Interference of gastrointestinal barriers with antibiotic susceptibility of foodborne pathogens: an in vitro case study of ciprofloxacin and tetracycline against Salmonella enterica and Listeria monocytogenes.

Minimum inhibitory concentrations (MIC) assays are often questioned for their representativeness. Especially when foodborne pathogens are tested, it is of crucial importance to also consider parameters of the human digestive system. Hence, the current study aimed to assess the inhibitory capacity of two antibiotics, ciprofloxacin and tetracycline, against Salmonella enterica and Listeria monocytogenes, under representative environmental conditions. More specifically, aspects of the harsh environment of the human gastrointestinal tract (GIT) were gradually added to the experimental conditions starting from simple aerobic lab conditions into an in vitro simulation of the GIT. In this way, the effects of parameters including the anoxic environment, physicochemical conditions of the GIT (low gastric pH, digestive enzymes, bile acids) and the gut microbiota were evaluated. The latter was simulated by including a representative consortium of selected gut bacteria species. In this study, the MIC of the two antibiotics against the relevant foodborne pathogens were established, under the previously mentioned environmental conditions. The results of S. enterica highlighted the importance of the anaerobic environment when conducting such studies, since the pathogen thrived under such conditions. Inclusion of physicochemical barriers led to exactly opposite results for S. enterica and L. monocytogenes since the former became more susceptible to ciprofloxacin while the latter showed lower susceptibility towards tetracycline. Finally, the inclusion of gut bacteria had a bactericidal effect against L. monocytogenes even in the absence of antibiotics, while gut bacteria protected S. enterica from the effect of ciprofloxacin.

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.

Simultaneous growth, survival and death: The trimodal behavior of Salmonella cells under osmotic stress giving rise to "Phoenix phenomenon".

Time-lapse microscopy methods were used to monitor growth, survival and death of Salmonella enterica serotype Agona individual cells on solid laboratory medium (tryptone soy agar) in the presence of various salt concentrations (0.5%, 3.5%, 4.5% and 5.7% NaCl). The results showed a highly heterogeneous behavior. As NaCl concentration increased, the distribution of the first division time was shifted to higher values and became wider. The mean first division time increased from 1.8 h at 0.5% NaCl to 5.48 h, 16.2 h, and 35.9 h at 3.5%, 4.5% and 5.7% NaCl, respectively. The concentration of NaCl in the growth medium also affected the ability of the cells to divide. The percentage of cells able to grow decreased from 88.9% at 0.5% NaCl to 66.5%, 32.8%, and 6.9% at 3.5%, 4.5% and 5.7% NaCl, respectively. In the latter case (5.7% NaCl), 74 cells out of 406 cells tested (18%) died with mean time to death 5.03 h and standard deviation 6.70 h. To investigate the effect of the behavior of individual cells on the dynamics of the whole population, simulation analysis was used. The simulation results showed that the simultaneous growth, survival and death of cells observed under osmotic stress can lead to a total population behavior known as the "Phoenix" phenomenon. The simulation findings were confirmed by validation experiments using both viable counts and time lapse microscopy. The results of the present study show the high heterogeneity of individual cell responses and the complexity in the behavior of microbial populations at conditions approaching the boundaries of growth.