is dedicated to the repair of oxidative DNA damage caused by reactive oxygen species generated by chemical and physical agents or by metabolism which can react with DNA and cause a variety of mutations. Epithelial cells are typically the first type of host cell to come into contact with potential microbial invaders. In this work, we have evaluated whether the adherence to human epithelial cells causes DNA damage and associated filamentation. Experiments concerning adherence to HEp-2 cells were carried out with mutants deficient in BER that were derived from Escherichia coli K-12. Since the removal of mannose during bacterial interaction with HEp-2 cells allows adhesion through mannose-sensitive adhesins, the experiments were also performed in the presence and the absence of mannose. Our results showed enhanced filamentation for the single xth (BW9091) and triple xth nfo nth (BW535) mutants in adherence assays with HEp-2 cells performed without d-mannose. The increased filamentation growth was inhibited by complementation of BER mutants with a wild type xth gene. Moreover, we measured SOS induction of bacteria adhered to HEp-2 cells in the presence and absence of d-mannose through of SOS-chromotest assay and we observed a higher β-galactosidase expression in the absence of mannose. In this context, data showed evidence that bacterial attachment to HEp-2 epithelial surfaces can generate DNA lesions and SOS induction.     

LamB-mediated adherence of enteropathogenic Escherichia coli to HEp-2 cells

Aims:  To establish the role of maltoporin (LamB) in adherence of enteropathogenic Escherichia coli (EPEC) to epithelial cells in vitro.
Methods and Results:  Three strains, wild type (WT) EPEC, a maltoporin (LamB) mutant ΔlamB , and DH5α were used to study adherence to cultured HEp-2 cells. Mutant ΔlamB was found to be deficient in adherence compared to WT EPEC. Adherence of ΔlamB was restored to wild type levels when complemented with the cloned lamB gene. The non–adherent strain DH5α also adhered to HEp-2 cells when it harboured the cloned lamB gene. The LamB protein was isolated from WT EPEC by electroelution and antibodies were raised in rabbits. The specificity of the antibodies was analysed by Western blotting. Anti-LamB antiserum reduced adherence of WT EPEC to HEp-2 cells. The LamB protein was coated on latex beads and the beads adhered to HEp-2 cells. Anti-LamB antiserum prevented bead adherence to HEp-2 cells. Multiple sequence alignment showed that the L9 loop of EPEC LamB had four amino acids different from the L9 loop of LamB from several other related pathogens.
Conclusions:  LamB serves as an alternative or additional adherence factor for EPEC.
Significance and Impact of the Study:  Adherence is an important component of the pathogenesis of noninvasive pathogens like EPEC. A putative adhesin such as LamB, which has already been found to be co-expressed with virulence factor EspB may be a potential vaccine candidate for control of EPEC and related pathogens.     

Adhesive properties of Enterobacter sakazakii to human epithelial and brain microvascular endothelial cells

Background  

Enterobacter sakazakii is an opportunistic pathogen that has been associated with sporadic cases and outbreaks causing meningitis, necrotizing enterocolitis and sepsis especially in neonates. However, up to now little is known about the mechanisms of pathogenicity in E. sakazakii. A necessary state in the successful colonization, establishment and ultimately production of disease by microbial pathogens is the ability to adhere to host surfaces such as mucous membranes, gastric and intestinal epithelial or endothelial tissue.     

Untreated and enzyme‐modified bovine whey products reduce association of Salmonella Typhimurium,Escherichia coli O157:H7 and Cronobacter malonaticus (formerly Enterobacter sakazakii) to CaCo‐2 cells

