Survival and biofilm formation of Listeria monocytogenes in simulated vaginal fluid: influence of pH and strain origin

Listeria monocytogenes, the agent responsible for listeriosis, can be transmitted from mother to fetus/neonates by vertical transmission, transplacentally or during passage through the birth canal. The purpose of this study was to investigate the survival and biofilm formation of L. monocytogenes (isolated from clinical cases or from food) in simulated vaginal fluid at different pH values (4.2, 5.5 and 6.5). The results demonstrated that this pathogen is inhibited by the normal vaginal pH, but may proliferate when it increases. Clinical strains were significantly more resistant to pH 4.2 than food isolates. Listeria monocytogenes survived and even grew at the higher pHs investigated, suggesting that fetus/neonates from women having increased vaginal pH values during pregnancy may be at a higher risk of listeriosis. All isolates tested were producers of biofilm at different pH values; however, L. monocytogenes produced higher quantities of biofilm in a nutrient-rich medium. No significant differences in biofilm production were detected between food and clinical isolates. As L. monocytogenes are biofilm producers, this increases the probability of occurrence of neonatal infection.     

Genome sequence of Listeria monocytogenes Scott A, a clinical isolate from a food-borne listeriosis outbreak

Listeria monocytogenes is an opportunistic food-borne pathogen and the causative agent of listeriosis in animals and humans. We present the genome sequence of Listeria monocytogenes Scott A, a widely distributed and frequently used serovar 4b clinical isolate from the 1983 listeriosis outbreak in Massachusetts.     

新生儿及产妇感染单核细胞增生李斯特菌分离株的血清型、耐药性及分子特征研究

为探讨广西南宁地区新生儿及产妇感染的单核细胞增生李斯特菌(Listeria monocytogenes, Lm)的血清型、药物敏感性及其分子流行病学特征,本研究回顾性收集2015-2017年广西壮族自治区妇幼保健院新生儿科及产科送检标本中分离的Lm,对其进行体外药物敏感性检测、血清学分型以及多位点序列分型(multilocus sequence typing, MLST)分析菌株间的同源性;同时分析患儿及其母亲的临床特征及危险因素。结果显示,广西南宁地区新生儿感染Lm发病率较低,2015-2017年发病率为0.091‰;所有分离的Lm分属4b(83.3%)和1/2a(16.7%)2个血清型;药物敏感性试验结果显示,Lm对青霉素、氨苄西林、复方新诺明及美罗培南均100%敏感,暂未发现耐药菌株;MLST分型共获得2个序列型(sequence types,ST),以ST­1型(83.3%)为主。其中分离自同一新生儿患者(Case 2)外周血(Lm2)、耳拭子(Lm3)及其母亲羊水(Lm4)、宫颈分泌物(Lm5)的4株菌具有相同的血清型、药物敏感性表型以及MLST分型。感染Lm的患儿主要表现为发热、肺炎、发绀、败血症及脑膜炎;而产妇感染则具有非特异性的临床特征。结果提示,广西南宁地区存在的Lm菌株为致病性较强的4b、1/2a血清型菌株;Lm可通过母婴垂直传播引起新生儿感染。因此,临床医师应重视孕产妇及新生儿Lm病原学检查、早期诊断和及时合理地使用抗生素预防、治疗,从而减少Lm引起的母婴感染。     

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.     

Murine pulmonary infection with Listeria monocytogenes: differential susceptibility of BALB/c, C57BL/6 and DBA/2 mice

