Induction of Salmonella Enteritidis into a Viable but Nonculturable State by Cinnamaldehyde and Its Resuscitation.

Trans-cinnamaldehyde (TC), a typical plant-derived compound, has been widely used in the control of foodborne pathogen contamination. Nevertheless, the risk associated with the occurrence of viable but nonculturable (VBNC) bacteria induced by TC remains unclear. The results of this study showed that Salmonella Enteritidis (S. Enteritidis) entered the VBNC state after being induced by TC at a minimum inhibitory concentration of 312.5 µg/mL and survived for at least 22 days under TC treatment. Enhanced resistance was found against heat treatment (75°C, 30 s), antibiotics (i.e., ampicillin, ceftriaxone sodium, chloramphenicol), and hydrogen peroxide (3%) in VBNC S. Enteritidis. A synergistic effect against VBNC S. Enteritidis occurred when TC was combined with acid treatment, including lactic acid and acetic acid (pH = 3.5). VBNC and resuscitated S. Enteritidis by sodium pyruvate treatment (100 mM) were found to retain the infectious ability to Caco-2 cells. Relative expression levels of the stress-related genes relA, spoT, ppx, lon, katG, sodA, dnaK, and grpE were upregulated in VBNC S. Enteritidis. Accumulation of reactive oxygen species (ROS) and protein aggregates was observed in VBNC cells. Besides, the resuscitation of VBNC cells was accompanied with clearance of ROS and protein aggregates. In summary, this study presents a comprehensive characterization of stress tolerance and resuscitation of VBNC S. Enteritidis induced by cinnamaldehyde, and the results provide useful information for the development of effective control strategy against VBNC pathogenic bacteria in food production.

Salmonella Enteritidis survival in different temperatures and nutrient solution pH levels in hydroponically grown lettuce.

Due to climate change, with contaminated and less fertile soils, and intense weather phenomena, a turn towards hydroponic vegetable production has been made. Hydroponic cultivation of vegetables is considered to be a clean, safe and environmentally friendly growing technique; however, incidence of microbial contamination i.e. foodborne pathogens, might occur, endangering human health. The aim of this study was to investigate the effects of different plant growth stages, pH (values 5, 6, 7, 8) and bacterial inoculum levels (3 and 6 log cfu/mL) on hydroponically cultivated lettuce spiked with Salmonella Enteritidis. The results revealed that the pH and inoculum levels affected the internalization and survival of the pathogen in the hydroponic environment and plant tissue. Younger plants were found to be more susceptible to pathogen internalization compared to older ones. Under the current growing conditions (hydroponics, pH and inoculum levels), no leaf internalization was observed at all lettuce growth stages, despite the bacterium presence in the hydroponic solution. Noticeably, bacteria load at the nutrient solution was lower in low pH levels. These results showed that bacterium presence initiates plant response as indicated by the increased phenols, antioxidants and damage index markers (H2O2, MDA) in order for the plant to resist contamination by the invader. Nutrient solution management can result in Taylor-made recipes for plant growth and possible controlling the survival and growth of S. Enteritidis by pH levels.

Florfenicol Enhances Colonization of a Salmonella enterica Serovar Enteritidis floR Mutant with Major Alterations to the Intestinal Microbiota and Metabolome in Neonatal Chickens.