Aims: Adhesion of a micro‐organism to a cell surface is often considered to be the first step in pathogenesis. Inhibiting this process may have therapeutic effects in vivo. This study investigates the inhibitory effects of various bovine whey products on the association of Salm. Typhimurium, E. coli O157:H7 and C. malonaticus (formerly Enterobacter sakazakii) to the human CaCo‐2 cell line. Invasion of CaCo‐2 cells by Salm. Typhimurium and C. malonaticus was also examined. Methods and Results: Infection assays were performed by incubating pathogenic acteria with CaCo‐2 cells in the presence of untreated (UT) or enzyme‐modified (EM) whey products. Associated micro‐organisms were directly quantified by plate counts. Invasion of CaCo‐2 cells by Salm. Typhimurium and C. malonaticus in the presence/absence of test materials was also quantified using gentamicin protection assays. At a concentration of 40 mg ml^?1, some UT whey products reduced association and invasion, but this effect was enhanced following hydrolysis with porcine pancreatic lipase. Conclusions: Both UT and EM sweet whey protein concentrates (WPCs) were found to be particularly effective inhibitors of association and invasion. All EM whey products significantly (P < 0·05) inhibited invasion of C. malonaticus into epithelial cells, causing a 2‐log reduction in the quantity of these micro‐organisms internalized. Significance and Impact of the Study: The present study suggests that whey products can inhibit association to and invasion of CaCo‐2 cells by selected micro‐organisms and may be useful in the treatment and/or prevention of foodborne infections.     

Bovine recto-anal junction squamous epithelial (RSE) cell adhesion assay for studying Escherichia coli O157 adherence

Aim: To develop a new adherence assay, using cattle recto‐anal junction squamous epithelial (RSE) cells, for evaluating bacterial adherence to cells of bovine origin. Methods and Results: Proof of concept was demonstrated using the human gastrointestinal pathogen Escherichia coli O157:H7, for which cattle are reservoirs. Adherence assays were conducted using both RSE and HEp‐2 cells, in the presence and absence of D+Mannose. E. coli O157 specifically adhered in a type I fimbriae‐independent manner to RSE cells in significantly higher numbers and also bound significantly higher numbers of RSE cells than diverse laboratory strains of nonpathogenic E. coli. Conclusion: The RSE cell adhesion assay output highly reproducible and interpretable results that compared very well with those obtained using the more extensively used HEp‐2 cell adherence assay. Significance and Impact of the study: The RSE cell adhesion assay provides a convenient means of directly defining and evaluating pathogen factors operating at the bovine recto‐anal junction. The RSE cell adhesion assay further has the potential for extrapolation to diverse bacteria, including food‐borne pathogens that colonize cattle via adherence to this particular anatomical site.     

16S rRNA gene based analysis of <Emphasis Type="Italic">Enterobacter sakazakii</Emphasis> strains from different sources and development of a PCR assay for identification

Background  

E. sakazakii is considered to be an opportunistic pathogen, implicated in food borne diseases causing meningitis or enteritis especially in neonates and infants. Cultural standard identification procedures for E. sakazakii include the observation of yellow pigmentation of colonies and a positive glucosidase activity. Up to now, only one PCR system based on a single available 16S rRNA gene sequence has been published for E. sakazakii identification. However, in our hands a preliminary evaluation of this system to a number of target and non-target strains showed significant specificity problems of this system. In this study full-length 16S rRNA genes of thirteen E. sakazakii strains from food, environment and human origin as well as the type strain ATCC 51329 were sequenced. Based on this sequence data a new specific PCR system for E. sakazakii was developed and evaluated.     

Aggregative adherent strains of Corynebacterium pseudodiphtheriticum enter and survive within HEp-2 epithelial cells

