Mary Anne Roshni Amalaradjou

Antibiofilm Effect of Trans-Cinnamaldehyde on Uropathogenic Escherichia coli

PURPOSE Urinary tract infections are the most common hospital acquired infections in humans, caused primarily by uropathogenic Escherichia coli. Indwelling urinary catheters for bladder drainage in humans become encrusted with uropathogenic E. coli biofilms that are resistant to common antibiotics, resulting in chronic infections. We studied the efficacy of the cinnamon ingredient trans-cinnamaldehyde (Sigma) for preventing uropathogenic E. coli biofilm. We also determined the efficacy of trans-cinnamaldehyde as an ingredient in catheter lock solution to inactivate preformed uropathogenic E. coli biofilm. MATERIALS AND METHODS Polystyrene plates and urinary catheters inoculated with uropathogenic E. coli (5 to 6.0 log cfu) were treated with trans-cinnamaldehyde (0%, 0.1%, 0.25% or 0.5%) at 37C. Catheters with uropathogenic E. coli biofilm were also treated with lock solution containing trans-cinnamaldehyde (0%, 1%, 1.25% or 1.5%). Uropathogenic E. coli biofilm on control and trans-cinnamaldehyde treated plates and catheters was determined on incubation days 0, 1, 3 and 5. Trans-cinnamaldehyde potential cytotoxity, if any, was determined in HTB-4 bladder epithelial cells (ATCC). RESULTS At all concentrations trans-cinnamaldehyde effectively prevented uropathogenic E. coli biofilm on plates and catheters. As a constituent in catheter lock solution, it inactivated uropathogenic E. coli biofilm on catheters. Trans-cinnamaldehyde produced no cytotoxic effects on human bladder epithelial cells at the tested concentrations. CONCLUSIONS Results suggest that trans-cinnamaldehyde may be applied as a catheter surface coating or as an ingredient in catheter lock solution to prevent urinary tract infection in humans.

Reduction of Salmonella enterica serovar enteritidis colonization in 20-day-old broiler chickens by the plant-derived compounds trans-cinnamaldehyde and eugenol

ABSTRACT The efficacies of trans-cinnamaldehyde (TC) and eugenol (EG) for reducing Salmonella enterica serovar Enteritidis colonization in broiler chickens were investigated. In three experiments for each compound, 1-day-old chicks (n = 75/experiment) were randomly assigned to five treatment groups (n = 15/treatment group): negative control (-ve S. Enteritidis, -ve TC, or EG), compound control (-ve S. Enteritidis, +ve 0.75% [vol/wt] TC or 1% [vol/wt] EG), positive control (+ve S. Enteritidis, -ve TC, or EG), low-dose treatment (+ve S. Enteritidis, +ve 0.5% TC, or 0.75% EG), and high-dose treatment (+ve S. Enteritidis, +ve 0.75% TC, or 1% EG). On day 0, birds were tested for the presence of any inherent Salmonella (n = 5/experiment). On day 8, birds were inoculated with ∼8.0 log10 CFU S. Enteritidis, and cecal colonization by S. Enteritidis was ascertained (n = 10 chicks/experiment) after 24 h (day 9). Six birds from each treatment group were euthanized on days 7 and 10 after inoculation, and cecal S. Enteritidis numbers were determined. TC at 0.5 or 0.75% and EG at 0.75 or 1% consistently reduced (P < 0.05) S. Enteritidis in the cecum (≥3 log10 CFU/g) after 10 days of infection in all experiments. Feed intake and body weight were not different for TC treatments (P > 0.05); however, EG supplementation led to significantly lower (P < 0.05) body weights. Follow-up in vitro experiments revealed that the subinhibitory concentrations (SICs, the concentrations that did not inhibit Salmonella growth) of TC and EG reduced the motility and invasive abilities of S. Enteritidis and downregulated expression of the motility genes flhC and motA and invasion genes hilA, hilD, and invF. The results suggest that supplementation with TC and EG through feed can reduce S. Enteritidis colonization in chickens.

Recombinant probiotic expressing Listeria adhesion protein attenuates Listeria monocytogenes virulence in vitro.