Florfenicol is an important antibiotic commonly used in poultry production to prevent and treat Salmonella infection. However, oral administration of florfenicol may alter the animals' natural microbiota and metabolome, thereby reducing intestinal colonization resistance and increasing susceptibility to Salmonella infection. In this study, we determined the effect of florfenicol (30 mg/kg of body weight) on gut colonization of neonatal chickens challenged with Salmonella enterica subsp. enterica serovar Enteritidis. We then analyzed the microbial community structure and metabolic profiles of cecal contents using microbial 16S amplicon sequencing and liquid chromatography-mass spectrometry (LC-MS) untargeted metabolomics, respectively. We also screened the marker metabolites using a multi-omics technique and assessed the effect of these markers on intestinal colonization by S. Enteritidis. Florfenicol administration significantly increased the loads of S. Enteritidis in cecal contents, spleen, and liver and prolonged the residence of S. Enteritidis. Moreover, florfenicol significantly affected cecal colony structures, with reduced abundances of Lactobacillus and Bacteroidetes and increased levels of Clostridia, Clostridium, and Dorea. The metabolome was greatly influenced by florfenicol administration, and perturbation in metabolic pathways related to linoleic acid metabolism (linoleic acid, conjugated linoleic acid [CLA], 12,13-EpOME, and 12,13-diHOME) was most prominently detected. We screened CLA and 12,13-diHOME as marker metabolites, which were highly associated with Lactobacillus, Clostridium, and Dorea. Supplementation with CLA maintained intestinal integrity, reduced intestinal inflammation, and accelerated Salmonella clearance from the gut and remission of enteropathy, whereas treatment with 12,13-diHOME promoted intestinal inflammation and disrupted intestinal barrier function to sustain Salmonella infection. Thus, these results highlight that florfenicol alters the intestinal microbiota and metabolism of neonatal chickens and promotes Salmonella infection mainly by affecting linoleic acid metabolism. IMPORTANCE Florfenicol is a broad-spectrum fluorine derivative of chloramphenicol frequently used in poultry to prevent/treat Salmonella. However, oral administration of florfenicol may lead to alterations in the microbiota and metabolome in the chicken intestine, thereby reducing colonization resistance to Salmonella infection, and the possible mechanisms linking antibiotics and Salmonella colonization in poultry have not yet been fully elucidated. In the current study, we show that increased colonization by S. Enteritidis in chickens administered florfenicol is associated with large shifts in the gut microbiota and metabolic profiles. The most influential linoleic acid metabolism is highly associated with the abundances of Lactobacillus, Clostridium, and Dorea in the intestine. The screened target metabolites in linoleic acid metabolism affect S. Enteritidis colonization, intestinal inflammation, and intestinal barrier function. Our findings provide a better understanding of the susceptibility of animal species to Salmonella after antibiotic intervention, which may help to elucidate infection mechanisms that are important for both animal and human health.

Development of a genomic DNA reference material for Salmonella enteritidis detection using polymerase chain reaction.

Several rapid methods based on nucleic acids can detect foodborne pathogens, such as Salmonella spp. However, a common reference that enables metrological traceability among measurement results is not available. Reference materials (RM) are thus key to guarantee methodological comparability. This study developed a candidate genomic DNA reference material for Salmonella enteritidis quantification to establish performance conditions and reference values for normalized RM production. The growth of Salmonella enteritidis ATCC® 13076 in Rappaport Vassiliadis selective medium was characterized, and we optimized a method of DNA extraction using cetrimonium bromide (CTAB) and LiCl. In a first stage six concentrations of DNA were prepared with and without yeast RNA (40 ng/µL) to evaluate its effect as a stabilizer in terms of homogeneity and short-term stability. Based on the findings, in a second stage two DNA concentrations were prepared and a reference value with its uncertainty was assigned based on the results of characterization, homogeneity, and stability studies using digital polymerase chain reaction and the gene targets, invA, ttr, and hilA. The material was stable for 9 months at 4 °C, with a expanded uncertainty contribution range of 11%-14%. The novel candidate RM is the first to be developed nationwide and will improve the quality of measurements in the area of food safety.

Inactivation of Salmonella Enteritidis on cherry tomatoes by ultrasound, lactic acid, detergent, and silver nanoparticles.

