Endogenous cytokines during a lethal infection with Listeria monocytogenes in mice

It has been demonstrated that endogenous cytokines including gamma interferon (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6) play protective roles but that IL-4 and IL-10 play detrimental roles in nonlethal Listeria monocytogenes infection in mice. In this paper, we studied the roles of endogenous cytokines in a lethal infection with L. monocytogenes in mice. TNF-alpha and IL-6 titres in the bloodstreams, spleens and livers paralleled bacterial numbers in the organs, and both these cytokines and the bacterial numbers peaked just before the mice died. The high titres of TNF-alpha notably detected in the circulation in lethal infection were different from those in nonlethal infection. The maximum production of IFN-gamma was observed before the peaks of TNF-alpha and IL-6, and IFN-gamma almost disappeared from the bloodstreams and organs just before the mice died. No notable difference of IFN-gamma titres between lethal infection and nonlethal infection in the specimens obtained from mice was observed. IL-10 was also detected in the bloodstreams earlier than the peaks of TNF-alpha and IL-6 during lethal infection, while IL-4 was never detected in the sera. The administration of monoclonal antibodies (mAbs) against TNF-alpha, IFN-gamma, IL-6, IL-4 or IL-10 failed to rescue mice from lethal L. monocytogenes infection, whereas anti-TNF-alpha mAb and anti-IFN-gamma mAb prevented mice from lethality by high-dose endotoxin shock. These results suggest that lethality in L. monocytogenes infection might not be determined solely by these cytokines.     

Roles of endogenous cytokines in liver apoptosis of mice in lethal Listeria monocytogenes infection

Various bacterial pathogens have been identified as mediators of apoptosis. Apoptosis reportedly shows both detrimental and beneficial effects on biological functions. We studied the role of liver apoptosis in lethal Listeria monocytogenes infection and the regulation of apoptosis by endogenous cytokines during infection. Apoptosis was observed in the spleen but not in the liver of infected mice, whereas the induction of liver necrosis was evident by rising levels of serum aminotransferases in these animals. Apoptosis was detected in the liver of L. monocytogenes-infected mice which had been treated with monoclonal antibody (mAb) against tumor necrosis factor-alpha (TNF-alpha) or interleukin-6 (IL-6), or in TNF-alpha(-/-) mice, but not in gamma- interferon (IFN-gamma)(-/-) mice or mice which had been treated with mAb against IL-4 or IL-10. Augmentation of liver apoptosis in mice treated with mAb against TNF-alpha or IL-6 or in TNF-alpha(-/-) mice correlated with the increase in bacterial numbers in the organ, while no augmentation of apoptosis was observed in the liver of IFN-gamma(-/-) mice irrespective of the marked increase in bacterial numbers in the organs, indicating that augmentation of liver apoptosis may not be merely due to the increase in bacterial growth in the organs. These results suggest that TNF-alpha and IL-6 may play an important role in protecting the liver from apoptosis in lethal L. monocytogenes infection.     

Effect of a matrix metalloproteinase inhibitor on host resistance against Listeria monocytogenes infection

Hydroxy acid-based matrix metalloproteinase (MMP) inhibitors have been shown to inhibit tumor infiltration and growth, endotoxin shock, and acute graft-versus-host disease. Blockade of the release of soluble tumor necrosis factor-alpha (TNF-alpha) and CD95 ligand (CD95L; FasL) from cell-associated forms is reportedly involved in the mechanism of the drug effect. We investigated the effect of a MMP inhibitor, KB-R7785, on host resistance against Listeria monocytogenes infection, in which TNF-alpha is essentially required for the defense, in mice. The administration of KB-R7785 exacerbated listeriosis, while the drug prevented lethal shock induced by lipopolysaccharide and D-galactosamine. KB-R7785 inhibited soluble TNF-alpha production in spleen cell cultures stimulated by heat-killed L. monocytogenes and the drug treatment reduced serum TNF-alpha levels in infected mice, whereas the compound was ineffective on the modulation of interferon-gamma and interleukin-10 production. The effect of KB-R7785 was considered to be dependent on TNF-alpha because the drug failed to affect L. monocytogenes infection in anti-TNF-alpha monoclonal antibody-treated mice and TNF-alpha knockout mice. Anti-CD95L monoclonal antibody was also ineffective on the infection. These results suggest that induction of infectious diseases, to which TNF-alpha is critical in host resistance, should be considered in MMP inhibitor-treated hosts.     

