Salmonella Persist in Activated Macrophages in T Cell-Sparse Granulomas but Are Contained by Surrounding CXCR3 Ligand-Positioned Th1 Cells.

Salmonella enterica (Se) bacteria cause persistent intracellular infections while stimulating a robust interferon-γ-producing CD4+ T (Th1) cell response. We addressed this paradox of concomitant infection and immunity by tracking fluorescent Se organisms in mice. Se bacteria persisted in nitric oxide synthase (iNOS)-producing resident and recruited macrophages while inducing genes related to protection from nitric oxide. Se-infected cells occupied iNOS+ splenic granulomas that excluded T cells but were surrounded by mononuclear phagocytes producing the chemokines CXCL9 and CXCL10, and Se epitope-specific Th1 cells expressing CXCR3, the receptor for these chemokines. Blockade of CXCR3 inhibited Th1 occupancy of CXCL9/10-dense regions, reduced activation of the Th1 cells, and led to increased Se growth. Thus, intracellular Se bacteria survive in their hosts by counteracting toxic products of the innate immune response and by residing in T cell-sparse granulomas, away from abundant Th1 cells positioned via CXCR3 in a bordering region that act to limit infection.

Time of arrival during plant disease progression and humidity additively influence Salmonella enterica colonization of lettuce.

The interplay between plant hosts, phytopathogenic bacteria, and enteric human pathogens in the phyllosphere has consequences for human health. Salmonella enterica has been known to take advantage of phytobacterial infection to increase its success on plants, but there is little knowledge of additional factors that may influence the relationship between enteric pathogens and plant disease. In this study, we investigated the role of humidity and the extent of plant disease progression on S. enterica colonization of plants. We found that high humidity was necessary for the replication of S. enterica on diseased lettuce, but not required for S. enterica ingress into the UV-protected apoplast. Additionally, the Xanthomonas hortorum pv. vitians (hereafter, X. vitians)-infected lettuce host was found to be a relatively hostile environment for S. enterica when it arrived prior to the development of watersoaking or following necrosis onset, supporting the existence of an ideal window during X. vitians infection progress that maximizes S. enterica survival. In vitro growth studies in sucrose media suggest that X. vitians may allow S. enterica to benefit from cross-feeding during plant infection. Overall, this study emphasizes the role of phytobacterial disease as a driver of S. enterica success in the phyllosphere, demonstrates how the time of arrival during disease progress can influence S. enterica's fate in the apoplast, and highlights the potential for humidity to transform an infected apoplast into a growth-promoting environment for bacterial colonizers. IMPORTANCE: Bacterial leaf spot of lettuce caused by Xanthomonas hortorum pv. vitians is a common threat to leafy green production. The global impact caused by phytopathogens, including X. vitians, is likely to increase with climate change. We found that even under a scenario where increased humidity did not enhance plant disease, high humidity had a substantial effect on facilitating Salmonella enterica growth on Xanthomonas-infected plants. High humidity climates may directly contribute to the survival of human enteric pathogens in crop fields or indirectly affect bacterial survival via changes to the phyllosphere brought on by phytopathogen disease.

Phenotypic and molecular characterisation of Salmonella spp. isolates in healthy poultry.

1. Epidemiological surveillance of Salmonella spp. serves as a primary tool for maintaining the health of poultry flocks. Characterising circulating serotypes is crucial for implementing control and prevention measures. This study conducted phenotypic and molecular characterisation of S. enterica Pullorum, S. enterica Heidelberg, and S. enterica Corvalis isolated from broiler chickens during slaughtering.2. All strains were susceptible to gentamicin, neomycin and norfloxacin. However, resistance rates exceeded 50% for ciprofloxacin and tiamulin, irrespective of the serotype. Approximately 64% of strains were classified as multidrug-resistant, with S. enterica Heidelberg strains exhibiting significantly higher overall resistance. The isolates demonstrated the ability to adhere and produce biofilm at a minimum of three temperatures, with S. enterica Pullorum capable of biofilm production at all temperatures encountered during poultry rearing.3. Each strain possessed between two and seven different virulence-associated genes. Genetic similarity, as indicated by pulsed field gel electrophoresis, exceeded 90% for all three serotypes and strains were classified in the R5 ribotype by PCR, regardless of serotype. Sequencing revealed high similarity among all strains, with homology ranging from 99.61 to 100% and all were classified to a single cluster.4. The results suggested a clonal relationship among the strains, indicating the possible circulation of a unique clonal group of S. enterica Pullorum in the southern region of Brazil.