Ultrasound (US) combined with chemical agents could represent an effective method for decontaminating fruits and vegetables. This study aimed to evaluate the use of US (40 kHz for 5 min) alone or with 1% lactic acid (LA), 1% commercial detergent (DET), or 6 mg/L silver nanoparticles (AgNP, average diameter 100 nm) as an alternative treatment to 200 mg/L sodium dichloroisocyanurate for inactivating Salmonella enterica serovar Enteritidis present on cherry tomatoes. The interfacial tension between sanitizing solutions and bacterial adhesion was investigated. Sanitizers in solutions with DET and AgNP had lower surface tension. All treatments, except that with DET, reduced Salmonella Enteritidis by more than one logarithmic cycle. There was no significant difference between the mean values of log colony-forming units (CFU)/g reduction in all treatments. Transmission electron microscopy revealed the loss of the Salmonella Enteritidis capsule following treatment with US and with US + LA. Salmonella Enteritidis counts (2.29 log CFU/g) in cherry tomatoes were markedly reduced to safe levels by treatment with the combination of AgNP and US + LA (2.37 log CFU/g).

High concentration of sodium chloride could induce the viable and culturable states of Escherichia coli O157:H7 and Salmonella enterica serovar Enteritidis.

In the present study, Escherichia coli O157:H7 and Salmonella enterica serovar Enteritidis were transferred into Luria-Bertani medium without NaCl (LBWS) and adjusted to various pHs (4, 5, 6 and 7) with lactic acid containing 0·75, 5, 10 and 30% NaCl, and stored at 25°C until the bacterial populations reached below detectable levels on tryptic soy agar (TSA). Although E. coli O157:H7 and S. Enteritidis did not grow on TSA when incubated in LBWS with 30% NaCl for 35 and 7 days, more than 60 and 70% of the bacterial cells were shown to be viable via fluorescent staining with SYTO9 and propidium iodide (PI), respectively, suggesting that a number of cells could be induced into the viable but nonculturable (VBNC) state. These bacteria that were induced into a VBNC state were transferred to a newly prepared tryptic soy broth (TSB) and then incubated at 37°C for several days. After more than 7 days, E. coli O157:H7 and S. Enteritidis regained their culturability. We, therefore, suggest that E. coli O157:H7 and S. Enteritidis entered the VBNC state under the adverse condition of higher salt concentrations and were revived when these conditions were reversed.

Quantitative proteomics implicates YggT in streptomycin resistance in Salmonella enterica serovar Enteritidis.

OBJECTIVES: To identify proteins that may be associated with antibiotic resistance in the multidrug-resistant Salmonella enterica D14, by constructing proteomic profiles using mass spectrometry-based label-free quantitative proteomics (LFQP). RESULTS: D14 was cultured with four antibiotics (ampicillin, nalidixic acid, streptomycin, and tetracycline) separately. Subsequently, the findings from an equal combination of the four cultures were compared with the profile of sensitive S. enterica 104. 2255 proteins, including 149 differentially up-regulated proteins, were identified. Many of these up-regulated proteins were associated with flagellar assembly and chemotaxis, two-component system, amino acid metabolism, ß-lactam resistance, and transmembrane transport. A subset of 10 genes was evaluated via quantitative real-time PCR (qPCR), followed by the construction of cheR, fliS, fliA, arnA, and yggT deletion mutants. Only the yggT-deleted D14 mutant showed decrease in streptomycin resistance, whereas the other deletions had no effect. Furthermore, complementation of yggT and the overexpression of yggT in S. enterica ATCC 14028 increased the streptomycin resistance. Additionally, spot dilution assay results confirmed that Salmonella strains, harboring yggT, exhibited an advantage in the presence of streptomycin. CONCLUSIONS: The above proteomic and mutagenic analyses revealed that yggT is involved in streptomycin resistance in S. enterica.

Antimicrobial activity of copper surfaces against biofilm formation by Salmonella Enteritidis and its potential application in the poultry industry.