A monoclonal antibody against T-cell receptor αβ induces endogenous cytokines and prevents mice from a lethal infection with Listeria monocytogenes

Abstract In vivo induction of cytokines by a monoclonal antibody (mAb) against T-cell receptor (TCR) αβ and the protective effect induced by the mAb on a lethal infection with Listeria monocytogenes were studied. Injection of anti-TCR αβ mAb induced rapid production of endogenous tumour necrosis factor in the spleens, and gamma interferon and interleukin-6 in the bloodstreams and spleens of mice. Administration of anti-CD4 mAb, anti-CD8 mAb, or anti-Thy1.2 mAb resulted in suppression of anti-TCR αβ mAb-induced endogenous cytokine production. Mice were protected against lethal L. monocytogenes infection when treated with anti-TCR αβ mAb. The protective effect was not demonstrated in CD4 + cell- or CD8 + cell-depleted mice. These results suggest that anti-TCR αβ mAb shows a protective effect on a lethal infection with L. monocytogenes in mice and that the mAb-induced endogenous cytokines might be involved in the effect of anti-TCR αβ mAb.     

Protection by dexamethasone from a lethal infection with Listeria monocytogenes in mice

Abstract The effects of dexamethasone (DEX) on a lethal infection with Listeria monocytogenes were studied in mice. Mice were completely protected against the lethal infection when treated with 3.3 mg per kg of DEX. The effect was observed only when DEX was injected before infection. The control mice died from day 3 to day 5 of infection, whereas DEX-treated mice could eliminate L. monocytogenes cells from the organs by day 11 of infection. High titres of endogenous tumour necrosis factor (TNF), interleukin-6 (IL-6) and gamma interferon (IFN-γ) were induced in the bloodstreams and organs of the drug-free mice. DEX suppressed IL-6 production, but augmented TNF and IFN-γ production within 24 h of infection, whereas production of all three endogenous cytokines was suppressed in the DEX-treated mice on day 3 of infection when the control mice began to die. These results suggest that DEX shows a protective effect on a lethal infection with L. monocytogenes in mice and that regulation of production of endogenous cytokines might be involved in the effect of DEX.     

Dysregulation of interleukin-10 and interleukin-12 are involved in the reduced host resistance to Listeria monocytogenes infection in alymphoplastic aly mutant mice

The aly is a unique spontaneous autosomal recessive mutation in mice that causes a systemic defect of lymph nodes and Peyer's patches and disorganized splenic and thymic structures with immunodeficiency. Our previous study demonstrated that resistance to Listeria monocytogenes infection and interferon-gamma (IFN-gamma) production are attenuated in the mutant mice. In this study, we investigated the mechanism of decrease in antilisterial resistance and IFN-gamma production in aly mice. Interleukin (IL)-12 production in response to heat-killed L. monocytogenes (HK-LM) was decreased but IL-10 production was increased in aly/aly macrophage cultures, compared with those in aly/+ macrophages. Nonadherent cells and macrophages obtained from the spleens of naive aly/+ mice and aly/aly mice were reconstituted and stimulated with HK-LM. IFN-gamma production was markedly decreased when macrophages derived from aly/aly mice were used. IFN-gamma production in aly/aly spleen cell cultures was recovered in the presence of anti-IL-10 monoclonal antibody (mAb) or recombinant IL-12. When aly/+ mice and aly/aly mice were injected with mAb against IL-10 or IL-12 p40, antilisterial resistance was inhibited by injection of anti-IL-12 p40 mAb, while anti-IL-10 mAb treatment augmented the resistance. Administration of anti-IFN-gamma mAb attenuated antilisterial resistance in aly/+ mice but not in aly/aly mice. The present results suggest that downregulation of IL-12 and upregulation of IL-10 in macrophages might be involved in the decrease in antilisterial resistance and IFN-gamma production in aly/aly mice in addition to the structural defect in lymphoid organs. Moreover, the results predict that an IL-12-dependent and IFN-gamma-independent mechanism may be also involved in the decrease in antilisterial resistance in aly/aly mice.     