Murine listeriosis is a paradigm to understand host pathogen interactions. Airway infections with Listeria monocytogenes, although representing a serious problem in early onset neonatal listeriosis, has not been investigated in detail in animal models so far. Here, the susceptibility of BALB/c, DBA/2 and C57BL/6 mice towards an intratracheal (i.t.) infection with virulent L. monocytogenes EGDe and the attenuated variant L. monocytogenes EGD hlyW491A(pERL3-CMVGFP) is reported. The course of infection was characterized by determination of bacterial numbers in the organs and assessment of the health condition of the mice. The distribution and cellular localization of Listeria in the airways was assessed by immunocytochemistry and confocal and electron microscopy. The differential susceptibility of inbred mouse strains to airway infections with L. monocytogenes could be assigned to the major virulence factor listeriolysin O. Resistant C57BL/6 mice were not affected by the two listerial strains. In contrast, BALB/c and DBA/2 mice showed differential susceptibility towards L. monocytogenes EGDe and attenuated bacteria, with all the mice being killed by the wild-type bacteria but rarely by the variant that secretes a listeriolysin of only 10% activity of that of the wild-type toxin. Thus, listeriolysin is a decisive factor for differential susceptibility against Listeria. After i.t. application, bacteria were predominantly localized in the peribronchiolar space and invaded alveolar macrophages but rarely lung epithelial cells. Dissemination from the lung into the deep organs started almost immediately after application, although a pulmonary bacterial reservoir remained during the first 4 days.     

Regulation of Listeria virulence: PrfA master and commander

Listeria monocytogenes is the causative agent of listeriosis, a severe foodborne infection. These bacteria live as soil saprotrophs on decaying plant matter but also as intracellular parasites, using the cell cytosol as a replication niche. PrfA, a regulatory protein, integrates a number of environmental cues that signal the transition between these two contrasting lifestyles, activating a set of key virulence factors during host infection. While a number of details concerning the general mode of action of this virulence master switch have been elucidated, others remain unsolved. Recent work has revealed additional mechanisms that contribute to L. monocytogenes virulence modulation, often via cross-talk with PrfA, or by regulating new genes involved in host colonization.     

Molecular typing by pulsed-field gel electrophoresis of Spanish animal and human Listeria monocytogenes isolates.

A total of 153 strains of Listeria monocytogenes isolated from different sources (72 from sheep, 12 from cattle, 18 from feedstuffs, and 51 from humans) in Spain from 1989 to 2000 were characterized by pulsed-field gel electrophoresis. The strains of L. monocytogenes displayed 55 pulsotypes. The 84 animal, 51 human, and 18 feedstuff strains displayed 31, 29, and 7 different pulsotypes, respectively, indicating a great genetic diversity among the Spanish L. monocytogenes isolates studied. L. monocytogenes isolates from clinical samples and feedstuffs consumed by the diseased animals were analyzed in 21 flocks. In most cases, clinical strains from different animals of the same flock had identical pulsotypes, confirming the existence of a listeriosis outbreak. L. monocytogenes strains with pulsotypes identical to those of clinical strains were isolated from silage, potatoes, and maize stalks. This is the first study wherein potatoes and maize stalks are epidemiologically linked with clinical listeriosis.     

Efficacy of a Lactococcus lactis ΔpyrG vaccine delivery platform expressing chromosomally integrated hly from Listeria monocytogenes

Listeria monocytogenes is a significant food-borne pathogen and the causative agent of listeriosis, a disease which manifests as meningitis in immunocompromised adults or infection of the fetus and miscarriage in pregnant women. We have previously used Lactococcus lactis, a GRAS (Generally Regarded As Safe) organism, as a vaccine vector against listeriosis by engineering plasmid-mediated expression of the immunodominant antigen from L. monocytogenes, listeriolysin O (LLO). However, the environmental release of an engineered vaccine vector carrying a replicating plasmid during clinical usage may raise safety concerns. Here we describe the integration of the LLO gene (hly) into the L. lactis chromosome through homologous double crossover to allow stable expression, in order to avoid the use of antibiotic selection markers and to eliminate the requirement for a plasmid-based system. The approach was designed to simultaneously eliminate the pyrG gene encoding the CTP synthase which is responsible for converting UTP to CTP in a unique step in the de novo pyrimidine synthesis in L. lactis. This gene was targeted in order to restrict bacterial replication outside of the host (biological containment). The resulting cytidine auxotroph was able to secrete LLO constitutively and could elicit LLO(91-99)-specific CD8(+) T lymphocytes in the murine infection model. Moreover, protection against lethal challenge with L. monocytogenes was accomplished after intraperitoneal (IP) vaccination with the constructed strain. The implications for the use of cytidine auxotropy in biological containment are discussed.     