As a consequence of developing antimicrobial resistance to disinfectants, copper, which exhibits antimicrobial activity, has been studied as a possible alternative to the use of stainless steel surfaces. The aim was to evaluate the antimicrobial activity of copper surfaces in preventing biofilm formation by Salmonella Enteritidis and to determine their corrosive capacity. Strains of S. Enteritidis were incubated at 4 °C, 12 °C, and 25 °C with 1 cm2 coupons of electrolytic copper (99.9% Cu), brass (70% Cu), copper coated with tin, and stainless steel (control). A planktonic cell-suspension assay was used, followed by serial dilutions and bacterial counts. The corrosion test was performed with two disinfectants: benzalkonium chloride and sodium hypochlorite (100, 200, and 400 ppm). There was a significant reduction in biofilm production (log10 CFU cm-2) on the copper (2.64 at 4 °C, 4.20 at 12 °C, 4.56 at 25 °C) and brass (2.79 at 4 °C, 3.49 at 12 °C, 4.55 at 25 °C) surfaces compared to the control (5.68 at 4 °C, 5.89 at 12 °C, 6.01 at 25 °C). The antimicrobial surfaces showed uniform corrosion similar to that of surfaces generally used. These results demonstrated the effectiveness of copper surfaces in reducing S. Enteritidis and suggest they can be used as a complementary antimicrobial to control for this pathogen.

Antimicrobial activity of ClO2 gas against Salmonella Enteritidis on almonds.

Nuts, including almonds, are occasionally contaminated with Salmonella spp. In this study, we used chlorine dioxide (ClO2) gas to inactivate S. enterica subsp. Enterica serovar Enteritidis on almonds. Almonds inoculated with a single strain of S. Enteritidis (8.95 log cfu/mL) were exposed to ClO2 gas generated from 1.0 or 1.5 mL ClO2 solution in a sealed container at 50 or 60 °C (43% relative humidity) for up to 10 h. The concentration of ClO2 gas peaked at 354-510 and 750-786 ppm within 0.5 h upon deposition of 1.0 and 1.5 mL of aqueous ClO2, respectively, and gradually decreased thereafter. Population of S. Enteritidis on almonds treated at 50 °C decreased to 1.70-2.32 log cfu/sample within 1 h of exposure to ClO2 gas and decreased to below the detection limit (1.7 log cfu/sample) at all ClO2 concentrations after 8 h. At 60 °C, the microbial population fell below the detection limit within 1 h, regardless of the volume of ClO2 solution supplied. Microbial survival on almonds treated with ClO2 gas and stored at 12 or 25 °C was observed for up to 8 weeks and the organism was not recovered from the almonds treated for 10 h and stored at 12 °C for 2-8 weeks. The lightness (L value) and redness (a value) of almonds treated for 10 h were not changed by ClO2 gas treatment, but yellowness (b value) increased. Results showed that Salmonella on almonds was successfully inactivated by ClO2 gas treatment and the microbial survival did not occur during storage.

Inhibition of phage-resistant bacterial pathogen re-growth with the combined use of bacteriophages and EDTA.

The combined effects of ethylenediaminetetraacetic acid (EDTA) and bacteriophage (phage) treatment of foodborne pathogens were investigated. Although viable counts for Campylobacter jejuni decreased by 1.5 log after incubation for 8 h in the presence of phage PC10, re-growth was observed thereafter. The combination of phage PC10 and 1 mM EDTA significantly inhibited the re-growth of C. jejuni. The viable counts for C. jejuni decreased by 2.6 log (P < 0.05) compared with that of the initial count after 24 h. Moreover, EDTA at 0.67 or 1.3 mM, combined with the specific lytic phages, also effectively inhibited the re-growth of phage-resistant cells of Campylobacter coli, Salmonella enterica serovar Enteritidis, and Salmonella enterica serovar Typhimurium. In addition, the combined effects of lytic phages and EDTA were investigated on the viability of Campylobacter in BHI broth at low temperatures followed by the optimum growth temperature. The re-growth of C. coli was significantly inhibited by the coexistence of 1.3 mM EDTA, and the viable counts of surviving bacteria was about the same as the initial viable count after the incubation. This is the first study demonstrating the combined use of lytic phages and EDTA is effective in inhibiting the re-growth of phage-resistant bacteria in Gram-negative bacteria.

