单增李斯特菌生物膜及其形成机制的研究进展

单增李斯特菌(Lm)是重要的人兽共患食源性病原菌。Lm生物膜与其致病性和耐药性密切相关。影响Lm生物膜形成的关键因子有鞭毛糖蛋白、胞外基质和群体感应系统等。鞭毛糖蛋白能促进菌体聚集,从而直接影响生物膜的形成。胞外DNA参与Lm粘附和生物膜早期的形成,并与胞外多糖和胞外结合蛋白一起构成生物膜胞外基质。Lm的Agr群体感应系统正调控生物膜形成,是一种集合毒力因子、耐药因子和生物膜的整体水平调控网络体系。     

Carbon Catabolite Control is Important for Listeria monocytogenes Biofilm Formation in Response to Nutrient Availability

The foodborne pathogen Listeria monocytogenes has the ability to develop biofilm in food-processing environment, which becomes a major concern for the food safety. The biofilm formation is strongly influenced by the availability of nutrients and environmental conditions, and particularly enhanced in poor minimal essential medium (MEM) containing glucose rather than in rich brain heart infusion (BHI) broth. To gain better insight into the conserved protein expression profile in these biofilms, the proteomes from biofilm- and planktonic-grown cells from MEM with 50?mM glucose or BHI were compared using two-dimensional polyacrylamide gel electrophoresis followed by MALDI-TOF/TOF analysis. 47 proteins were successfully identified to be either up (19 proteins) or down (28 proteins) regulated in the biofilm states. Most (30 proteins) of them were assigned to the metabolism functional category in cluster of orthologous groups of proteins. Among them, up-regulated proteins were mainly associated with the pentose phosphate pathway and glycolysis, whereas a key enzyme CitC involved in tricarboxylic acid cycle was down-regulated in biofilms compared to the planktonic states. These data implicate the importance of carbon catabolite control for L. monocytogenes biofilm formation in response to nutrient availability.     

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.     

The growth and resistance to sodium hypochlorite of Listeria monocytogenes in a steady-state multispecies biofilm

A constant-depth film fermenter (CDFF) was used to culture a steady-state multispecies biofilm consisting of one strain each of Listeria monocytogenes, Pseudomonas fragi and Staphylococcus xylosus. These bacteria were initially grown together in a conventional chemostat to achieve a steady state before being inoculated into the CDFF over an 18-h period. A dilute tryptone soya broth (TSB) medium was supplied to the CDFF and the biofilm allowed to develop over a 28-d period. This mature biofilm was then subjected to increasing levels of sodium hypochlorite solution to measure any antimicrobial effect. The three organisms were seen to reach a steady state after 6 d in the chemostat before being transferred to the CDFF where the mature multispecies biofilm reached steady state at 17 d. Listeria monocytogenes in both planktonic and biofilm growth stabilized at 1. 8 and 1.5%, respectively, of the total plate counts, while Ps. fragi and Staph. xylosus were the predominant organisms in the biofilm at 59% and 39.5%, respectively, of the total microbial population. Steady-state biofilms in the CDFF were exposed to increasing strengths of sodium hypochlorite; 200, 500 and 1000 p.p.m. free chlorine, but a substantial two-log cycle drop in bacterial numbers was only achieved at 1000 p.p.m. free chlorine. In planktonic culture all three organisms were completely eliminated when exposed to 10 p.p.m. free chlorine for a 30-s period.     