Methods for Fixing Biofilms of Staphylococcus aureus and Salmonella enterica for Microscopic Examination.

Different methods for fixing biofilms of Staphylococcus aureus and Salmonella enterica for light and electron microscopy were compared. Paraformaldehyde fixation did not preserve biofilm integrity during dehydration; Ito-Karnovsky fixation revealed cell morphology, but did not preserve the matrix. Ruthenium red combined with aldehydes allowed the matrix to be preserved and visualized. An analysis of the ultrastructure of S. aureus and S. enterica cells in biofilms and suspensions at various fixations is presented. The ultrastructure of the biofilm matrix has been described.

Intracellular niche-specific profiling reveals transcriptional adaptations required for the cytosolic lifestyle of Salmonella enterica.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a zoonotic pathogen that causes diarrheal disease in humans and animals. During salmonellosis, S. Typhimurium colonizes epithelial cells lining the gastrointestinal tract. S. Typhimurium has an unusual lifestyle in epithelial cells that begins within an endocytic-derived Salmonella-containing vacuole (SCV), followed by escape into the cytosol, epithelial cell lysis and bacterial release. The cytosol is a more permissive environment than the SCV and supports rapid bacterial growth. The physicochemical conditions encountered by S. Typhimurium within the epithelial cytosol, and the bacterial genes required for cytosolic colonization, remain largely unknown. Here we have exploited the parallel colonization strategies of S. Typhimurium in epithelial cells to decipher the two niche-specific bacterial virulence programs. By combining a population-based RNA-seq approach with single-cell microscopic analysis, we identified bacterial genes with cytosol-induced or vacuole-induced expression signatures. Using these genes as environmental biosensors, we defined that Salmonella is exposed to oxidative stress and iron and manganese deprivation in the cytosol and zinc and magnesium deprivation in the SCV. Furthermore, iron availability was critical for optimal S. Typhimurium replication in the cytosol, as well as entC, fepB, soxS, mntH and sitA. Virulence genes that are typically associated with extracellular bacteria, namely Salmonella pathogenicity island 1 (SPI1) and SPI4, showed increased expression in the cytosol compared to vacuole. Our study reveals that the cytosolic and vacuolar S. Typhimurium virulence gene programs are unique to, and tailored for, residence within distinct intracellular compartments. This archetypical vacuole-adapted pathogen therefore requires extensive transcriptional reprogramming to successfully colonize the mammalian cytosol.

A kinase-independent function of PAK is crucial for pathogen-mediated actin remodelling.

The p21-activated kinase (PAK) family regulate a multitude of cellular processes, including actin cytoskeleton remodelling. Numerous bacterial pathogens usurp host signalling pathways that regulate actin reorganisation in order to promote Infection. Salmonella and pathogenic Escherichia coli drive actin-dependent forced uptake and intimate attachment respectively. We demonstrate that the pathogen-driven generation of both these distinct actin structures relies on the recruitment and activation of PAK. We show that the PAK kinase domain is dispensable for this actin remodelling, which instead requires the GTPase-binding CRIB and the central poly-proline rich region. PAK interacts with and inhibits the guanine nucleotide exchange factor ß-PIX, preventing it from exerting a negative effect on cytoskeleton reorganisation. This kinase-independent function of PAK may be usurped by other pathogens that modify host cytoskeleton signalling and helps us better understand how PAK functions in normal and diseased eukaryotic cells.

Does the immune system of milk increase activity for infants experiencing infectious disease episodes in Kilimanjaro, Tanzania?

