Gut microbiota of the small intestine as an antimicrobial barrier against foodborne pathogens: Impact of diet on the survival of S. Typhimurium and L. monocytogenes during in vitro digestion.

The human gastrointestinal tract employs an assortment of chemical, enzymatic and immune barriers to impede pathogen colonization. An essential component of these barriers is the gut microbiota, which infers protection against ingested pathogens through its colonization resistance mechanisms. Specifically, the gut microbiota of the distal small intestine (ileum) renders a crucial line of defense, given that this location is regarded as an important interaction site. This study aimed to evaluate the impact of the ileal microbiota on the survival of the foodborne pathogens Salmonella enterica serotype Typhimurium and Listeria monocytogenes, utilizing an in vitro digestion model system. Moreover, the effect of diet on the gut microbiota colonization resistance mechanisms was assessed, by comparing a healthy (high fiber/low sugar) and a western diet (low fiber/high sugar). For S. Typhimurium, the results revealed that the digestion of a healthy diet led to a similar inactivation compared to the western diet, with the values of total log reduction being 0.83 and 0.82 log(CFU), respectively; yet the lack of readily accessible nutrients in the healthy diet combined with the acidic shock during gastric digestion caused the induction of stress tolerance to the pathogen. This resulted in increased pathogen survival in the presence of gut microbiota, with S. Typhimurium proliferating during the ileal phase with a maximum specific growth rate of 0.16 1/h. On the contrary, for L. monocytogenes, the healthy diet was associated with a greater inactivation than the western diet (total log reduction values: 3.08 and 1.30 log(CFU), respectively), which appeared strongly influenced by the encounter of the pathogen with the gut microbiota. Regarding the latter, the species Escherichia coli and Bacteroides thetaiotaomicron appeared to be the most prevalent in most cases. Finally, it was also demonstrated that the ileal microbiota colonization resistance mechanisms largely relied on competitive responses. The obtained knowledge of this research can contribute to the development and/or complementation of defensive strategies against pathogen infection, while also underlining the value of in vitro approaches.

Influence of Hurdle Technology on Foodborne Pathogen Survival in the Human Gastrointestinal Tract.

The application of several sublethal stresses in hurdle technology can exert microbial stress resistance, which, in turn, might enable foodborne pathogens to overcome other types of lethal stresses, such as the gastrointestinal barriers. The present study evaluated the survival of Salmonella Typhimurium and Listeria monocytogenes during simulated digestion, following exposure to combinations of water activity (aw), pH and storage temperature stresses. The results revealed that both pathogens survived their passage through the simulated gastrointestinal tract (GIT) with their previous habituation to certain hurdle combinations inducing stress tolerance. More specifically, the habituation to a low temperature or to a high pH resulted in the increased stress tolerance of Salmonella, while for Listeria, the cells appeared stress tolerant after exposure to a high temperature or to a low pH. Nonetheless, both pathogens expressed increased sensitivity after habituation to growth-limiting hurdle combinations. The survival of stress-tolerant pathogenic cells in the human GIT poses major public health issues, since it can lead to host infection. Consequently, further research is required to obtain a deeper understanding of the adaptive stress responses of foodborne bacteria after exposure to combinations of sublethal hurdles to improve the existing food safety systems.

Effect of food structure and buffering capacity on pathogen survival during in vitro digestion.

Even though a plethora of barriers are employed by the human gastrointestinal tract (GIT) to cope with invading pathogens, foodborne diseases are still a common problem. The survival of food pathogens in the GIT is known to depend on food carrier properties. The aim of this study was to investigate the influence of food buffering capacity and food structure on the survival of Salmonella Typhimurium and Listeria monocytogenes during simulated digestion, following contamination of different food model systems that had different combinations of fat and protein content. The results illustrated the strong protective properties of proteins, acting either as a strong buffering agent or as a physical barrier against gastric acidity, for both pathogens. In comparison, fat manifested a lower buffering capacity and weaker protective effects against the two pathogens. Intriguingly, a low fat content was often linked with increased microbial resistance. Nonetheless, both pathogens survived their transit through the simulated GIT in all cases, with S. Typhimurium exhibiting growth during intestinal digestion and L.monocytogenes demonstrating a healthy residual population at the end of the intestinal phase. These results corroborate the need for a deeper understanding regarding the mechanisms with which food affects bacterial survival in the human GIT.