Human listeriosis and animal models

Human listeriosis is a potentially fatal foodborne infection caused by Listeria monocytogenes, an opportunistic psychrophile bacterium that is widespread in the environment. It has only recently emerged as a significant cause of human infection in industrialized countries, owing to appearance of a vulnerable population of immunocompromised individuals, and the concomitant development of large-scale agro-industrial plants and refrigerated food. Here we review the main clinical features of human listeriosis and highlight specificities and similarities with animal listeriosis in diverse species. Finally, we present some of the critical determinants for the choice of an appropriate animal model to study human listeriosis.     

Real-time PCR assay to differentiate Listeriolysin S-positive and -negative strains of Listeria monocytogenes

Due to the severity of the food-borne infection listeriosis, strict legislation governs the detectable and permissible limits at which Listeria monocytogenes is permitted in foods. These requirements, coupled with the ubiquitous nature of L. monocytogenes strains and the potential for epidemic outbreaks, mean that the pathogen can devastate affected sectors of the food industry. Although almost all L. monocytogenes strains have the potential to cause listeriosis, those implicated in the vast majority of epidemics belong to a subset of strains belonging to evolutionary lineage I. It has been established that a significant proportion of these strains, including those implicated in the majority of outbreaks, produce an additional hemolysin, designated listeriolysin S (LLS), which may be responsible for the enhanced virulence of these strains. In order to ultimately establish this definitively, it is important to first be able to rapidly discriminate between LLS-positive and -negative strains. Here, after essential genes within the LLS-encoding cluster, Listeria pathogenicity island 3, were identified by deletion mutagenesis, a real-time PCR assay which targets one such gene, llsX, was developed as a means of identifying LLS-positive L. monocytogenes. The specificity of the assay was validated against a panel of 40 L. monocytogenes strains (20 of which were LLS positive) and 25 strains representative of other Listeria species. Furthermore, 1 CFU of an LLS-positive strain per 25 g/ml of spiked foods was detected in less than 30 h when the assay was coupled with culture enrichment. The detection limit of this assay was 10 genome equivalents.     

Antibodies to listeriolysin O reflect the acquired resistance to Listeria monocytogenes in experimentally infected goats

We induced experimental listeriosis in goats by two sequential oral inoculations of Listeria monocytogenes serovar 1/2a at 8 months' interval. Immunoblot analysis with the goat sera demonstrated listeriolysin O (LLO) as the principal protein antigen of L. monocytogenes. Pre-existing antibodies to LLO were, depending on their initial level, associated with either mild clinical symptoms of short duration or the total absence of clinical symptoms. Similarly, the presence and development of such antibodies corresponded with the disappearance pattern of L. monocytogenes from the gastrointestinal tract. These findings suggest that an association exists between antibodies to LLO and acquired resistance to Listeria infections.     

Mast cells initiate early anti-Listeria host defences

The Gram-positive bacterium Listeria monocytogenes ( L. m. ) is the aetiological agent of listeriosis. The early phase listeriosis is characterized by strong innate host responses that play a major role in bacterial clearance. This is emphasized by the fact that mice deficient in T and B cells have a remarkable ability to control infection. Mast cells, among the principal effectors of innate immunity, have largely been studied in the context of hyper-reactive conditions such as allergy and autoimmune diseases. In the present study, we evaluated the significance of mast cells during the early phase of listeriosis. Compared with controls, mice depleted of mast cells showed hundred-fold higher bacterial burden in spleen and liver and were significantly impaired in neutrophil mobilization. Although L. m. interacts with and triggers mast cell degranulation, bacteria were hardly found within such cells. Mainly neutrophils and macrophages phagozytosed L. m . Thus, mast cells control infection not via direct bacterial uptake, but by initiating neutrophils influx to the site of infection. We show that this is initiated by pre-synthesized TNF-α, rapidly secreted by mast cell upon activation by L. m . We also show that upon recruitment, neutrophils also become activated and additionally secrete TNF-α thus amplifying the anti- L. m. inflammatory response.     