INTRODUCTION: Multiple studies have reported that milk immune content increases for infants experiencing infectious disease (ID) episodes, suggesting that the immune system of milk (ISOM) offers enhanced protection when needed to combat ID. METHODS: To test the hypothesis that ISOM content and/or activity increases during an infant's ID episode, we characterized milk secretory immunoglobulin A (sIgA; a major ISOM constituent) and in vitro interleukin-6 (IL-6) responses to Salmonella enterica and Escherichia coli, as system-level biomarkers of ISOM activity, in a prospective study among 96 mother-infant dyads in Kilimanjaro, Tanzania. RESULTS: After control for covariates, no milk immune variables (sIgA, Coef: 0.03; 95% CI -0.25, 0.32; in vitro IL-6 response to S. enterica, Coef: 0.23; 95% CI: -0.67, 1.13; IL-6 response to E. coli, Coef: -0.11; 95% CI: -0.98, 0.77) were associated with prevalent ID (diagnosed at the initial participation visit). Among infants experiencing an incident ID (diagnosed subsequent to the initial participation), milk immune content and responses were not substantially higher or lower than the initial visit (sIgA, N: 61; p: 0.788; IL-6 response to S. enterica, N: 56; p: 0.896; IL-6 response to E. coli, N: 36; p: 0.683); this was unchanged by exclusion of infants with ID at the time of initial participation. CONCLUSION: These findings are not consistent with the hypothesis that milk delivers enhanced immune protection when infants experience ID. In environments with a high burden of ID, dynamism may be less valuable to maternal reproductive success than stability in the ISOM.

Effect of culture method on storage, plasma, and dry heat treatment resistance of Salmonella enterica serovar Typhimurium on black pepper.

The purpose of this study was to determine the effect of the culture method on the resistance of Salmonella Typhimurium in low water activity foods to storage, plasma, and dry heat. Whole black peppers were used as the model food. S. Typhimurium cultured in liquid broth (tryptic soy broth) or solid agar (tryptic soy agar) and inoculated on whole black pepper was stored or treated with cold plasma or dry heat. Inactivation of S. Typhimurium cultured in liquid medium was higher in all the treatments. Liquid-cultured S. Typhimurium showed higher DPPP = O (diphenyl-1-pyrenylphosphine oxide) values compared to the solid-cultured S. Typhimurium after plasma or dry heat treatment. Furthermore, the unsaturated fatty acid and saturated fatty acid ratio (USFA/SFA) was significantly (P < 0.05) reduced from 0.41 to 0.29 when S. Typhimurium was cultured on solid agar. These results suggested that the use of food-borne pathogens cultured on solid agar is more suitable for low water activity food pasteurization studies.

Specific Bacterial Cell Wall Components Influence the Stability of Coxsackievirus B3.

Enteric viruses infect the mammalian gastrointestinal tract and lead to significant morbidity and mortality worldwide. Data indicate that enteric viruses can utilize intestinal bacteria to promote viral replication and pathogenesis. However, the precise interactions between enteric viruses and bacteria are unknown. Here, we examined the interaction between bacteria and coxsackievirus B3, an enteric virus from the picornavirus family. We found that bacteria enhance the infectivity of coxsackievirus B3 (CVB3) in vitro. Notably, specific bacteria are required, as Gram-negative Salmonella enterica, but not Escherichia coli, enhanced CVB3 infectivity and stability. Investigating the cell wall components of both S. enterica and E. coli revealed that structures in the O-antigen or core of lipopolysaccharide, a major component of the Gram-negative bacterial cell wall, were required for S. enterica to enhance CVB3. To determine if these requirements were necessary for similar enteric viruses, we investigated if S. enterica and E. coli enhanced infectivity of poliovirus, another enteric virus in the picornavirus family. We found that while E. coli did not enhance the infectivity of CVB3, E. coli enhanced poliovirus infectivity. Overall, these data indicate that distinct bacteria enhance CVB3 infectivity and stability, and specific enteric viruses may have differing requirements for their interactions with specific bacterial species. IMPORTANCE Previous data indicate that several enteric viruses utilize bacteria to promote intestinal infection and viral stability. Here, we show that specific bacteria and bacterial cell wall components are required to enhance infectivity and stability of coxsackievirus B3 in vitro. These requirements are likely enteric virus specific, as the bacteria for CVB3 differ from poliovirus, a closely related virus. Therefore, these data indicate that specific bacteria and their cell wall components dictate the interaction with various enteric viruses in distinct mechanisms.