Small-molecule modulators of Listeria monocytogenes biofilm development

Listeria monocytogenes is an important food-borne pathogen whose ability to form disinfectant-tolerant biofilms on a variety of surfaces presents a food safety challenge for manufacturers of ready-to-eat products. We developed here a high-throughput biofilm assay for L. monocytogenes and, as a proof of principle, used it to screen an 80-compound protein kinase inhibitor library to identify molecules that perturb biofilm development. The screen yielded molecules toxic to multiple strains of Listeria at micromolar concentrations, as well as molecules that decreased (≤ 50% of vehicle control) or increased (≥ 200%) biofilm formation in a dose-dependent manner without affecting planktonic cell density. Toxic molecules-including the protein kinase C antagonist sphingosine-had antibiofilm activity at sub-MIC concentrations. Structure-activity studies of the biofilm inhibitory compound palmitoyl-d,l-carnitine showed that while Listeria biofilm formation was inhibited with a 50% inhibitory concentration of 5.85 ± 0.24 μM, d,l-carnitine had no effect, whereas palmitic acid had stimulatory effects. Saturated fatty acids between C(9:0) and C(14:0) were Listeria biofilm inhibitors, whereas fatty acids of C(16:0) or longer were stimulators, showing chain length specificity. De novo-synthesized short-chain acyl carnitines were less effective biofilm inhibitors than the palmitoyl forms. These molecules, whose activities against bacteria have not been previously established, are both useful probes of L. monocytogenes biology and promising leads for the further development of antibiofilm strategies.     

Comparison of protein patterns of Listeria monocytogenes grown in biofilm or in planktonic mode by proteomic analysis

The proteome of a Listeria monocytogenes strain isolated from a food plant was investigated to study the differential protein pattern expressed by biofilms and planktonic bacteria. The approach used in this study was a combination of two-dimensional electrophoresis, matrix-assisted laser desorption ionization-time of flight and database searches for the protein identification. Thirty-one proteins varied significantly between the two growth conditions. Twenty-two and nine proteins were up- and down-regulated respectively and nine proteins were successfully identified. The variations of the protein patterns indicated that the biofilm development is probably controlled by specific regulation of protein expression involved at various levels of cellular physiology.     

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.     

Biofilm formation by Salmonella spp. and Listeria monocytogenes on plastic surface

AIMS: To investigate the biofilm formation by 122 Salmonella spp. and 48 Listeria monocytogenes strains on a plastic surface. METHODS: Quantification of biofilm formation was performed in brain heart infusion (BHI), trypcase soya broth (TSB), meat broth (MB) and 1/20 diluted trypcase soya broth (1/20-TSB) in plastic microtitre plates. RESULTS: All tested Salmonella spp. and L. monocytogenes strains produced biofilm in a suitable medium. However, the quantities of biofilm produced by Salmonella spp. were greater than those produced by tested L. monocytogenes strains. The nutrient content of the medium significantly influenced the quantity of produced biofilm. Diluted TSB was the most effective in promoting biofilm production by Salmonella spp., followed by TSB, while the least quantity of biofilm was formed in BHI and MB. L. monocytogenes produced the highest quantities of biofilm in BHI, followed by TSA, then MB, and the least quantities of biofilm were produced in 1/20-TSB. CONCLUSIONS: Salmonella spp. produces more biofilm in nutrient-poor medium, while L. monocytogenes produce more biofilm in nutrient-rich medium.     

利用转座子Tn917构建单核细胞增生李斯特菌菌膜形成突变株

单核细胞增生李斯特菌菌膜形成相关基因和调控因子的分离和鉴定是阐明其菌膜形成分子机理的基础。利用原生质体转化这一方式,将带有转座子Tn917的质粒pTV1OK成功地转进了单核细胞增生李斯特菌。通过诱导Tn917转座,得到单核细胞增生李斯特菌Tn917插入突变库,转座率为10-7。经96孔细胞培养板筛选发现,菌株LM49形成菌膜能力明显大于野生型。该菌株在细胞培养板中培养４d后形成的紫色圆环的颜色明显深于野生型。用Tn917特异引物进行PCR扩增,结果显示只有以该突变株的DNA为模板才能得到相应大小的扩增产物,证实该菌株基因组中有Tn917插入。Tn917的插入使菌株LM49的菌膜形成能力增强。     

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.     

