Darinka Vučković

Murine model of pregnancy‐associated Listeria monocytogenes infection

Listeria monocytogenes has been recognized as a significant pathogen, occurring worldwide, capable of causing animal and human infections. In its most severe form, listeriosis is an invasive disease that affects immunocompromised patients. Additionally, pregnant women represent a high-risk group for L. monocytogenes infection. Abortion, stillbirth or severe neonatal infection can be the serious outcome of such an infection. In an experimental murine model of pregnancy-associated listeriosis we studied the impact of L. monocytogenes on the maternal immune response and pregnancy outcome. In comparison to virgin animals, pregnant mice mounted lower levels of protective cytokines and were unable to eliminate the pathogen. The impaired maternal immune response that has been found both on the systemic and local level, facilitated bacterial multiplication in the liver, placenta and ultimately in the fetal tissues. This resulted in severe necrotizing hemorrhagic hepatitis and Listeria-induced placental necrosis, increasing the incidence of postimplantation loss and poor pregnancy outcome.

Stress response and pathogenic potential of Campylobacter jejuni cells exposed to starvation

Campylobacter jejuni is a Gram-negative, fragile, spiral bacterium, known worldwide to be a major cause of acute human enteritis. Like many other food-borne bacteria, campylobacters must be able to survive under diverse conditions both inside the host and in the environment. Understanding stress response mechanisms provides information necessary for improving food processing and strategies that enhance food safety as well as clarifying the pathogenesis of campylobacteriosis. We investigated the relation between stress response to starvation and pathogenic potential in C. jejuni. Starvation changed the morphology and physiology of C. jejuni cells. However, the lower metabolic activity of 5-h-starved culture was not a dormant state, but probably a viable but non-culturable (VBNC) form of the cells, since starved C. jejuni induced heat stress resistance. The health hazard potential of starved cells is still unclear. We showed that, in spite of starvation, C. jejuni survived in vitro within Caco-2 enterocites up to 4 days and caused systemic campylobacteriosis in vivo in a mouse model. However, bacterial numbers in investigated organs were significantly lower and the infection was resolved sooner. Our results show that nutrient insufficiency is responsible for C. jejuni transformation, influencing but not abolishing its survival and virulence properties while in the VBNC state.

Plasma cytokine response in mice with bacterial infection

BACKGROUND: Exposure to microorganisms elicts the production of cytokines. These soluble factors enhance several innate immune functions and regulate the ensuing specific immune response aimed at limiting the spread of infection. AIM: This study was undertaken to quantify the plasma levels of pro-inflammatory cytokines during the course of primary Listeria monocytogenes and Campylobacter jejuni infection. Using an in vivo infection the relationship between endogenous cytokines and the bacterial number in the liver of infected animals was examined. METHODS: C57BL/6 mice were infected by the intraperitoneal route. At different time points we determined the number of colony-forming units of bacteria in the liver of infected animals and paralled these with the plasma levels of interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) measured by enzyme immunoassays. RESULTS: L. monocytogenes infection lasted 10-11 days. IFN-gamma production occurred in the early phase but was more pronounced after day 4, following the appearance of specific immunity. The duration of experimental campylobacteriosis was 15 days. Early IFN-gamma production was not significant but a progressive rise of this cytokine in plasma was seen during the second week post infection. Mice produced measurable amounts of plasma TNF-alpha immediately after being given viable L. monocytogenes, peaking on day 2-3 when the greatest number of bacteria was present in the examined organs. During C. jejuni infection plasma TNF-alpha was produced in a similar manner, but the highest concentrations were found a few days later than in listeriosis, in correlation with the different course of campylobacteriosis. The quantity of IL-6 increased and decreased in concordance with clearance of L monocytogenes and the clinical status of the animals. C. jejuni did not promote the induction of this cytokine. This is to some extent an unusual finding. With respect to the role of IL-6 in Th2 responses and antibody production, the appearance of this cytokine in campylobacteriosis was more expected. DISCUSSION: During systemic bacterial infection, a network of pro-inflammatory cytokines is activated and blood levels of these cytokines are elevated, albeit inconsistently, with large individual variations and depending on microbial characteristics and structure.