Cross-feeding modulates the rate and mechanism of antibiotic resistance evolution in a model microbial community of Escherichia coli and Salmonella enterica.

With antibiotic resistance rates on the rise, it is critical to understand how microbial species interactions influence the evolution of resistance. In obligate mutualisms, the survival of any one species (regardless of its intrinsic resistance) is contingent on the resistance of its cross-feeding partners. This sets the community antibiotic sensitivity at that of the 'weakest link' species. In this study, we tested the hypothesis that weakest link dynamics in an obligate cross-feeding relationship would limit the extent and mechanisms of antibiotic resistance evolution. We experimentally evolved an obligate co-culture and monoculture controls along gradients of two different antibiotics. We measured the rate at which each treatment increased antibiotic resistance, and sequenced terminal populations to question whether mutations differed between mono- and co-cultures. In both rifampicin and ampicillin treatments, we observed that resistance evolved more slowly in obligate co-cultures of E. coli and S. enterica than in monocultures. While we observed similar mechanisms of resistance arising under rifampicin selection, under ampicillin selection different resistance mechanisms arose in co-cultures and monocultures. In particular, mutations in an essential cell division protein, ftsI, arose in S. enterica only in co-culture. A simple mathematical model demonstrated that reliance on a partner is sufficient to slow the rate of adaptation, and can change the distribution of adaptive mutations that are acquired. Our results demonstrate that cooperative metabolic interactions can be an important modulator of resistance evolution in microbial communities.

Spatio-temporal modeling of the crowding conditions and metabolic variability in microbial communities.

The metabolic capabilities of the species and the local environment shape the microbial interactions in a community either through the exchange of metabolic products or the competition for the resources. Cells are often arranged in close proximity to each other, creating a crowded environment that unevenly reduce the diffusion of nutrients. Herein, we investigated how the crowding conditions and metabolic variability among cells shape the dynamics of microbial communities. For this, we developed CROMICS, a spatio-temporal framework that combines techniques such as individual-based modeling, scaled particle theory, and thermodynamic flux analysis to explicitly incorporate the cell metabolism and the impact of the presence of macromolecular components on the nutrients diffusion. This framework was used to study two archetypical microbial communities (i) Escherichia coli and Salmonella enterica that cooperate with each other by exchanging metabolites, and (ii) two E. coli with different production level of extracellular polymeric substances (EPS) that compete for the same nutrients. In the mutualistic community, our results demonstrate that crowding enhanced the fitness of cooperative mutants by reducing the leakage of metabolites from the region where they are produced, avoiding the resource competition with non-cooperative cells. Moreover, we also show that E. coli EPS-secreting mutants won the competition against the non-secreting cells by creating less dense structures (i.e. increasing the spacing among the cells) that allow mutants to expand and reach regions closer to the nutrient supply point. A modest enhancement of the relative fitness of EPS-secreting cells over the non-secreting ones were found when the crowding effect was taken into account in the simulations. The emergence of cell-cell interactions and the intracellular conflicts arising from the trade-off between growth and the secretion of metabolites or EPS could provide a local competitive advantage to one species, either by supplying more cross-feeding metabolites or by creating a less dense neighborhood.

The expanded specificity and physiological role of a widespread N-degron recognin.

All cells use proteases to maintain protein homeostasis. The proteolytic systems known as the N-degron pathways recognize signals at the N terminus of proteins and bring about the degradation of these proteins. The ClpS protein enforces the N-degron pathway in bacteria and bacteria-derived organelles by targeting proteins harboring leucine, phenylalanine, tryptophan, or tyrosine at the N terminus for degradation by the protease ClpAP. We now report that ClpS binds, and ClpSAP degrades, proteins still harboring the N-terminal methionine. We determine that ClpS recognizes a type of degron in intact proteins based on the identity of the fourth amino acid from the N terminus, showing a strong preference for large hydrophobic amino acids. We uncover natural ClpS substrates in the bacterium Salmonella enterica, including SpoT, the essential synthase/hydrolase of the alarmone (p)ppGpp. Our findings expand both the specificity and physiological role of the widespread N-degron recognin ClpS.