A protocol for the cultivation and monitoring of ileal gut microbiota surrogates.

AIMS: This research aimed to develop and validate a cultivation and monitoring protocol that is suitable for a surrogate microbial community that accounts for the gut microbiota of the ileum of the small intestine. METHODS AND RESULTS: Five bacterial species have been selected as representatives of the ileal gut microbiota and a general anaerobic medium (MS-BHI, as minimally supplemented brain heart infusion) has been constructed and validated against BCCM/LGM recommended and commercial media. Moreover, appropriate selective/differential media have been investigated for monitoring each ileal gut microbiota surrogate. Results showed that MS-BHI was highly efficient in displaying individual and collective behaviour of the ileal gut microbiota species, when compared with other types of media. Likewise, the selective/differential media managed to identify and describe the behaviour of their targeted species. CONCLUSIONS: MS-BHI renders a highly efficient, inexpensive and easy-to-prepare cultivation and enumeration alternative for the surrogate ileal microbiota species. Additionally, the selective/differential media can identify and quantify the bacteria of the surrogate ileal microbial community. SIGNIFICANCE AND IMPACT OF STUDY: The selected gut microbiota species can represent an in vitro ileal community, forming the basis for future studies on small intestinal microbiota. MS-BHI and the proposed monitoring protocol can be used as a standard for gut microbiota studies that utilize conventional microbiological techniques.

Behavior of the Surviving Population of Listeria monocytogenes and Salmonella Typhimurium Biofilms Following a Direct Helium-Based Cold Atmospheric Plasma Treatment.

Although the Cold Atmospheric Plasma (CAP) technology proved promising for inactivation of biofilms present on abiotic food contact surfaces, more research is required to examine the behavior of the CAP surviving biofilm-associated cells. It was therefore examined whether (i) CAP treated (Listeria monocytogenes and Salmonella Typhimurium) biofilm-associated cells were able to further colonize the already established biofilms during a subsequent incubation period and (ii) isolates of the surviving population became less susceptible toward CAP when the number of biofilm development-CAP treatment cycles increased. For this purpose, a direct treatment was applied using a helium-based Dielectric Barrier Discharge electrode configuration. Results indicated that the surviving population was able to further colonize the already established biofilms, since the cell density of the CAP treated + incubated biofilms equaled the initial density of the untreated biofilms. For the L. monocytogenes biofilms, also the total biomass proved to further increase, which might result in an even further increased resistance. The susceptibility of the biofilm-associated cells proved to be influenced by the specific number of CAP treatment cycles, which might potentially result in an overestimation of the CAP treatment efficacy and, consequently, an increased risk of food contamination.

The effect of ultrasound treatment in combination with nisin on the inactivation of Listeria innocua and Escherichia coli.

Ultrasound, alone or in combination with natural antimicrobials, is a novel food processing technology of interest to replace traditional food decontamination methods, as it is milder than classical sterilisation (heat treatment) and maintains desirable sensory characteristics. However, ultrasound efficacy can be affected by food structure/composition, as well as the order in which combined treatments are applied. More specifically, treatments which target different cell components could result in enhanced inactivation if applied in the appropriate order. The microbial properties i.e. Gram positive/Gram negative can also impact the treatment efficacy. This work presents a systematic study of the combined effect of ultrasound and nisin on the inactivation of the bacteria Listeria innocua (Gram positive) and Escherichia coli (Gram negative), at a range of cavitation conditions (44, 500, 1000 kHz). The order of treatment application was varied, and the impact of system structure was also investigated by varying the concentration of Xanthan gum used to create the food model systems (0 - 0.5% w/v). Microbial inactivation kinetics were monitored, and advanced microscopy and flow cytometry techniques were utilised to quantify the impact of treatment on a cellular level. Ultrasound was shown to be effective against E. coli at 500 kHz only, with L. innocua demonstrating resistance to all frequencies studied. Enhanced inactivation of E. coli was observed for the combination of nisin and ultrasound at 500 kHz, but only when nisin was applied before ultrasound treatment. The system structure negatively impacted the inactivation efficacy. The combined effect of ultrasound and nisin on E. coli was attributed to short-lived destabilisation of the outer membrane as a result of sonication, allowing nisin to penetrate the cytoplasmic membrane and facilitate cell inactivation.