Host defense peptides identified in human apolipoprotein B as novel food biopreservatives and active coating components.

The effectiveness of three novel "host defence peptides" identified in human Apolipoprotein B (ApoB) as novel antimicrobial and antibiofilm agents to be employed in food industry is reported. ApoB-derived peptides have been found to exert significant antimicrobial effects towards Salmonella typhimurium ATCC® 14028 and Salmonella enteritidis 706 RIVM strains. Furthermore, they have been found to retain antimicrobial activity under experimental conditions selected to simulate those occurring during food storage, transportation and heat treatment, and have been found to be endowed with antibiofilm properties. Based on these findings, to evaluate the applicability of ApoB-derived peptides as food biopreservatives, coating solutions composed by chitosan (CH) and an ApoB-derived peptide have been prepared and found to be able to prevent Salmonella cells attachment to different kinds of surfaces employed in food industry. Finally, obtained coating solution has been demonstrated to hinder microbial proliferation in chicken meat samples. Altogether, obtained findings indicate that ApoB-derived peptides are promising candidates as novel biopreservatives for food packaging.

Label-Free Immunoassay for Multiplex Detections of Foodborne Bacteria in Chicken Carcass Rinse with Surface Plasmon Resonance Imaging.

The frequent outbreaks of foodborne pathogens have stimulated the demand of biosensors capable of rapid and multiplex detection of contaminated food. In this study, surface plasmon resonance imaging (SPRi) was used in simultaneous label-free detection of multiple foodborne pathogens, mainly Salmonella spp. and Shiga-toxin producing Escherichia coli (STEC), in commercial chicken carcass rinse. The antibodies were immobilized on the same SPRi sensor chip as a label-free immunoassay. Their immobilization concentrations were optimized to be ranging from 0.25 to 1.0 mg/mL, and independent of pH values. This label-free immunoassay achieved 106 colony-forming unit (CFU)/mL limit of detection for Salmonella, which was further improved to 1.0 CFU/mL with overnight bacteria enrichment. The injected samples with different bacteria, Salmonella Enteritidis, STEC, and Listeria monocytogenes, have been identified by the same biochip. Moreover, the SPRi signals revealed complex interference effects among coexisting bacteria species in heterogeneous bacteria solutions. This SPRi-based immunoassay demonstrates the great potential in high-throughput screening of multiple pathogenic bacteria coexisting in chicken carcass rinse. The reliability of antibody immobilization and cross-reactions of different antibodies on the same biochip are the major challenges of practical application of SPRi.

Novel Insight into the Effects of CpxR on Salmonella enteritidis Cells during the Chlorhexidine Treatment and Non-Stressful Growing Conditions.

The development and spread of antibiotics and biocides resistance is a significant global challenge. To find a solution for this emerging problem, the discovery of novel bacterial cellular targets and the critical pathways associated with antimicrobial resistance is needed. In the present study, we investigated the role of the two most critical envelope stress response regulators, RpoE and CpxR, on the physiology and susceptibility of growing Salmonella enterica serovar enteritidis cells using the polycationic antimicrobial agent, chlorhexidine (CHX). It was shown that deletion of the cpxR gene significantly increased the susceptibility of this organism, whereas deletion of the rpoE gene had no effect on the pathogen's susceptibility to this antiseptic. It has been shown that a lack of the CpxR regulator induces multifaceted stress responses not only in the envelope but also in the cytosol, further affecting the key biomolecules, including DNA, RNA, and proteins. We showed that alterations in cellular trafficking and most of the stress responses are associated with a dysfunctional CpxR regulator during exponential growth phase, indicating that these physiological changes are intrinsically associated with the lack of the CpxR regulator. In contrast, induction of type II toxin-antitoxin systems and decrease of abundances of enzymes and proteins associated with the recycling of muropeptides and resistance to polymixin and cationic antimicrobial peptides were specific responses of the ∆cpxR mutant to the CHX treatment. Overall, our study provides insight into the effects of CpxR on the physiology of S. Enteritidis cells during the exponential growth phase and CHX treatment, which may point to potential cellular targets for the development of an effective antimicrobial agent.