Corynebacterium pseudodiphtheriticum is a well-known human pathogen that mainly causes respiratory disease and is associated with high mortality in compromised hosts. Little is known about the virulence factors and pathogenesis of C. pseudodiphtheriticum. In this study, cultured human epithelial (HEp-2) cells were used to analyse the adherence pattern, internalisation and intracellular survival of the ATCC 10700 type strain and two additional clinical isolates. These microorganisms exhibited an aggregative adherence-like pattern to HEp-2 cells characterised by clumps of bacteria with a "stacked-brick" appearance. The differences in the ability of these microorganisms to invade and survive within HEp-2 cells and replicate in the extracellular environment up to 24 h post infection were evaluated. The fluorescent actin staining test demonstrated that actin polymerisation is involved in the internalisation of the C. pseudodiphtheriticum strains. The depolymerisation of microfilaments by cytochalasin E significantly reduced the internalisation of C. pseudodiphtheriticum by HEp-2 cells. Bacterial internalisation and cytoskeletal rearrangement seemed to be partially triggered by the activation of tyrosine kinase activity. Although C. pseudodiphtheriticum strains did not demonstrate an ability to replicate intracellularly, HEp-2 cells were unable to fully clear the pathogen within 24 h. These characteristics may explain how some C. pseudodiphtheriticum strains cause severe infection in human patients.     

Influence of mucosal cell origin on the in vitro adherence of Candida albicans: Are mucosal cells from different sources equivalent?

The influence of collecting mucosal cells from various anatomical sites, and varying the date of collection and cell donor on adhesion of Candida albicans to human epithelial cells was examined by using an in vitro adherence assay. Examination of buccal mucosal cells from twenty-four donors showed statistically significant differences in the number of attached yeasts between individuals. Sex did not exert a significant influence on adhesion. Examination of buccal mucosal cells from ten donors collected on five different dates revealed that yeast attachment to mucosal epithelial cells varied significantly within subjects across time. Epithelial cells from some donors manifested greater date-to-date variations in yeast adhesion than others. Adherence of Candida to mucosal cells from three anatomical sites (mouth, vagina and urinary tract) collected from ten different donors was also tested. Yeast adherence to buccal cells was highest, lowest using urinary tract cells, while vaginal epithelium was intermediate. Adherence to mucosal cells from three sites was significantly different both within and between individuals although some subjects manifested larger variations than others. These data suggest that the in vitro adherence of Candida albicans is influenced by mucosal cell donor, date of collection and body site of origin. Mucosal cells from different sources do not appear to be equivalent in receptiveness to C. albicans and this might explain some of the discrepancies observed when adhesion studies performed by different investigators are compared. The existing need for a more uniform methodology with which to pursue studies on fungal attachment to mucosal surfaces is emphasized.     

Isolation and PCR detection of <Emphasis Type="Italic">Enterobacter sakazakii</Emphasis> in South African food products,specifically infant formula milks

Enterobacter sakazakii has recently been identified as an opportunistic pathogen. The current culture-dependent detection methods for these bacteria are time-consuming and in this study a PCR method for the detection of E. sakazakii in South African food products, including an internal amplification control (IAC) was developed. DNA was isolated and amplified from the products and they were plated on selective growth media after pre-enrichment and enrichment in Enterobacteriaceae enrichment broth. Four of the 22 products tested positive for the presence of E. sakazakii, confirmed by PCR detection and growth on selective media. The PCR method proved effective in detecting E. sakazakii in South African products after three days and could serve as an alternative for traditional microbiological techniques.     

Real‐time PCR method for the quantification of Burkholderia cepacia complex attached to lung epithelial cells and inhibition of that attachment

Aims: To develop a rapid method to quantify the attachment of the cystic fibrosis pathogen, Burkholderia multivorans, to lung epithelial cells (16HBE14o^?) using real‐time PCR with a view to monitoring potential inhibition of lung cell attachment. Methods and Results: Mammalian and bacterial DNA were purified from bacteria attached to lung epithelial cells. The relative amount of bacteria attached was determined by amplification of the recA gene relative to the human GAPDH gene, in the presence of SYBR Green^®. The method was thoroughly validated and shown to correlate well with traditional plating techniques. Inhibition of bacterial attachment with simple sugars was then evaluated by real‐time PCR. Of the sugars examined, pre‐incubation of B. multivorans with lactose, mannose and xylitol all decreased bacterial adherence to 16HBE14o^? cells, while glucose and galactose had no significant effect. Pre‐incubation with lactose had the greatest effect, resulting in reduced adhesion to 35% of untreated controls. Conclusions: This method can be used to quickly and effectively screen novel agents with higher affinities for bacterial adhesins. Significance and Impact of the Study: This method will enable the rapid development of novel agents to inhibit colonization by this pathogen from the environment.     