Bias in the Listeria monocytogenes enrichment procedure: lineage 2 strains outcompete lineage 1 strains in University of Vermont selective enrichments

Listeria monocytogenes can be isolated from a range of food products and may cause food-borne outbreaks or sporadic cases of listeriosis. L. monocytogenes is divided into three genetic lineages and 13 serotypes. Strains of three serotypes (1/2a, 1/2b, and 4b) are associated with most human cases of listeriosis. Of these, strains of serotypes 1/2b and 4b belong to lineage 1, whereas strains of serotype 1/2a and many other strains isolated from foods belong to lineage 2. L. monocytogenes is isolated from foods by selective enrichment procedures and from patients by nonselective methods. The aim of the present study was to investigate if the selective enrichment procedure results in a true representation of the subtypes of L. monocytogenes present in a sample. Eight L. monocytogenes strains (four lineage 1 strains and four lineage 2 strains) and one Listeria innocua strain grew with identical growth rates in the nonselective medium brain heart infusion (BHI), but differed in their growth rate in the selective medium University of Vermont medium I (UVM I). When coinoculated in UVM I, some strains completely outgrew other strains. This outcome was dependent on the lineage of L. monocytogenes rather than the individual growth rate of the strains. When inoculated at identical cell densities in UVM I, L. innocua outcompeted L. monocytogenes lineage 1 strains but not lineage 2 strains. In addition, lineage 2 L. monocytogenes strains outcompeted lineage 1 L. monocytogenes strains in all combinations tested, indicating a bias in strains selected by the enrichment procedures. Bias also occurred when coinoculating two lineage 2 or lineage 1 strains; however, it did not appear to correlate with origin (clinical versus food). Identical coinoculation experiments in BHI suggested that the selective compounds in UVM I and II influenced this bias. The results of the present study demonstrate that the selective procedures used for isolation of L. monocytogenes may not allow a true representation of the types present in foods. Our results could have a significant impact on epidemiological studies, as lineage 1 strains, which are often isolated from clinical cases of listeriosis, may be suppressed during enrichment by other L. monocytogenes lineages present in a food sample.     

单核细胞增生李斯特菌的检测技术进展

单核细胞增生李斯特菌（Listeria monocytogenes）是一类人畜共患的食源性致病菌。近年来其检测技术取得了迅猛的发展,本文对目前使用的基于培养、免疫学和分子生物学技术的三大类单核细胞增生李斯特菌检测方法进行了综述,同时对单核细胞增生李斯特菌检测的新策略进行了展望。     

The protein secretion systems in Listeria: inside out bacterial virulence

Listeria monocytogenes, the etiologic agent of listeriosis, remains a serious public health concern with its frequent occurrence in food coupled with a high mortality rate. The capacity of a bacterium to secrete proteins to or beyond the bacterial cell surface is of crucial importance in the understanding of biofilm formation and bacterial pathogenesis to further develop defensive strategies. Recent findings in protein secretion in Listeria together with the availability of complete genome sequences of several pathogenic L. monocytogenes strains, as well as nonpathogenic Listeria innocua Clip11262, prompted us to summarize the listerial protein secretion systems. Protein secretion would rely essentially on the Sec (Secretion) pathway. The twin-arginine translocation pathway seems encoded in all but one sequenced Listeria. In addition, a functional flagella export apparatus, a fimbrilin-protein exporter, some holins and a WXG100 secretion system are encoded in listerial genomes. This critical review brings new insights into the physiology and virulence of Listeria species.     

Listeria monocytogenes: epidemiology, human disease, and mechanisms of brain invasion

Listeria monocytogenes is a facultative intracellular bacterium that has predilection for causing central nervous systemic infections in humans and domesticated animals. This pathogen can be found worldwide in the food supply and most L. monocytogenes infections are acquired through ingestion of contaminated food. The main clinical syndromes caused by L. monocytogenes include febrile gastroenteritis, perinatal infection, and systemic infections marked by central nervous system infections with or without bacteremia. Experimental infection of mice has been used for over 50 years as a model system to study the pathogenesis of this organism including the mechanisms by which it invades the brain. Data from this model indicate that a specific subset of monocytes, distinguished in part by high expression of the Ly-6C antigen, become parasitized in the bone marrow and have a key role in transporting intracellular bacteria across the blood-brain barriers and into the central nervous system. This Minireview will summarize recent epidemiologic and clinical information regarding L. monocytogenes as a human pathogen and will discuss current in vitro and in vivo data relevant to the role of parasitized monocytes and the pathogenetic mechanisms that underlie its formidable ability to invade the central nervous system.     