Reduction of Salmonella contamination on the surface of chicken skin using bacteriophage.

BACKGROUND: Enteric infections caused by Salmonella spp. remain a major public health burden worldwide. Chickens are known to be a major reservoir for this zoonotic pathogen. The presence of Salmonella in poultry farms and abattoirs is associated with financial costs of treatment and a serious risk to human health. The use of bacteriophages as a biocontrol is one possible intervention by which Salmonella colonization of chickens could be reduced. In a prior study, phages Eϕ151 and Tϕ7 significantly reduced broiler chicken caecal colonization by S. Enteritidis and S. Typhimurium respectively. METHODS: Salmonella-free Ross broiler chickens were orally infected with S. Enteritidis P125109 or S. Typhimurium 4/74. After 7 days of infection, the animals were euthanased, and 25cm2 sections of skin were collected. The skin samples were sprayed with a phage suspension of either Eϕ151 (S. Enteritidis), Tϕ7 phage suspension (S. Typhimurium) or SM buffer (Control). After incubation, the number of surviving Salmonellas was determined by direct plating and Most Probable Number (MPN). To determine the rate of reduction of Salmonella numbers on the skin surface, a bioluminescent S. Typhimurium DT104 strain was cultured, spread on sections of chicken breast skin, and after spraying with a Tϕ11 phage suspension, skin samples were monitored using photon counting for up to 24 h. RESULTS: The median levels of Salmonella reduction following phage treatment were 1.38 log10 MPN (Enteritidis) and 1.83 log10 MPN (Typhimurium) per skin section. Treatment reductions were significant when compared with Salmonella recovery from control skin sections treated with buffer (p < 0.0001). Additionally, significant reduction in light intensity was observed within 1 min of phage Tϕ11 spraying onto the skin contaminated with a bioluminescent Salmonella recombinant strain, compared with buffer-treated controls (p < 0.01), implying that some lysis of Salmonella was occurring on the skin surface. CONCLUSIONS: The results of this study suggest that phages may be used on the surface of chicken skin as biocontrol agents against Salmonella infected broiler chicken carcasses. The rate of bioluminescence reduction shown by the recombinant Salmonella strain used supported the hypothesis that at least some of the reduction observed was due to lysis occurred on the skin surface.

Changes in cellular structure of heat-treated Salmonella in low-moisture environments.

AIMS: Salmonella cells desiccated in an environment with low-water activity (aw ) show longer survival times and enhanced resistance to heat. However, little is known about the cellular ultrastructure of Salmonella in low-aw environment in relation to the survival and persistence during desiccation. MATERIALS AND RESULTS: In this study, Salmonella Enteritidis strain PT30 was dehydrated by exposure to air or by mixing with wheat flour (aw 0·30 at room temperature) for 7 days followed by heat treatment at 80°C for 10, 20, 60 min respectively. Transmission electron microscopy (TEM) was employed to examine and compare the ultrastructure of heat-treated S. Enteritidis cells after desiccation with the cells suspended in trypticase soy broth (TSB). Cells suspended in TSB broth showed disrupted ribosomes, congregated proteins and denatured DNA. However, no significant alterations were observed in the ultrastructure of the desiccated cells after heat treatment. The number of desiccated S. Enteritidis cells decreased by <1·5 log CFU per gram after 80°C treatment for 60 min, however, cells suspended in TSB declined more than 5 log10 CFU per mL at 80°C within 5 min. CONCLUSIONS: A drastic difference in the number of survivors and cellular ultrastructure was observed between vegetative and air or food-dried S. Enteritidis cells after subjecting to heat treatment at 80°C. No significant ultrastructure changes were observed in desiccated cells after heat treatment except for roughening and corrugating surfaces. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides a direct comparison to illustrate how desiccation influences the cell ultrastructure before/after heat treatment, which will aid in better understanding of the fundamental mechanism underlying the increased thermal resistance of Salmonella cells in low-aw environment.