Copper tolerance in bacteria requires the activation of multiple accessory pathways.

Copper is a required micronutrient for bacteria and an essential cofactor for redox-active cuproenzymes. Yet, excess copper is extremely toxic, and is exploited as a bacteriocide in medical and biotechnological applications and also by the mammalian immune system. To evade copper toxicity, bacteria not only control intracellular copper homeostasis, but they must also repair the damage caused by excess copper. In this review, we summarize the bacterial cell-wide response to copper toxicity in Enterobacteria. Tapping into the abundant research data on two key organisms, Escherichia coli and Salmonella enterica, we show that copper resistance requires both the direct copper homeostatic response and also the indirect accessory pathways that deal with copper-induced damage. Since patterns of copper response are conserved through the Proteobacteria, we propose a cell-wide view of copper detoxification and copper tolerance that can be used to identify novel targets for copper-based antibacterial therapeutics.

Genetic Determinants of Stress Resistance in Desiccated Salmonella enterica.

Enteric pathogens, including Salmonella, are capable of long-term survival after desiccation and resist heat treatments that are lethal to hydrated cells. The mechanisms of dry-heat resistance differ from those of wet-heat resistance. To elucidate the mechanisms of dry-heat resistance in Salmonella, screening of the dry-heat resistance of 108 Salmonella strains, representing 39 serotypes, identified the 22 most resistant and the 8 most sensitive strains for comparative genome analysis. A total of 289 genes of the accessory genome were differently distributed between resistant and sensitive strains. Among these genes, 28 proteins with a putative relationship to stress resistance were selected for to quantify relative gene expression before and after desiccation and expression by solid-state cultures on agar plates relative to cultures growing in liquid culture media. Of these 28 genes, 15 genes were upregulated (P < 0.05) after desiccation or by solid-state cultures on agar plates. These 15 genes were cloned into the low-copy-number vector pRK767 under the control of the lacZ promoter. The expression of 6 of these 15 genes increased (P < 0.05) resistance to dry heat and to treatment with pressure of 500 MPa. Our finding extends the knowledge of mechanisms of stress resistance in desiccated Salmonella to improve control of this bacterium in dry food. IMPORTANCE This study directly targeted an increasing threat to food safety and developed knowledge and targeted strategies that can be used by the food industry to help reduce the risk of foodborne illness in their dry products and thereby reduce the overall burden of foodborne illness. Genomic and physiological analyses have elucidated mechanisms of bacterial resistance to many food preservation technologies, including heat, pressure, disinfection chemicals, and UV light; however, information on bacterial mechanisms of resistance to dry heat is scarce. Mechanisms of tolerance to desiccation likely also contribute to resistance to dry heat, but this assumption has not been verified experimentally. It remains unclear how mechanisms of resistance to wet heat relate to dry-heat resistance. Thus, this study will fill a knowledge gap to improve the safety of dry foods.

Identification of Natural Mutations Responsible for Altered Infection Phenotypes of Salmonella enterica Clinical Isolates by Using Cell Line Infection Screens.