Mechanisms of pathogenesis in Listeria monocytogenes infection. I. Influence of iron

Sword, C. P. (The University of Kansas, Lawrence). Mechanisms of pathogenesis in Listeria monocytogenes infection. I. Influence of iron. J. Bacteriol. 92: 536-542. 1966.-The effects of ferric and ferrous iron as well as other cations on Listeria infection in mice were studied. Iron compounds caused a reduction in the ld(50) dose of Listeria, and, when added to a synthetic medium, proved stimulatory for in vitro growth of the organism. Bacterial counts on spleen and liver tissue from iron-treated mice showed that iron injections caused more rapid growth of bacteria and resulted in higher numbers of organisms in the tissue. The reticuloendothelial system did not appear to be impaired by this treatment. Immunized animals were not affected by iron treatment during challenge. Mice with experimentally induced hemolytic anemia showed increased susceptibility to listeriosis, whereas those treated with Desferal, a specific iron-chelating agent, appeared more resistant. Iron proved stimulatory for the avirulent strain, 9037-7, and resulted in an ld(50) of 1.3 x 10(4) organisms in iron-treated animals. Growth of L. monocytogenes and mortality from experimental infection appeared to be correlated with availability of iron to the bacteria. The results suggest that host iron metabolism may play a part in the onset and progress of Listeria infections.     

Influence of staphylococcal enterotoxin B (SEB) on the course of murine listeriosis

Abstract Confrontation of the immune system with bacterial superantigens leads to an initial activation of the immune system followed by a state of profound immunosuppression. To investigate the role of a superantigen in an acute infection with a facultatively intracellular bacterium, we have studied the effect of staphylococcal enterotoxin B on the course of murine listeriosis. Intraperitoneal injection of SEB led to a statistically significant growth restriction of Listeria monocytogenes in the organs of mice infected intravenously or intraperitoneally when treatment with SEB and infection with L. monocytogenes were given simultaneously or when the mice were treated two days before infection. No effect of SEB on murine listeriosis was found when SEB was given more than two days before infection or one or more days after infection. We conclude that initial immunostimulation by SEB which is indicated by a massive liberation of all interleukins measured (IL1α, IL6, TNFα, IL2, IFNγ, IL4) is responsible for the growth restriction of L. monocytogenes in the organs of treated mice. Apoptosis of Vβ8 positive T cells which was accompanied by a 30% reduction of these cells at day 7 after treatment seems to be totally compensated.     

Inhibition of calpain blocks the phagosomal escape of Listeria monocytogenes

Listeria monocytogenes is a gram-positive facultative intracellular bacterium responsible for the food borne infection listeriosis, affecting principally the immunocompromised, the old, neonates and pregnant women. Following invasion L. monocytogenes escapes the phagosome and replicates in the cytoplasm. Phagosome escape is central to L. monocytogenes virulence and is required for initiating innate host-defence responses such as the secretion of the cytokine interleukin-1. Phagosome escape of L. monocytogenes is reported to depend upon host proteins such as γ-interferon-inducible lysosomal thiol reductase and the cystic fibrosis transmembrane conductance regulator. The host cytosolic cysteine protease calpain is required in the life cycle of numerous pathogens, and previous research reports an activation of calpain by L. monocytogenes infection. Thus we sought to determine whether host calpain was required for the virulence of L. monocytogenes. Treatment of macrophages with calpain inhibitors blocked escape of L. monocytogenes from the phagosome and consequently its proliferation within the cytosol. This was independent of any direct effect on the production of bacterial virulence factors or of a bactericidal effect. Furthermore, the secretion of interleukin-1β, a host cytokine whose secretion induced by L. monocytogenes depends upon phagosome escape, was also blocked by calpain inhibition. These data indicate that L. monocytogenes co-opts host calpain to facilitate its escape from the phagosome, and more generally, that calpain may represent a cellular Achilles heel exploited by pathogens.     

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.