A Population Balance Model to Describe the Evolution of Sublethal Injury.

The detection and quantification of sublethal injury (SI) of pathogenic microorganisms has become a common procedure when assessing the efficiency of microbial inactivation treatments. However, while a plethora of studies investigates SI in function of time, no suitable modelling procedure for SI data has been proposed thus far. In this study, a new SI model structure was developed that relies on existing microbial inactivation models. This model is based on the description of inactivation kinetics between the subpopulations of healthy, sublethally injured and dead cells. The model was validated by means of case studies on previously published results, modelled by different inactivation models, i.e., (i) log-linear inactivation; (ii) biphasic inactivation; and (iii) log-linear inactivation with tailing. Results were compared to those obtained by the traditional method that relies on calculating SI from independent inactivation models on non-selective and selective media. The log-linear inactivation case study demonstrated that the SI model is equivalent to the use of independent models when there can be no mistake in calculating SI. The biphasic inactivation case study illustrated how the SI model avoids unrealistic calculations of SI that would otherwise occur. The final case study on log-linear inactivation with tailing clarified that the SI model provides a more mechanistic description than the independent models, in this case allowing the reduction of the number of model parameters. As such, this paper provides a comprehensive overview of the potential and applications for the newly presented SI model.

The impact of food model system structure on the inactivation of Listeria innocua by cold atmospheric plasma and nisin combined treatments.

Novel processing methods such as cold atmospheric plasma (CAP) and natural antimicrobials like nisin, are of interest to replace traditional food decontamination approaches as, due to their mild nature, they can maintain desirable food characteristics, i.e., taste, texture, and nutritional content. However, the microbial growth characteristics (planktonic growth/surface colonies) and/or the food structure itself (liquid/solid surface) can impact the inactivation efficacy of these novel processing methods. More specifically, cells grown as colonies on a solid(like) surface experience a completely different growth environment to cells grown planktonically in liquid, and thus could display a different response to novel processing treatments through stress adaptation and/or cross protection mechanisms. The order in which combined treatments are applied could also impact their efficacy, especially if the mechanisms of action are complementary. This work presents a fundamental study on the efficacy of CAP and nisin, alone and combined, as affected by food system structure. More specifically, Listeria innocua was grown planktonically (liquid broth) or on a viscoelastic Xanthan gum gel system (1.5% w/v) and treated with CAP, nisin, or a combination of the two. Both the inactivation system, i.e., liquid versus solid(like) surface and the growth characteristics, i.e., planktonic versus colony growth, were shown to impact the treatment efficacy. The combination of nisin and CAP was more effective than individual treatments, but only when nisin was applied before the CAP treatment. This study provides insight into the environmental stress response/adaptation of L. innocua grown on structured systems in response to natural antimicrobials and novel processing technologies, and is a step towards the faster delivery of these food decontamination methods from the bench to the food industry.

Visible Light as an Antimicrobial Strategy for Inactivation of Pseudomonas fluorescens and Staphylococcus epidermidis Biofilms.