In Vivo Modulation of Campylobacter jejuni Virulence in Response to Environmental Stress

Campylobacters have developed a number of mechanisms for responding to environmental conditions, although the different virulence properties of these cells following exposure to stress are still poorly understood. We analyzed in vitro stress responses and the consequent in vivo modulation of Campylobacter jejuni pathogenicity in BALB/c mice, as a result of the exposure of the C. jejuni to environmental stress (starvation, oxidative stress, heat shock). In vitro, the influence of starvation and oxidative stress was milder than that of heat shock, although the majority of the stress conditions influenced the survival of C. jejuni. During starvation, C. jejuni viability was maintained longer than its culturability. Additionally, starvation elicited transformation of stressed bacteria to coccoid forms. In contrast, bacteria exposed to oxygen remained culturable, but their viability decreased. Pre-starvation did not contribute to improved survival of C. jejuni cells during oxygen exposure. Changes in bacteria numbers and the levels of several cytokines (interleukins 6 and 10, tumor necrosis factor-α, interferon-γ) were followed in vivo, in liver homogenates from the mice intravenously infected with either control (untreated) or stressed C. jejuni. The systemic infection with the control or stressed C. jejuni occurred with different production dynamics of the cytokines investigated. Starvation was the most powerful stress factor, which significantly decreased infectious potential of C. jejuni during the first 3 days postinfection. The most pronounced differences in cytokine production were found in interferon-γ and interleukin-10 production, which indicates that these have roles in the immune response to C. jejuni infection. These in vivo studies of environmental impact on bacterial virulence reveal that microbial adaptation during stress challenge is crucial not just for pathogen survival out of the host, but also during host-pathogen interactions, and thus for the bacterial pathogenicity.

Reduction of microbiological risk in minced meat by a combination of natural antimicrobials

BACKGROUND Responsibility for food safety must be taken through the entire food-production chain, to avoid consumer cross-contamination. The antimicrobial activities of an Alpinia katsumadai seed extract and epigallocatechin gallate (EGCG), and their combination, were evaluated against individual food-borne pathogenic strains of Listeria monocytogenes, Escherichia coli and Campylobacter jejuni, individually and as a cocktail, in chicken-meat juice and sterile minced meat as food models, and in minced meat with the naturally present microflora, as an actual food sample. RESULTS The antimicrobial combination of the A. katsumadai extract and EGCG was the most efficient for C. jejuni growth inhibition, followed by inhibition of L. monocytogenes, which was reduced more efficiently in the bacterial cocktail than as an individual strain. The antimicrobial combination added to minced meat at refrigeration temperatures used in the food chain (8 °C) revealed inhibition of these pathogens and inhibition of the naturally present bacteria after 5 days. CONCLUSIONS The antibacterial efficiencies of the tested combinations are influenced by storage temperature. Food safety can be improved by using the appropriate combination of natural antimicrobials to reduce the microbiological risk of minced meat.

Virulence genes and cytokine profile in systemic murine Campylobacter coli infection

Campylobacter coli are one of the most common bacteria in bacterial gastroenteritis and acute enterocolitis in humans. However, relatively little is known regarding the mechanisms of pathogenesis and host response to C. coli infections. To investigate the influence of genetic changes, we first used PCR to demonstrate the presence of the known virulence genes cadF, virB11, cdtB, cdtC and ceuE in the clinical isolate C. coli 26536, which was isolated from the liver of infected BALB/c mice. Sequence analyses of the cadF, virB11, cdtB and ceuE genes in C. coli 26536 confirmed the stability in these virulence genes during their transmission through the host. We further investigated C. coli infection for the bacterial clearance from the liver and spleen of infected mice, and for their immune response. C. coli persisted well in both organs, with better survival in the liver. We also determined the levels of several pro-inflammatory cytokines (i.e., interleukin [IL]-6, IL-12, interferon-γ, tumor necrosis factor-α) and the anti-inflammatory cytokine IL-10 in plasma and in liver homogenates from the infected mice, using enzyme-linked immunosorbent assays. The lowest levels among these cytokines were for tumor necrosis factor-α in the plasma and IL-6 in the liver on days 1, 3 and 8 post-infection. The most pronounced production was for IL-10, in both plasma (days 1 and 8 post-infection) and liver (day 8 post-infection), which suggests that it has a role in healing of the organ inflammation. Our findings showed dynamic relationships between pro- and anti-inflammatory cytokines and thus contribute toward clarification of the healing processes involved in the resolution of C. coli infections.