The initial steps of Salmonella pathogenesis involve adhesion to and invasion into host epithelial cells. While well-studied for Salmonella enterica serovar Typhimurium, the factors contributing to this process in other, host-adapted serovars remains unexplored. Here, we screened clinical isolates of serovars Gallinarum, Dublin, Choleraesuis, Typhimurium, and Enteritidis for adhesion to and invasion into intestinal epithelial cell lines of human, porcine, and chicken origins. Thirty isolates with altered infectivity were used for genomic analyses, and 14 genes and novel mutations associated with high or low infectivity were identified. The functions of candidate genes included virulence gene expression regulation and cell wall or membrane synthesis and components. The role of several of these genes in Salmonella adhesion to and invasion into cells has not previously been investigated. The genes dksA (encoding a stringent response regulator) and sanA (encoding a vancomycin high-temperature exclusion protein) were selected for further analyses, and we confirmed their roles in adhesion to and invasion into host cells. Furthermore, transcriptomic analyses were performed for S Enteritidis and S Typhimurium, with two highly infective and two marginally infective isolates for each serovar. Expression profiles for the isolates with altered infection phenotypes revealed the importance of type 3 secretion system expression levels in the determination of an isolate's infection phenotype. Taken together, these data indicate a new role in cell host infection for genes or gene variants previously not associated with adhesion to and invasion into the epithelial cells.IMPORTANCESalmonella is a foodborne pathogen affecting over 200 million people and resulting in over 200,000 fatal cases per year. Its adhesion to and invasion into intestinal epithelial cells represent one of the first and key steps in the pathogenesis of salmonellosis. Still, around 35 to 40% of bacterial genes have no experimentally validated function, and their contribution to bacterial virulence, including adhesion and invasion, remains largely unknown. Therefore, the significance of this study is in the identification of new genes or gene allelic variants previously not associated with adhesion and invasion. It is well established that blocking adhesion and/or invasion would stop or hamper bacterial infection; therefore, the new findings from this study could be used in future developments of anti-Salmonella therapy targeting genes involved in these key processes. Such treatment could be a valuable alternative, as the prevalence of antibiotic-resistant bacteria is increasing very rapidly.

Immune priming depends on age, sex and Wolbachia in the interaction between Armadillidium vulgare and Salmonella.

The protection conferred by a first infection upon a second pathogenic exposure (i.e. immune priming) is an emergent research topic in the field of invertebrate immunity. Immune priming has been demonstrated in various species, but little is known about the intrinsic factors that may influence this immune process. In this study, we tested whether age, gender and the symbiotic bacterium Wolbachia affect the protection resulting from immune priming in A. vulgare against S. enterica. We firstly primed young and old, symbiotic and asymbiotic males and females, either with a non-lethal low dose of S. enterica, LB broth or without injection (control). Seven days post-injection, we performed a LD50 injection of S. enterica in all individuals and we monitored their survival rates. We demonstrated that survival capacities depend on these three factors: young and old asymbiotic individuals (males and females) expressed immune priming (S. enterica-primed individuals survived better than LB-primed and non-primed), with a general decline in the strength of protection in old females, but not in old males, compared to young. When Wolbachia is present, the immune priming protection was observed in old, but not in young symbiotic individuals, even if the Wolbachia load on entire individuals is equivalent regardless to age. Our overall results showed that the immune priming protection in A. vulgare depends on individuals' states, highlighting the need to consider these factors both in mechanistical and evolutionary studies focusing on invertebrate's immunity.

A mathematical model of flagellar gene regulation and construction in Salmonella enterica.

Millions of people worldwide develop foodborne illnesses caused by Salmonella enterica (S. enterica) every year. The pathogenesis of S. enterica depends on flagella, which are appendages that the bacteria use to move through the environment. Interestingly, populations of genetically identical bacteria exhibit heterogeneity in the number of flagella. To understand this heterogeneity and the regulation of flagella quantity, we propose a mathematical model that connects the flagellar gene regulatory network to flagellar construction. A regulatory network involving more than 60 genes controls flagellar assembly. The most important member of the network is the master operon, flhDC, which encodes the FlhD4C2 protein. FlhD4C2 controls the construction of flagella by initiating the production of hook basal bodies (HBBs), protein structures that anchor the flagella to the bacterium. By connecting a model of FlhD4C2 regulation to a model of HBB construction, we investigate the roles of various feedback mechanisms. Analysis of our model suggests that a combination of regulatory mechanisms at the protein and transcriptional levels induce bistable FlhD4C2 levels and heterogeneous numbers of flagella. Also, the balance of regulatory mechanisms that become active following HBB construction is sufficient to provide a counting mechanism for controlling the total number of flagella produced.

Heat stress impairs egg production in commercial laying hens infected by fowl typhoid.