The increase of antimicrobial resistance is challenging the scientific community to find solutions to eradicate bacteria, specifically biofilms. Light-Emitting Diodes (LED) represent an alternative way to tackle this problem in the presence of endogenous or exogenous photosensitizers. This work adds to a growing body of research on photodynamic inactivation using visible light against biofilms. Violet (400 nm), blue (420 nm), green (570 nm), yellow (584 nm) and red (698 nm) LEDs were used against Pseudomonas fluorescens and Staphylococcus epidermidis. Biofilms, grown on a polystyrene surface, were irradiated for 4 h. Different irradiance levels were investigated (2.5%, 25%, 50% and 100% of the maximum irradiance). Surviving cells were quantified and the inactivation kinetic parameters were estimated. Violet light could successfully inactivate P. fluorescens and S. epidermidis (up to 6.80 and 3.69 log10 reduction, respectively), while blue light was effective only against P. fluorescens (100% of maximum irradiance). Green, yellow and red irradiation neither increased nor reduced the biofilm cell density. This is the first research to test five different wavelengths (each with three intensities) in the visible spectrum against Gram-positive and Gram-negative biofilms. It provides a detailed study of the potential of visible light against biofilms of a different Gram-nature.

Inactivation of L. monocytogenes and S. typhimurium Biofilms by Means of an Air-Based Cold Atmospheric Plasma (CAP) System.

Previous (biofilm) inactivation studies using Cold Atmospheric Plasma (CAP) focused on helium (with or without the addition of oxygen) as feeding gas since this proved to result in a stable and uniform plasma. In industry, the use of helium gas is expensive and unsafe for employees. Ambient air is a possible substitute, provided that similar inactivation efficacies can be obtained. In this research, 1 and 7 day-old (single/dual-species) model biofilms containing L. monocytogenes and/or S. typhimurium cells were treated with an air-based Surface Barrier Discharge (SBD) plasma set-up for treatment times between 0 and 30 min. Afterwards, cell densities were quantified via viable plate counts, and predictive models were applied to determine the inactivation kinetics and the efficacy. Finally, the results were compared to previously obtained results using a helium-based SBD and DBD (Dielectric Barrier Discharge) system. This study has demonstrated that the efficacy of the air-based CAP treatment depended on the biofilm and population type, with log-reductions ranging between 1.5 and 2.5 log10(CFU/cm2). The inactivation efficacy was not significantly influenced by the working gas, although the values were generally higher for the air-based system. Finally, this study has demonstrated that the electrode configuration was more important than the working gas composition, with the DBD electrode being the most efficient.

Dual-Species Model Biofilm Consisting of Listeria monocytogenes and Salmonella Typhimurium: Development and Inactivation With Cold Atmospheric Plasma (CAP).

Most environmental biofilms contain a variety of species. These species can establish cooperative and competitive interactions, possibly resulting in an increase or a decrease in antimicrobial resistance. Therefore, results obtained following inactivation of single-species biofilms by means of different technologies (e.g., Cold Atmospheric Plasma, CAP) should be validated for multi-species biofilms. First, a strongly adherent and mature Listeria monocytogenes and S. Typhimurium dual-species biofilm was developed by altering different incubation conditions, i.e., growth medium, incubation temperature, inoculum ratio of L. monocytogenes and S. Typhimurium cells, and incubation time. Adherence and maturity were quantified by means of optical density measurements and viable plate counts, respectively. Secondly, both the (1 day old) reference biofilm and a more mature 7 days old biofilm were treated for different CAP treatment times (0-30 min). Viable plate counts were again used to determine the (remaining) cell density. For both the biofilm development and inactivation, predictive models were applied to describe the growth/inactivation kinetics. Finally, the kinetics of the [1 and 7 day(s) old] dual-species biofilms were compared with those obtained for the corresponding single-species biofilms. Results implied that a strongly adherent and mature reference dual-species biofilm was obtained following 24 h of incubation at 25°C using 20-fold diluted TSB and an inoculum ratio of 1:1. Main observations regarding CAP inactivation were: (i) the dual-species biofilm age had no influence on the CAP efficacy, although a longer treatment time was required for the oldest biofilm, (ii) for the 1 day old biofilms, CAP treatment became less efficient for S. Typhimurium inactivation when this species was part of the dual-species biofilm, while L. monocytogenes inactivation was not influenced by the biofilm type, and (iii) for the 7 days old biofilms, CAP inactivation of both species became more efficient when they were part of the dual-species biofilms. It can be concluded that the efficacy of the CAP treatment is altered when cells become part of a dual-species biofilm, which is quite important with respect to a possible application of CAP for biofilm inactivation within the food industry.