Background Listeria monocytogenes, an intracellular foodborne pathogen, infects immunocompromised hosts. The primary route of transmission is through contaminated food. In the gastrointestinal tract, it traverses the epithelial barrier through intracellular or paracellular routes. Strategies to prevent L. monocytogenes entry can potentially minimize infection in high-risk populations. Listeria adhesion protein (LAP) aids L. monocytogenes in crossing epithelial barriers via the paracellular route. The use of recombinant probiotic bacteria expressing LAP would aid targeted clearance of Listeria from the gut and protect high-risk populations from infection. Methodology/Principal Findings The objective was to investigate the ability of probiotic bacteria or LAP-expressing recombinant probiotic Lactobacillus paracasei (LbpLAP) to prevent L. monocytogenes adhesion, invasion, and transwell-based transepithelial translocation in a Caco-2 cell culture model. Several wild type probiotic bacteria showed strong adhesion to Caco-2 cells but none effectively prevented L. monocytogenes infection. Pre-exposure to LbpLAP for 1, 4, 15, or 24 h significantly (P<0.05) reduced adhesion, invasion, and transepithelial translocation of L. monocytogenes in Caco-2 cells, whereas pre-exposure to parental Lb. paracasei had no significant effect. Similarly, LbpLAP pre-exposure reduced L. monocytogenes translocation by as much as 46% after 24 h. LbpLAP also prevented L. monocytogenes-mediated cell damage and compromise of tight junction integrity. Furthermore, LbpLAP cells reduced L. monocytogenes-mediated cell cytotoxicity by 99.8% after 1 h and 79% after 24 h. Conclusions/Significance Wild type probiotic bacteria were unable to prevent L. monocytogenes infection in vitro. In contrast, LbpLAP blocked adhesion, invasion, and translocation of L. monocytogenes by interacting with host cell receptor Hsp60, thereby protecting cells from infection. These data show promise for the use of recombinant probiotics in preventing L. monocytogenes infection in high-risk populations.

Inactivation of Salmonella spp. on tomatoes by plant molecules

The efficacy of carvacrol (CAR), trans-cinnamaldehyde (TC), eugenol (EUG) and β-resorcylic acid (BR) as a wash treatment for reducing Salmonella spp. on tomatoes was investigated. Plum tomatoes inoculated with a six-serotype mixture of Salmonella (10⁸CFU) were subjected to washing in sterile deionized water (control) or deionized water containing chlorine (100 ppm), CAR (0.25 and 0.75%), TC (0.5 and 0.75%), EUG (0.25 and 0.75%), or BR (0.75 and 1.0%) for 15 sec, 1 min, and 3 min. The plant molecules were more effective (P<0.05) in reducing Salmonella on tomatoes compared to washing in water and chlorine. Both concentrations of CAR and TC, and 0.75% EUG decreased Salmonella counts on tomatoes by~6.0 log CFU/ml at 1 min. Both concentrations of BR decreased the pathogen on tomatoes to undetectable levels at 3 min of exposure. Washing of tomatoes in deionized water and chlorine for 3 min reduced Salmonella by ca. 2.0 and 4.0 log CFU/ml, respectively. No Salmonella was detected in the wash water containing the plant molecules or chlorine, whereas a substantial population of the pathogen survived in the control wash water. Moreover, none of the dipping treatments had any effect on the red color of tomatoes (P>0.05). Results indicate that CAR, TC, EUG and BR could effectively be used to kill Salmonella on tomatoes, but additional studies on sensory and quality characteristics of tomatoes treated with plant molecules are warranted.

Inactivation of Escherichia coli O157:H7 in apple juice and apple cider by trans-cinnamaldehyde.

This study investigated the antimicrobial effect of low concentrations of trans-cinnamaldehyde (TC) on Escherichia coli O157:H7 in apple juice and apple cider. A five-strain mixture of E. coli O157:H7 was inoculated into apple juice or cider at approximately 6.0 log CFU/ml, followed by the addition of TC (0%v/v, 0.025%v/v, 0.075%v/v and 0.125%v/v). The inoculated apple juice samples were incubated at 23 degrees C and 4 degrees C for 21 days, whereas the cider samples were stored only at 4 degrees C. The pH of apple juice and cider, and E. coli O157:H7 counts were determined on days 0, 1, 3, 5, 7, 14 and 21. TC was effective (P<0.05) in inactivating E. coli O157:H7 in apple juice and apple cider. At 23 degrees C, 0.125 and 0.075%v/v TC completely inactivated E. coli O157:H7 in apple juice (negative by enrichment) on days 1 and 3, respectively. At 4 degrees C, 0.125 and 0.075%v/v TC decreased the pathogen counts in the juice and cider to undetectable levels on days 3 and 5, respectively. Results indicate that low concentrations of TC could be used as an effective antimicrobial to inactivate E. coli O157:H7 in apple juice and apple cider.