Salmonella Gallinarum (SG) is an avian-restricted pathogen that causes fowl typhoid in poultry. Although it has been reported frequently over many decades in poultry flocks worldwide, the microorganism is more commonly associated with poultry in developing countries, particularly those with high ambient temperatures, where the acute form of the disease results in considerable economic losses. A more detailed investigation of environmental factors that affect the course of disease may assist in identifying effective prevention and control measures. Heat stress is known to impair the immunological response to a variety of pathogens and clearly may be an important contributory factor in the prevalence of disease in countries with warm or hot climates. Thus, the objective of the present study was to evaluate the effects of heat stress on chickens infected with SG. For this, light and semi-heavy commercial laying hens were distributed randomly within four groups as follows: infected and non-infected groups in rooms held at ambient temperature, and infected and non-infected groups under heat stress. Clinical signs, egg production, and mortality were recorded daily. Bacteriological counts in liver and spleen samples were estimated at 2, 5, 7, and 14 days post-infection. The results showed that both SG infection and heat stress had similar effects on egg production and a synergistic effect of the two stressors was observed. The data show an interaction between disease and heat stress which could point towards environmental and biosecurity approaches to resolving the possible 30% fall in production observed in such countries.

Inhibitory effect of different chicken-derived lactic acid bacteria isolates on drug resistant Salmonella SE47 isolated from eggs.

Lactic Acid Bacteria (LAB) regulate and maintain the stability of healthy microbial flora, inhibit the adhesion of pathogenic bacteria and promote the colonization of beneficial micro-organisms. The drug resistance and pathogenicity of Salmonella enteritis SE47 isolated from retail eggs were investigated. Meanwhile, Enterococcus faecalis L76 and Lactobacillus salivarius LAB35 were isolated from intestine of chicken. With SE47 as indicator bacteria, the diameters of L76 and LAB35 inhibition zones were 12 mm and 8·5 mm, respectively, by agar inhibition circle method, which indicated that both of them had inhibitory effect on Salmonella, and L76 had better antibacterial effect; two chicken-derived lactic acid bacteria isolates and Salmonella SE47 were incubated with Caco-2. The adhesion index of L76 was 17·5%, which was much higher than that of LAB35 (10·21%) and SE47 (4·89%), this experiment shows that the higher the bacteriostatic effect of potential probiotics, the stronger the adhesion ability; then Caco-2 cells were incubated with different bacteria, and the survival of Caco-2 cells was observed by flow cytometry. Compared with Salmonella SE47, the results showed that lactic acid bacteria isolates could effectively protect Caco-2 cells; finally, after different bacteria incubated Caco-2 cells, according to the cytokine detection kit, the RNA of Caco-2 cells was extracted and transcribed into cDNA, then detected by fluorescence quantitative PCR, the results showed that L76 could protect Caco-2 cells from the invasion of Salmonella SE47, with less cell membrane rupture and lower expression of MIF and TNF genes. Therefore, the lactic acid bacteria isolates can effectively inhibit the adhesion of Salmonella and protect the integrity of intestinal barrier.

Biovolume and spatial distribution of foodborne Gram-negative and Gram-positive pathogenic bacteria in mono- and dual-species biofilms.

The objective of this study was to characterize the biofilms formed by Salmonella enterica serotype Agona, Listeria monocytogenes, methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE) after 12, 48, 72, 120 and 240 h of incubation at 10 °C. Biofilms containing a single species, together with dual-species biofilms in which S. enterica and a Gram-positive bacterium existed in combination, were formed on polystyrene and evaluated by using confocal laser scanning microscopy (CLSM). All strains were able to form biofilm. The greatest biovolume in the observation field of 14,161 µm2 was observed for mono-species biofilms after 72 h, where biovolumes of 94,409.0 µm3 ± 2131.0 µm3 (S. enterica), 58,418.3 µm3 ± 5944.9 µm3 (L. monocytogenes), 68,020.8 µm3 ± 5812.3 µm3 (MRSA) and 59,280.0 µm3 ± 4032.9 µm3 (VRE) were obtained. In comparison with single-species biofilms, the biovolume of S. enterica was higher in the presence of MRSA or VRE after 48, 72 and 120 h. In dual-species biofilms, the bacteria showed a double-layer distribution pattern, with S. enterica in the top layer and Gram-positive bacteria in the bottom layer. This spatial disposition should be taken into account when effective strategies to eliminate biofilms are being developed.