Effect of acid-adaptation on Listeria monocytogenes survival and translocation in a murine intragastric infection model

Acid tolerance response mechanisms can greatly influence Listeria monocytogenes survival in low pH foods. In the present paper, the effect of acid-adaptation together with control of gastric pH level on L. monocytogenes survival and translocation was analyzed after intragastric inoculation in the BALB/c mouse model. Our results showed that acid-adaptation led to an increase in resistance to the first barrier constituted by the low gastric pH and that inoculation at alkaline pH had a synergistic effect. It resulted in a higher live bacterial load reaching the next intestinal compartments and was correlated with increased translocation rates to the mesenteric lymph nodes, both at the frequency and quantitative levels. Our results in this murine model suggest that acid-adaptation of L. monocytogenes in low pH foods, together with control of gastric pH level through dietary practices, or use of inhibitors of gastric acid secretion, may be potential aggravating risk factors to food-borne listeriosis.     

Serological response in rabbits to Listeria monocytogenes after oral or intragastric inoculation

Abstract The serological response in rabbits against Listeria monocytogenes after oral or intragastric inoculation was investigated. Both the number of sero-positive and the average serum titres were hgher in animals inoculated by the oral route. This difference was especially marked in rabbits inoculated with the lower dose (1×10³ colony-forming units (cfu)), which developed a strong serological response (average serum titre of 1280 after 4 inoculations) in most of the inoculated animals (80%), without any clinical signs. The implication of these results in the epidemiology of listeriosis is discussed.     

Listeria monocytogenes induces host DNA damage and delays the host cell cycle to promote infection

Listeria monocytogenes (Lm) is a human intracellular pathogen widely used to uncover the mechanisms evolved by pathogens to establish infection. However, its capacity to perturb the host cell cycle was never reported. We show that Lm infection affects the host cell cycle progression, increasing its overall duration but allowing consecutive rounds of division. A complete Lm infectious cycle induces a S-phase delay accompanied by a slower rate of DNA synthesis and increased levels of host DNA strand breaks. Additionally, DNA damage/replication checkpoint responses are triggered in an Lm dose-dependent manner through the phosphorylation of DNA-PK, H2A.X, and CDC25A and independently from ATM/ATR. While host DNA damage induced exogenously favors Lm dissemination, the override of checkpoint pathways limits infection. We propose that host DNA replication disturbed by Lm infection culminates in DNA strand breaks, triggering DNA damage/replication responses, and ensuring a cell cycle delay that favors Lm propagation.     

The risk of Listeria monocytogenes infection in beef cattle operations

Aim:  To determine the prevalence of Listeria monocytogenes and associated risk factors among beef operations (cow-calf and feedlot) in central and southern California.
Methods and Results:  A repeated cross-sectional study where faecal and environmental samples were collected from 50 operations three times a year at different seasons was carried out. Samples were tested for presence of L. monocytogenes using a combination of enrichment and polymerase chain reaction tests. Data on putative risk factors were also collected. Listeria monocytogenes was detected in faecal samples from cows, calves and other animals on calf-cow operations at proportions of 3·1%, 3·75% and 2·5%, respectively. The organism was detected in 5·3% of cut-grass, 5·3% of soil, 14·3% of irrigation ditches, 3·1% of the ponds and 6·5% of water troughs samples. Listeria monocytogenes was less common in faecal (0·3%) and soil (0·75%) samples collected from feedlots.
Conclusions:  Listeria monocytogenes was present at a higher proportion among cow-calf operations than feedlots. There was no significant seasonal variation in the occurrence of this pathogen within the two types of operations.
Significance and Impact of the Study:  If risk mitigation strategies were implemented to reduce the public health risk these should focus in cow-calf operations.     

Ecogenetics of antibiotic resistance in Listeria monocytogenes

The acquisition process of antibiotic resistance in an otherwise susceptible organism is shaped by the ecology of the species. Unlike other relevant human pathogens, Listeria monocytogenes has maintained a high rate of susceptibility to the antibiotics used for decades to treat human and animal infections. However, L. monocytogenes can acquire antibiotic resistance genes from other organisms’ plasmids and conjugative transposons. Ecological factors could account for its susceptibility. L. monocytogenes is ubiquitous in nature, most frequently including reservoirs unexposed to antibiotics, including intracellular sanctuaries. L. monocytogenes has a remarkably closed genome, reflecting limited community interactions, small population sizes and high niche specialization. The L. monocytogenes species is divided into variants that are specialized in small specific niches, which reduces the possibility of coexistence with potential donors of antibiotic resistance. Interactions with potential donors are also hampered by interspecies antagonism. However, occasional increases in population sizes (and thus the possibility of acquiring antibiotic resistance) can derive from selection of the species based on intrinsic or acquired resistance to antibiotics, biocides, heavy metals or by a natural tolerance to extreme conditions. High-quality surveillance of the emergence of resistance to the key drugs used in primary therapy is mandatory.     