VirAB在单核细胞增生李斯特菌耐药性及生物被膜形成中的作用

【背景】单核细胞增生李斯特菌(Listeria monocytogenes,Lm)对一些临床常用抗生素、乳酸链球菌素(Nisin)等抗菌药物的敏感性下降,然而其背后的机制仍未完全阐明。【目的】调查转运蛋白VirAB在Lm对抗菌药物的耐药性及生物被膜形成中的作用。【方法】利用同源重组技术构建Lm基因缺失突变株,比较野生株和缺失株对抗菌药物的耐药性;利用微孔板法观测突变株生物被膜形成能力的变化;利用平板泳动法研究菌株的泳动能力。【结果】与野生株相比,virAB缺失突变株对头孢类抗生素、Nisin和溴化乙锭的敏感性增加;当培养基中分别添加亚致死浓度的苯扎氯铵、卡那霉素和四环素时,突变株均表现出不同程度的生长缺陷。缺失virAB后菌株形成生物被膜的能力下降。【结论】VirAB在Lm对头孢类等抗菌药物的耐药及生物被膜形成方面具有重要作用。     

Variation in biofilm formation among strains of Listeria monocytogenes

Contamination of food by Listeria monocytogenes is thought to occur most frequently in food-processing environments where cells persist due to their ability to attach to stainless steel and other surfaces. Once attached these cells may produce multicellular biofilms that are resistant to disinfection and from which cells can become detached and contaminate food products. Because there is a correlation between virulence and serotype (and thus phylogenetic division) of L. monocytogenes, it is important to determine if there is a link between biofilm formation and disease incidence for L. monocytogenes. Eighty L. monocytogenes isolates were screened for biofilm formation to determine if there is a robust relationship between biofilm formation, phylogenic division, and persistence in the environment. Statistically significant differences were detected between phylogenetic divisions. Increased biofilm formation was observed in Division II strains (serotypes 1/2a and 1/2c), which are not normally associated with food-borne outbreaks. Differences in biofilm formation were also detected between persistent and nonpersistent strains isolated from bulk milk samples, with persistent strains showing increased biofilm formation relative to nonpersistent strains. There were no significant differences detected among serotypes. Exopolysaccharide production correlated with cell adherence for high-biofilm-producing strains. Scanning electron microscopy showed that a high-biofilm-forming strain produced a dense, three-dimensional structure, whereas a low-biofilm-forming strain produced a thin, patchy biofilm. These data are consistent with data on persistent strains forming biofilms but do not support a consistent relationship between enhanced biofilm formation and disease incidence.     

Spatial competition with Lactococcus lactis in mixed-species continuous-flow biofilms inhibits Listeria monocytogenes growth

Surfaces in industrial settings provide a home for resident biofilms that are likely to interact with the attachment, growth and survival of pathogens such as Listeria monocytogenes. Experimental results have indicated that L. monocytogenes cells were inhibited by the presence of a model resident flora (Lactococcus lactis) in dual-species continuous flow-biofilms, and are spatially restricted to the lower biofilm layers. Using a new, simplified individual-based model (IBM) that simulates bacterial cell growth in a three-dimensional space, the spatial arrangements of the two species were reconstructed and their cell counts successfully predicted. This model showed that the difference in generation times between L. monocytogenes and L. lactis cells during the initial stages of dual-species biofilm formation was probably responsible for the species spatialization observed and the subsequent inhibition of growth of the pathogen.     

Listeria monocytogenes virulence proteins induce surface expression of Fas ligand on T lymphocytes

Virulence factors secreted by Listeria monocytogenes are known to interfere with host cellular signalling pathways. We investigated whether L. monocytogenes modulates T-cell receptor signalling by examining surface expression of proteins known to be upregulated on activated T cells. In vitro culture of murine splenocytes with L. monocytogenes resulted in a specific and dose-dependent upregulation of Fas ligand (FasL). Induction of FasL expression was also observed for pathogenic Listeria ivanovii but not for non-pathogenic Listeria innocua, indicating involvement of Listeria virulence protein(s). Examination of L. monocytogenes strains deficient in different virulence genes demonstrated that FasL upregulation was dependent on the expression of two secreted proteins: listeriolysin O (LLO) and phosphatidylcholine-preferring phospholipase C (PC-PLC). Treatment of cells with purified proteins demonstrated that LLO was sufficient for inducing FasL, while PC-PLC synergized with LLO for the induction of FasL expression. FasL-expressing cells induced by L. monocytogenes were capable of killing Fas-expressing target cells. Furthermore, L. monocytogenes infection results in upregulation of FasL on T cells in mice. These results describe a novel function for LLO and PC-PLC and suggest that L. monocytogenes may use these virulence factors to modulate the host immune response.     