Stress Response and Virulence of Heat-Stressed Campylobacter jejuni

Thermotolerant Campylobacter spp. frequently cause bacterial gastroenteritis in humans commonly infected through the consumption of undercooked poultry meat. We examined Campylobacter jejuni heat-stress responses in vitro after exposure to 48°C and 55°C. The in vivo modulation of its pathogenicity was also investigated using BALB/c mice intravenously infected with stressed C. jejuni. Regardless of the bacterial growth phase, the culturability and viability of C. jejuni in vitro was reduced after exposure to 55°C. This correlated with the altered protein profile and decreased virulence properties observed in vivo. Heat stress at 48°C elicited the transition to more resistant bacterial forms, independent of morphological changes or the appearance of shorter spiral and coccoid cells. This treatment did not cause marked changes in bacterial virulence properties in vivo. These results indicated that the characteristics and pathogenicity of C. jejuni in response to heat stress are temperature dependent. Further studies on the responses of C. jejuni to stresses used during food processing, as well as the modulation of its virulence, are important for a better understanding of its contamination and infective cycle, and will, thus, contribute to improved safety in the food production chain.

Reduced contamination and infection via inhibition of adhesion of foodborne bacteria to abiotic polystyrene and biotic amoeba surfaces

Adhesion of foodborne pathogens to materials of industrial surfaces is an important step in their transmission through the food chain. Adhesion is also a prerequisite for bacterial colonisation within a host, to enable intracellular invasion. We define a strategy to reduce contamination and infection by Listeria monocytogenes, Campylobacter jejuni and Escherichia coli using an ethanol extract of Alpinia katsumadai seeds (AlpE) and epigallocatechin gallate (EGCG) as anti- adhesive agents. We show for the first time that AlpE and EGCG reduce adhesion of individual cultures to polystyrene (AlpE, up to 10.6% ; EGCG, up to 39.7%) and to the amoeba Acanthamoeba castellanii (AlpE, up to 52.6% ; EGCG, up to 53.4%). The combination of AlpE/EGCG significantly reduced C. jejuni adherence to the abiotic (45.5%) and biotic (52.2%) surfaces. Thus, using natural agents from plants at low doses, we can potentially reduce the primary source of food contamination and a frequent source of infections.

Salmonella enterica arthritis in a rheumatoid arthritis patient under anti-tumor necrosis factor therapy

Anti-tumour necrosis factor (TNF) monoclonal antibodies have become an invaluable treatment against chronic inflammatory diseases such as rheumatoid arthritis (RA). However, due to increased risk of opportunistic infections, patients receiving anti-TNF therapy should be closely monitored for serious infections. Here, we describe a case of acute Salmonella_enteritidis infection of a joint arthroplasty that previously was functioning well, in a patient receiving infliximab treatment for RA. After prolonged antimicrobial chemotherapy and interrupted infliximab treatment, reimplantation of a new prosthesis was successfully performed two years after Salmonella septic arthritis. Therefore, because of the possibility of extraintestinal salmonellosis, screening for fecal colonization could be advisable in patients undergoing anti-TNF treatment. Moreover we emphasize the importance of appropriate counselling of these patients concerning food hygiene.

Systemic cytokine response during listeria monocytogenes infection in pregnant BALB/C Mice

Pregnancy has an important impact on the maternal immune system, associated with a shift in the Th1/Th2 cytokine balance towards a type‐2 response. However, listeria induced Th1 type cytokines are essential in host protection against this facultative intracellular bacterium. We have analyzed and compared the kinetics of maternal serum concentrations of IFN‐γ, IL‐10, TNF‐α, IL‐6 and KC by ELISA. Serum IFN‐γ levels in infected pregnant mice were significantly reduced in comparison to infected virgin animals, while IL‐10 levels were increased, being significantly higher on days 3 and 6 p.i. as compared to infected virgin mice. Upon L. monocytogenes infection, circulating TNF‐α concentrations were below the detection limit. However, on day 6 p.i., in pregnant mice elevated levels of TNF‐α were noticed, correlating to the severity of disease. Increased concentrations of IL‐6 have been shown to follow the kinetics of bacterial numbers in the liver of infected animals. Murine chemokine KC production in maternal circulation followed a similar pattern as IL‐6.