毒力基因调控蛋白PrfA促进单核细胞增生李斯特菌生物被膜的形成

单核细胞增生李斯特菌(Listeria monocytogenes,LM)是重要的革兰氏阳性食源性致病菌,易在食品以及各种食品加工、运输和保藏设备的接触面形成生物被膜,从而具有更强的抗逆性而难以彻底清除,因此成为食品卫生安全的重要隐患.PrfA是LM毒力基因转录表达的重要调控因子,通过比较研究LM野生株(EGD和EGDe)、PrfA缺失株(EGDAprfA和EGDeAprfA)、无害李斯特菌(Listeria innocua,LI),携带组成性表达PrfA蛋白的重组无害李斯特菌(LI-pERL3-prfA*)以及重组单核细胞增生李斯特菌(EGDeΔprfA-pERL3-prfA*)生物被膜形成能力的差异,探讨LM重要的毒力调控蛋白PrfA对生物被膜形成的影响.实验结果显示:LM野生株具有较强的生物被膜形成能力,而LI形成生物被膜的能力最弱;PrfA的缺失能降低LM生物被膜的形成能力;组成性高量表达PrfA蛋白可以回复EGDeΔprfA的生物被膜形成能力,但对LI没有增强作用.以上实验结果表明:PrfA在LM生物被膜形成中具有重要的促进作用.     

Listeria monocytogenes infection in pregnant mice: Abnormalities in the function of non-adherent accessory light density dendritic cells

Abstract Pregnant A/J mice were found to be more susceptible to the lethal effect of Listeria monocytogenes bacteria than virgin females. However, during the first four days of post-infection there was no difference in the elimination of Listeria from the spleens of pregnant and virgin mice. This suggests that the increase in the susceptibility of pregnant mice to pathogenic activity of L. monocytogenes was related to the diminution in Listeria -specific cellular reactions. Indeed, we found that non-adherent light density dendritic cells (DCs) from pregnant mice showed a marked reduction in the ability to form clusters with L. monocytogenes immune T lymphocytes and it is known that cell cluster formation between antigen presenting cells (APC) and responding T cells is required for antigen recognition as well as for cell proliferation. DCs from pregnant mice also demonstrated the decrease and an instability in the expression of H-2 class II molecules which play a crucial role in the recognition of exogenous antigens. The abnormalities demonstrated in the function of the light density dendritic cells from the spleens of pregnant mice could compromise cellular reactions to L. monocytogenes bacteria possibly resulting in increased susceptibility of pregnant mice to experimental listeriosis.     

In vitro synergistic effect of gentamicin with the anti-inflammatory agent diclofenac against Listeria monocytogenes

Aims:  A total of nine Listeria monocytogenes strains (seven serotypes) were studied to ascertain whether the non-steroidal anti-inflammatory drug diclofenac (Dc) used in combination with the conventional antilisterial antibiotic gentamicin (Gm) or ampicillin (Am) synergistically augments the efficacy of the antibiotic in vitro .
Methods and Results:  The effect of combination was evaluated by the checkerboard method to obtain a fractional inhibitory concentration (FIC) index followed by kill curves. Dc was synergistic with Gm (FIC 0·37) and there was indifference with Am (FIC 1) against L. monocytogenes ATCC 51774. The magnitude of the differences between killing by a single agent and the combination observed at 24 h was significant ( P  < 0·05) for Dc plus Gm but not Dc plus Am.
Conclusions:  Thus, the ability of extended antibiotic therapy may be improved with the help of this synergistic drug pair in listeriosis.
Significance and Impact of the Study:  Such findings may indicate parallel administration of anti-inflammatory and anti listeriosis drugs.     