Efficacy of oregano and rosemary essential oils to affect morphology and membrane functions of noncultivable sessile cells of Salmonella Enteritidis 86 in biofilms formed on stainless steel.

AIMS: This study evaluated the efficacy of essential oil from Origanum vulgare L. (oregano; OVEO) and Rosmarinus officinalis L. (rosemary; ROEO) to inactivate sessile cells of Salmonella enterica serovar Enteritidis 86 (SE86) in young and mature biofilms formed on stainless steel. METHODS AND RESULTS: Ultrastructural alterations and damage in different physiological functions caused by OVEO and ROEO in noncultivable sessile cells of SE86 were investigated using scanning electron microscopy and flow cytometry. OVEO (2·5 µl ml-1 ) and ROEO (40 µl ml-1 ) were effective to eradicate young and mature biofilms formed by SE86 sessile cells on stainless steel surfaces; however, the efficacy varied with exposure time. OVEO and ROEO caused alterations in morphology of SE86 sessile cells, inducing the occurrence of bubbles or spots on cell surface. OVEO and ROEO compromised membrane polarization, permeability and efflux activity in noncultivable SE86 sessile cells. These findings show that OVEO and ROEO act by a multitarget mechanism on SE86 membrane functions. CONCLUSIONS: ROEO and OVEO showed efficacy to eradicate SE86 sessile cells in preformed biofilms on stainless steel, displaying a time-dependent effect and multitarget action mode on bacterial cell membrane. SIGNIFICANCE AND IMPACT OF THE STUDY: The study provides for the first time the effects of OVEO and ROEO on morphology and physiological functions of noncultivable sessile cells of S. Enteritidis biofilms preformed on stainless steel surfaces.

Effect of sub-lethal chemical disinfection on the biofilm forming ability, resistance to antibiotics and expression of virulence genes of Salmonella Enteritidis biofilm-surviving cells.

Although disinfection procedures are widely implemented in food environments, bacteria can survive and present increased virulence/resistance. Since little is known about these phenomena regarding biofilms, this study aimed to investigate the effect of chemical disinfection on biofilm-derived cells of Salmonella Enteritidis. Using a reference strain (NCTC 13349) and a food isolate (350), biofilm susceptibility to benzalkonium chloride (BAC), sodium hypochlorite (SH) and hydrogen peroxide (HP) was evaluated and biofilms were exposed to sub-lethal concentrations of each disinfectant. Biofilm-derived cells were characterized for their biofilm forming ability, antibiotic resistance and expression of virulence-associated genes. Except for a few instances, disinfectant exposure did not alter antibiotic susceptibility. However, SH and HP exposure enhanced the biofilm forming ability of Salmonella Enteritidis NCTC 13349. After BAC and HP exposure, biofilm-derived cells presented a down-regulation of rpoS. Exposure to BAC also revealed an up-regulation of invA, avrA and csgD on Salmonella Enteritidis NCTC 13349. The results obtained suggest that biofilm-derived cells that survive disinfection may represent an increased health risk.

Antibacterial activity and action mode of chlorogenic acid against Salmonella Enteritidis, a foodborne pathogen in chilled fresh chicken.