Effect of trans-cinnamaldehyde on inhibition and inactivation of Cronobacter sakazakii biofilm on abiotic surfaces.

The efficacy of trans-cinnamaldehyde (TC), an ingredient in cinnamon oil, for inhibiting biofilm synthesis (560 and 750 μM TC) and inactivating mature biofilms (23 and 38 mM TC) of Cronobacter sakazakii was investigated at 24 and 12 °C in the presence and absence of reconstituted infant formula on polystyrene plates, stainless steel coupons, feeding bottle coupons, and enteral feeding tube coupons. Additionally, TCs effect on the expression of genes critical for biofilm formation in C. sakazakii was determined by reverse transcription quantitative PCR. TC inhibited and inactivated C. sakazakii biofilms on all matrices tested at both temperatures. C. sakazakii was reduced by >4.0 and 3.0 log CFU/ml after 96 h of exposure to 38 mM and 750 μM TC, respectively. Reverse transcription quantitative PCR results revealed that TC significantly (P≤0.05) down-regulated biofilm-associated genes in C. sakazakii. TC could potentially be used to control C. sakazakii biofilms on infant formula feeding equipment and preparatory areas.

Plant-derived antimicrobials reduce Listeria monocytogenes virulence factors in vitro, and down-regulate expression of virulence genes.

Listeria monocytogenes (LM) is a major foodborne pathogen causing septicemia, meningitis and death in humans. LM infection is preceded by its attachment to and invasion of human intestinal epithelium followed by systemic spread. The major virulence factors in LM include motility, hemolysin and lecithinase production. Reducing LM attachment to and invasion of host tissue and production of virulence factors could potentially control listeriosis in humans. This study investigated the efficacy of sub-inhibitory concentrations (SICs, concentrations not inhibiting bacterial growth) of three, generally regarded as safe (GRAS)-status, plant-derived antimicrobial compounds in reducing LM attachment to and invasion of human colon adenocarcinoma (Caco-2) and human brain microvascular endothelial cells (HBMEC). Additionally, the effect of these compounds on the aforementioned LM virulence factors was studied. The compounds and their respective SICs used relative to their MICs were trans-cinnamaldehyde (TC 0.50mM, 0.75mM with the MIC of 0.90mM), carvacrol (CR 0.50mM, 0.65mM with the MIC of 0.75mM), and thymol (TY 0.33mM, 0.50mM with the MIC of 0.60mM). All three-plant antimicrobials reduced LM adhesion to and invasion of Caco-2 and HBMEC (p<0.05). The compounds also decreased LM motility, hemolysin production and lecithinase activity (p<0.05). Real-time PCR data revealed that TC, CR, and TY down-regulated the expression of LM virulence genes by >3.0 folds compared to controls (p<0.05). Results suggest that TC, CR, and TY could potentially be used to control LM infection; however, in vivo studies are necessary to validate these results.

Inactivation of Enterobacter sakazakii in reconstituted infant formula by trans-cinnamaldehyde

Enterobacter sakazakii is an emerging pathogen which causes a life-threatening form of meningitis, necrotizing colitis and meningoencephalitis in neonates and children. Epidemiological studies implicate dried infant formula as the principal source of the pathogen. Trans-cinnamaldehyde is a major component of bark extract of cinnamon. It is classified as generally recognized as safe (GRAS) by the U.S. Food and Drug Administration, and is approved for use in food (21 CFR 182.60). The objective of this study was to determine the antibacterial effect of trans-cinnamaldehyde on E. sakazakii in reconstituted infant formula. A 5-strain mixture of E. sakazakii was inoculated into 10 ml samples of reconstituted infant formula (at 6.0 log CFU/ml) containing 0%, 0.15%, 0.3% or 0.5% trans-cinnamaldehyde. The samples were incubated at 37, 23, 8 or 4 degrees C for 0, 6, 10 and 24 h, and the surviving populations of E. sakazakii at each sampling time were enumerated. In addition, potential cytotoxicity of trans-cinnamaldehyde, if any, was determined on human embryonic intestinal cells (INT-407). The treatments containing trans-cinnamaldehyde significantly reduced (P<0.05) the population of E. sakazakii, compared to the controls. Trans-cinnamaldehyde (0.5%) reduced the pathogen to undetectable levels by 4 h of incubation at 37 or 23 degrees C and 10 h of incubation at 8 or 4 degrees C, respectively. Trans-cinnamaldehyde produced no cytotoxic effects on human embryonic intestinal cells at the tested concentrations. Results indicate that trans-cinnamaldehyde could potentially be used to kill E. sakazakii in reconstituted infant formula, however sensory studies are warranted before recommending its use.