Influence of immunomodulation on the development of Listeria monocytogenes infection in aged guinea pigs

We investigated the impact of immunomodulation on the development of listeriosis within an aged population of guinea pigs after an intragastric challenge with Listeria monocytogenes. Supplementation with vitamin E for 35 days significantly increased the level of cytotoxic T cells (CD8(+)), while treatment with cyclosporin A resulted in a 25% decrease of CD8(+) T cells. In the animals receiving the low dose (10(2) CFU) of L. monocytogenes, 50% of the control-group animals became infected. Only 22% of animals receiving the orthomolecular dose of vitamin E became infected, whereas animals that were immunosuppressed had an infection rate of 89%. In the immunosuppressed group three animals (16%) developed listerial infection with a quantifiable bacterial level of 0.3-3 log CFU g(-1) of organ in the spleen and liver. In the high-dose study, the population of L. monocytogenes was consistently 1 log CFU g(-1) lower in the spleen or liver of the vitamin E-supplemented group, compared with the control and cyclosporin A-treated animals. At day 4, a significant increase in the levels of CD8(+) during listerial infection occurred in vitamin E-supplemented animals, suggesting an increased ability to produce CD8(+) T cells. The results suggest that immunomodulation of the host can influence listerial infection within an aged population of guinea pigs.     

Determination of natural resistance of mice fed dietary lipids to experimental infection induced by Listeria monocytogenes

Current understanding based on the effect of dietary lipid manipulation upon immune system function indicates that fatty acids are involved in the modulation of the immune response through different and complex pathways. Reduction of several immune parameters by fatty acid action may be applied in the treatment of diseases characterised by an overactivation of the immune system. As a consequence, a reduction of host resistance against infectious agents has been reported in animals fed dietary lipids. The present study confirms the action of dietary lipids on the survival of mice infected with the pathogenic bacterium Listeria monocytogenes. A significant increase in peritoneal cells from mice fed a hydrogenated coconut oil diet was found, while a significant reduction of bacterial recovery from spleens of these mice was observed in this group. In addition, both eicosanoid and phospholipase inhibitors did not promote any modification of lymphocyte proliferation from mice fed olive oil or fish oil.     

Protective effect of Lactobacillus sakei 2a against experimental challenge with Listeria monocytogenes in gnotobiotic mice

AIM: Lactobacillus sakei 2a isolated from sausage and presenting an in vitro antagonistic activity against Listeria monocytogenes Scott A was tested for a protective effect in mice experimentally challenged with the enterobacteria. METHODS AND RESULTS: In the experimental group, germ-free mice (n = 24) were inoculated intragastrically with 0.1 ml of a suspension containing 10(8) colony forming units (CFU) of Lact. sakei and 4 days later the animals were challenged intragastrically with 0.1 ml of a suspension containing 10(8) CFU of L. monocytogenes. Control group (n = 24) was only inoculated with the bacterial pathogen. Faecal counts showed that L. monocytogenes reached similar population levels (10(9) CFU g(-1) of contents) in both the groups. Animals in the control group showed lower (P = 0.0004) survival frequency (58.3%) when compared with the experimental one (100%). Anatomopathological examination confirmed the mortality data. CONCLUSIONS: Lactobacillus sakei 2a can survive in the mammal digestive tract where showed a protective effect against L. monocytogenes. This phenomenon was not due to an antagonistic activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Use of Lact. sakei 2a as a meat starter could inhibit not only L. monocytogenes growth in the fermented product but also pathogen virulence in the gastrointestinal tract.     

Omega-3 polyunsaturated fatty acid impairment of early host resistance against Listeria monocytogenes infection is independent of neutrophil infiltration and function

The primary objective of this study was to determine whether the n-3 PUFA-mediated changes in host response to a Listeria monocytogenes infection (e.g., cytokine production and bacterial clearance) were dependent upon neutrophils. Balb/c mice were fed one of two semi-purified diets that contained either 0 or 41 g of n-3 PUFA/kg. After 4 week, mice were injected with a neutrophil-depleting (RB6-8C5) or isotype-control antibody 24 h prior to infection. Bacterial clearance from the liver and spleen at 3 days post-challenge was measured and the concentration of five pro-inflammatory cytokines in sera 24 h post-infection were determined using a novel protein multiplexing kit. We found that neutrophil depletion impaired bacterial clearance independent of the effect of n-3 PUFA. Interestingly, we observed a rather complex interaction between neutrophil-depletion and n-3 PUFA intake on in vivo pro-inflammatory cytokine production. For example, neutrophil depletion elevated circulating IL-6 and MCP-1 (2- to 5-fold; p<0.05) in n-3 PUFA-fed mice, but less so or not at all in mice fed the control diet. In summary, our data suggest that n-3 PUFA-mediated reduction of host resistance to L. monocytogenes is independent of neutrophil activity.