The biochemical differentiation of Enterobacter sakazakii genotypes

Background  

Enterobacter sakazakii is an emergent pathogen that has been associated with neonatal infections through contaminated powdered infant milk formula. The species was defined by Farmer et al. (1980) who described 15 biogroups according to the biochemical characterization of 57 strains. This present study compares genotypes (DNA cluster groups based on partial 16S rDNA sequence analysis) with the biochemical traits for 189 E. sakazakii strains.     

Aspergillus fumigatus adhesion factors in dormant conidia revealed through comparative phenotypic and transcriptomic analyses

Aspergillus fumigatus is an important fungal pathogen of humans. Inhaled conidia of A. fumigatus adhere to pulmonary epithelial cells, causing opportunistic infection. However, little is known about the molecular mechanism of the adherence of resting conidia. Fungal molecules adhesive to host cells are presumed to be displayed on the conidial surface during conidial formation as a result of changes in gene expression. Therefore, we exhaustively searched for adhesion molecules by comparing the phenotypes and the gene expression profiles of A. fumigatus strains that have conidia showing either high or low adherence to human pulmonary A549 cells. Morphological observation suggested that strains that produce conidia of reduced size, hydrophobicity, or number show decreased adherence to A549 cells. K‐means cluster analyses of gene expression revealed 31 genes that were differentially expressed in the high‐adherence strains during conidial formation. We knocked out three of these genes and showed that the conidia of AFUA_4G01030 (encoding a hypothetical protein) and AFUA_4G08805 (encoding a haemolysin‐like protein) knockout strains had significantly reduced adherence to host cells. Furthermore, the conidia of these knockout strains had lower hydrophobicity and fewer surface spikes compared to the control strain. We suggest that the selectively expressed gene products, including those we identified experimentally, have composite synergistic roles in the adhesion of conidia to pulmonary epithelial cells.     

Monoclonal antibodies directed against extracellular matrix proteins reduce the adherence of Candida albicans to HEp-2 cells

The presence of the extracellular matrix (ECM) proteins collagen types I and IV, laminin and fibronectin on the surface of HEp-2 cells was confirmed by flow cytometry using monoclonal antibodies. Monoclonal antibodies directed against these ECM proteins reduced the adherence of C. albicans ATCC 44990 to HEp-2 cells, the greatest reductions being evident in assays which incorporated anti-collagen type IV monoclonal antibody. The ability of sugaramines to inhibit the adherence of C. albicans to a variety of cell types has been demonstrated previously and the most significant reduction in C. albicans – HEp-2 adherence was in assays which incorporated 0.2M galactosamine. The combination of anti-collagen IV monoclonal antibody and galactosamine reduced the adherence of C. albicans to HEp-2 cells by approximately 70% (p < 0.05). This revised version was published online in June 2006 with corrections to the Cover Date.     

Multilocus sequence typing of Cronobacter sakazakii and Cronobacter malonaticus reveals stable clonal structures with clinical significance which do not correlate with biotypes

Background  

The Cronobacter genus (Enterobacter sakazakii) has come to prominence due to its association with infant infections, and the ingestion of contaminated reconstituted infant formula. C. sakazakii and C. malonaticus are closely related, and are defined according their biotype. Due to the ubiquitous nature of the organism, and the high severity of infection for the immunocompromised, a multilocus sequence typing (MLST) scheme has been developed for the fast and reliable identification and discrimination of C. sakazakii and C. malonaticus strains. It was applied to 60 strains of C. sakazakii and 16 strains of C. malonaticus, including the index strains used to define the biotypes. The strains were from clinical and non-clinical sources between 1951 and 2008 in USA, Canada, Europe, New Zealand and the Far East.