Combined Effect of Cold Atmospheric Plasma and Hydrogen Peroxide Treatment on Mature Listeria monocytogenes and Salmonella Typhimurium Biofilms.

Cold Atmospheric Plasma (CAP) is a promising novel method for biofilm inactivation as log-reduction values up to 4.0 log10 (CFU/cm2) have been reported. Nevertheless, as the efficacy of CAP itself is not sufficient for complete inactivation of mature biofilms, the hurdle technology could be applied in order to obtain higher combined efficacies. In this study, CAP treatment was combined with a mild hydrogen peroxide (H2O2) treatment for disinfection of 1 and 7 day(s) old Listeria monocytogenes and Salmonella Typhimurium biofilms. Three different treatment sequences were investigated in order to determine the most effective treatment sequence, i.e., (i) first CAP, then H2O2, (ii) first H2O2, then CAP, and (iii) a simultaneous treatment of CAP and H2O2. Removal of the biofilm, induction of sub-lethal injury, and H2O2 breakdown due to the presence of catalase within the biofilms were investigated in order to comment on their possible contribution to the combined inactivation efficacy. Results indicated that the preferred treatment sequence was dependent on the biofilm forming species, biofilm age, and applied H2O2 concentration [0.05 or 0.20% (v/v)]. At the lowest H2O2 concentration, the highest log-reductions were generally observed if the CAP treatment was preceded by the H2O2 treatment, while at the highest H2O2 concentration, the opposite sequence (first CAP, then H2O2) proved to be more effective. Induction of sub-lethal injury contributed to the combined bactericidal effect, while the presence of catalase within the biofilms resulted in an increased resistance. In addition, high log-reductions were partially the result of biofilm removal. The highest overall log-reductions [i.e., up to 5.42 ± 0.33 log10 (CFU/cm2)] were obtained at the highest H2O2 concentration if CAP treatment was followed by H2O2 treatment. As this resulted in almost complete inactivation of the L. monocytogenes and S. Typhimurium biofilms, the combined treatment of CAP and H2O2 proved to be a promising method for disinfection of abiotic surfaces.

Inactivation of Single Strains of Listeria monocytogenes and Salmonella Typhimurium Planktonic Cells Biofilms With Plasma Activated Liquids.

Recent research has proven the ability of cold atmospheric plasma (CAP) for assuring food safety. A more flexible and transportable alternative is the use of plasma activated liquids (PAL), which are also known to have antimicrobial properties. However, within the context of food safety, little is known on its potential regarding decontamination. This research therefore focusses on identifying the impact of (i) the microbial species and its cell type (planktonic cells or biofilms), (ii) the CAP settings (i.e., gas composition and generation time) and (iii) PAL related factors (treatment time and PAL age) on the technologies efficacy. Cell densities were monitored using the plate counting technique for which the results were analyzed by means of predictive inactivation models. Moreover, the pH and the concentrations of long-lived species (i.e., hydrogen peroxide, nitrite, and nitrate) were measured to characterize the PAL solutions. The results indicated that although the type of pathogen impacted the efficacy of the treatment, mainly the cell mode had an important effect. The presence of oxygen in the operating gas ensured the generation of PAL solutions with a higher antimicrobial activity. Moreover, to ensure a good microbial inactivation, PAL generation times needed to be sufficiently long. Both CAP related factors resulted in a higher amount of long-lived species, enhancing the inactivation. For 30 min. PAL generation using O2, this resulted in log reductions up to 3.9 for biofilms or 5.8 for planktonic cells. However, loss of the PAL activity for stored solutions, together with the frequent appearance of a tailing phase in the inactivation kinetics, hinted at the importance of the short-lived species generated. Different factors, related to (i) the pathogen and its cell mode, (ii) the CAP settings and (iii) PAL related factors, proved to impact the antimicrobial efficacy of the solutions and should be considered with respect to future applications of the PAL technology.