Trans-Cinnamaldehyde Decreases Attachment and Invasion of Uropathogenic Escherichia Coli in Urinary Tract Epithelial Cells by Modulating Virulence Gene Expression

PURPOSE Uropathogenic Escherichia coli is the primary bacterium causing urinary tract infection in humans. Attachment and invasion of urinary tract epithelial cells by UPEC is the first critical step in establishing a successful urinary tract infection. We investigated the efficacy of subinhibitory concentrations of trans-cinnamaldehyde to inhibit uropathogenic E. coli attachment and invasion of human uroepithelial cells. We also determined the trans-cinnamaldehyde effect on uropathogenic E. coli genes encoding virulence factors critical for uroepithelial cell bacterial attachment and invasion. MATERIALS AND METHODS Polystyrene 24-well plates seeded with uroepithelial cells were inoculated with uropathogenic E. coli (about 6.0 log cfu) and subinhibitory concentrations of trans-cinnamaldehyde (0, 325, 560 and 750 μM), and incubated for 60 minutes at 37C. Uroepithelial cells were washed and lysed to enumerate adhered uropathogenic E. coli populations. For the invasion assay uroepithelial cells were treated with gentamicin after incubation and lysed to enumerate invaded uropathogenic E. coli. Also, the trans-cinnamaldehyde effect on uropathogenic E. coli genes encoding attachment and invasion associated virulence factors was determined by real-time quantitative polymerase chain reaction. RESULTS Trans-cinnamaldehyde significantly decreased uroepithelial cell attachment and invasion by uropathogenic E. coli (p <0.05). Real-time quantitative polymerase chain reaction revealed that trans-cinnamaldehyde significantly decreased the expression of major genes involved in uropathogenic E. coli attachment and invasion of host tissue (p <0.05). The down-regulating effect of trans-cinnamaldehyde on these genes potentially translated into decreased ability of uropathogenic E. coli to attach and invade bladder cells. CONCLUSIONS Trans-cinnamaldehyde may potentially be used as a safe, effective antimicrobial to control uropathogenic E. coli infection. Followup studies in animal models are warranted.

Prophylactic Supplementation of Caprylic Acid in Feed Reduces Salmonella Enteritidis Colonization in Commercial Broiler Chicks

Salmonella Enteritidis is a major foodborne pathogen for which chickens serve as reservoir hosts. Reducing Salmonella Enteritidis carriage in chickens would reduce contamination of poultry meat and eggs with this pathogen. We investigated the prophylactic efficacy of feed supplemented with caprylic acid (CA), a natural, generally recognized as safe eight-carbon fatty acid, for reducing Salmonella Enteritidis colonization in chicks. One hundred commercial day-old chicks were randomly divided into five groups of 20 birds each: CA control (no Salmonella Enteritidis, CA), positive control (Salmonella Enteritidis, no CA), negative control (no Salmonella Enteritidis, no CA), and 0.7 or 1% CA. Water and feed were provided ad libitum. On day 8, birds were inoculated with 5.0 log CFU of Salmonella Enteritidis by crop gavage. Six birds from each group were euthanized on days 1, 7, and 10 after challenge, and Salmonella Enteritidis populations in the cecum, small intestine, cloaca, crop, liver, and spleen were enumerated. The study was replicated three times. CA supplementation at 0.7 and 1% consistently decreased Salmonella Enteritidis populations recovered from the treated birds. Salmonella Enteritidis counts in the tissue samples of CA-treated chicks were significantly lower (P < 0.05) than those of control birds on days 7 and 10 after challenge. Feed intake and body weight did not differ between the groups. Histological examination revealed no pathological changes in the cecum and liver of CA-supplemented birds. The results suggest that prophylactic CA supplementation through feed can reduce Salmonella Enteritidis colonization in day-old chicks and may be a useful treatment for reducing Salmonella Enteritidis carriage in chickens.