The study evaluated the antibacterial activity of chlorogenic acid (CA) against Salmonella Enteritidis S1, a foodborne pathogen in chilled fresh chicken. Its minimum inhibitory concentration for S. Enteritidis S1 was 2 mM. 1 MIC CA treatment reduced the viable count of S. Enteritidis S1 by 3 log cfu/g in chilled fresh chicken. Scanning electron microscopy examination indicated that CA induced the cell envelope damage of S. Enteritidis S1. Following this, 1-N-Phenylnaphthylamine assay and LPS content analysis indicated that CA induced the permeability of outer membrane (OM). Confocal laser scanning microscopy examination further demonstrated that CA acted on the inner membrane (IM). To support this, the release of intracellular protein and ATP after CA treatment was also observed. CA also suppressed the activities of malate dehydrogenase and succinate dehydrogenase, two main metabolic enzymes in TCA cycle and electron transport chain. Thus, damage of intracelluar and outer membranes as well as disruption of cell metabolism resulted in cell death eventually. The finding suggested that CA has the potential to be developed as a preservative to control S. Enteritidis associated foodborne diseases.

Salmonella Pathogenicity Island 1 Is Expressed in the Chicken Intestine and Promotes Bacterial Proliferation.

Salmonella enterica serovar Enteritidis is a common cause of foodborne illness in the United States. The bacterium can be transmitted to humans via contaminated chicken meat and eggs, and virulence in humans requires type III secretion system 1 (TTSS-1), encoded on Salmonella pathogenicity island 1 (SPI-1). Chickens often carry S Enteritidis subclinically, obscuring the role of SPI-1 in facilitating bacterial colonization. To evaluate the role of SPI-1 in the infection of chicks by Salmonella, we created and utilized strains harboring a stable fluorescent reporter fusion designed to quantify SPI-1 expression within the intestinal tracts of animals. Using mutants unable to express TTSS-1, we demonstrated the important role of the secretion system in facilitating bacterial colonization. We further showed that coinoculation of an SPI-1 mutant with the wild-type strain increased the number of mutant organisms in intestinal tissue and contents, suggesting that the wild type rescues the mutant. Our results support the hypothesis that SPI-1 facilitates S Enteritidis colonization of the chicken and make SPI-1 an attractive target in preventing Salmonella carriage and colonization in chickens to reduce contamination of poultry meat and eggs by this foodborne pathogen.

Influence of catecholamines on biofilm formation by Salmonella Enteritidis.

Salmonella spp. are the main pathogens responsible for foodborne disease worldwide. Bacterial communities use the quorum sensing system to control biofilm formation. These systems function through the secretion of substances, called auto-inducers (AI), into the environment. AI-3 is structurally similar to epinephrine (EPI) and norepinephrine (NOR) -catecholamines secreted by eukaryotic cells to communicate with each other. In this context, this work aimed to evaluate the effect of EPI and NOR on biofilm formation by S. Enteritidis at 12 °C and 25 °C. Also, we detected the presence of the csgD, adrA, and fimA genes in these strains. Biofilm formation was investigated at two temperatures (12 °C and 25 °C) using a microtiter plate assay, under four different treatments (50 mM EPI, 100 mM EPI, 50 mM NOR; 100 mM NOR) and a control group. PCR was used to detect the virulence genes associated with biofilm production. A greater number of biofilm producer isolates were observed at 25 °C than at 12 °C, regardless of the treatment. The number of biofilms forming strains at 12 °C was significantly higher in the treatment with norepinephrine at 100 µM. The proportion of non-producer and biofilm producer strains at 25 °C did not differ significantly among the treatments. All strains presented the three genes (csgD, adrA, and fimA). The approach carried out in this work is a precursor in veterinary medicine, focusing on both public and poultry health, and evaluates the influence of catecholamines on the formation of biofilms with S. Enteritidis, an important pathogen with zoonotic potential. Norepinephrine seems to be more efficient at stimulating biofilm formation by S. Enteritidis strains at 12 °C. csgD, fimA, and adrA were detected in all strains.