Effect of cell immobilization on the growth dynamics of Salmonella Typhimurium and Escherichia coli at suboptimal temperatures.

Predictive microbiology has recently acknowledged the impact of the solid(like) food structure on microbial behavior. The presence of this solid(like) structure causes microorganisms to grow as colonies and no longer planktonically as in liquid. In this paper, the growth dynamics of Salmonella Typhimurium and Escherichia coli were studied as a function of temperature, considering different growth morphologies, i.e., (i) planktonic cells, (ii) immersed colonies and (iii) surface colonies. For all three growth morphologies, both microorganisms were grown in petri dishes. While E. coli was grown under optimal pH and water activity (aw), for S. Typhimurium pH and aw were adapted to 5.5 and 0.990. In order to mimic a solid(like) environment, 5% (w/v) gelatin was added. All petri dishes were incubated under static conditions at temperatures in the range [8.0°C-22.0°C]. Cell density was determined via viable plate counting. This work demonstrates that the growth morphology (planktonic vs. colony) has a negligible effect on the growth dynamics as a function of temperature. The observation of almost equal growth rates for planktonic cultures and colonies is in contrast to literature where, mostly, a difference is observed, i.e., µplanktonic cells≥µimmersed colonies≥µsurface colonies. This difference might be due to shaking of the liquid culture in these studies, which results in a nutrient and oxygen rich environment, in contrast to the diffusion-limited gel system. Experiments also indicate that lag phases for solid(like) systems are similar to those for the planktonic cultures, as can be found in literature for similar growth conditions. Considering the maximum cell density, no clear trend was deducted for either of the microorganisms. This study indicates that the growth parameters in the suboptimal temperature range do not depend on the growth morphology. For the considered experimental conditions, models previously developed for liquid environments can be used for solid(like) systems.

Tissue specific analysis reveals a differential organization and regulation of both ethylene biosynthesis and E8 during climacteric ripening of tomato.

BACKGROUND: Solanum lycopersicum or tomato is extensively studied with respect to the ethylene metabolism during climacteric ripening, focusing almost exclusively on fruit pericarp. In this work the ethylene biosynthesis pathway was examined in all major tomato fruit tissues: pericarp, septa, columella, placenta, locular gel and seeds. The tissue specific ethylene production rate was measured throughout fruit development, climacteric ripening and postharvest storage. All ethylene intermediate metabolites (1-aminocyclopropane-1-carboxylic acid (ACC), malonyl-ACC (MACC) and S-adenosyl-L-methionine (SAM)) and enzyme activities (ACC-oxidase (ACO) and ACC-synthase (ACS)) were assessed. RESULTS: All tissues showed a similar climacteric pattern in ethylene productions, but with a different amplitude. Profound differences were found between tissue types at the metabolic and enzymatic level. The pericarp tissue produced the highest amount of ethylene, but showed only a low ACC content and limited ACS activity, while the locular gel accumulated a lot of ACC, MACC and SAM and showed only limited ACO and ACS activity. Central tissues (septa, columella and placenta) showed a strong accumulation of ACC and MACC. These differences indicate that the ethylene biosynthesis pathway is organized and regulated in a tissue specific way. The possible role of inter- and intra-tissue transport is discussed to explain these discrepancies. Furthermore, the antagonistic relation between ACO and E8, an ethylene biosynthesis inhibiting protein, was shown to be tissue specific and developmentally regulated. In addition, ethylene inhibition by E8 is not achieved by a direct interaction between ACO and E8, as previously suggested in literature. CONCLUSIONS: The Ethylene biosynthesis pathway and E8 show a tissue specific and developmental differentiation throughout tomato fruit development and ripening.