The differential adherence capabilities of two Listeria monocytogenes strains in monoculture and multispecies biofilms as a function of temperature

AIMS: To determine the differential adherence capabilities at three different temperatures of Listeria monocytogenes Scott A, a clinical food pathogen, and L. monocytogenes FM876, a persistent strain from a milk-processing environment, to stainless steel. METHODS AND RESULTS: Differential adherence was investigated by submerging stainless steel coupons in both 48-h Listeria monocultures and mixed cultures additionally containing Staphylococcus xylosus DP5H and Pseudomonas fragi ATCC 4973. Immunofluorescent microscopy and image analysis techniques were utilized to identify and quantify the L. monocytogenes cells adhering to the steel at 4 degrees C, 18 degrees C and 30 degrees C. The monoculture biofilms consistently contained greater L. monocytogenes numbers than the multispecies biofilms, with the persistent strain FM876 showing significantly greater adherence than strain Scott A. Optimum adherence occurred at 18 degrees C in monoculture biofilms. CONCLUSION: L. monocytogenes strains exhibit differential, temperature-dependent, adherence to stainless steel. SIGNIFICANCE AND IMPACT OF THE STUDY: These results demonstrate temperature dependent biofilm adherence and support previous findings that persistent strains exhibit increased adherence capability.     

Flagellar motility is critical for Listeria monocytogenes biofilm formation

The food-borne pathogen Listeria monocytogenes attaches to environmental surfaces and forms biofilms that can be a source of food contamination, yet little is known about the molecular mechanisms of its biofilm development. We observed that nonmotile mutants were defective in biofilm formation. To investigate how flagella might function during biofilm formation, we compared the wild type with flagellum-minus and paralyzed-flagellum mutants. Both nonmotile mutants were defective in biofilm development, presumably at an early stage, as they were also defective in attachment to glass during the first few hours of surface exposure. This attachment defect could be significantly overcome by providing exogenous movement toward the surface via centrifugation. However, this centrifugation did not restore mature biofilm formation. Our results indicate that it is flagellum-mediated motility that is critical for both initial surface attachment and subsequent biofilm formation. Also, any role for L. monocytogenes flagella as adhesins on abiotic surfaces appears to be either minimal or motility dependent under the conditions we examined.     

单增李斯特菌生物膜形成调控机制的研究进展

单增李斯特菌是一种重要的食源性病原菌。单增李斯特菌的分布和存活与其形成生物膜的能力有关,生物膜对逆性环境有抵抗力,细菌会从生物膜中分离导致食品持续性的污染。生物膜的形成、成熟和结构取决于多种外部和内部因素,并且多种调控机制起着重要作用。文中旨在阐述单增李斯特菌生物膜形成过程中的调控机制(包括胞内作用、胞间作用和种间作用),以控制食品加工环境中致病性生物膜的形成,从而为食品安全提供新的干预策略。     

Microtiter plate assay for assessment of Listeria monocytogenes biofilm formation

Listeria monocytogenes has the ability to form biofilms on food-processing surfaces, potentially leading to food product contamination. The objective of this research was to standardize a polyvinyl chloride (PVC) microtiter plate assay to compare the ability of L. monocytogenes strains to form biofilms. A total of 31 coded L. monocytogenes strains were grown in defined medium (modified Welshimer's broth) at 32 degrees C for 20 and 40 h in PVC microtiter plate wells. Biofilm formation was indirectly assessed by staining with 1% crystal violet and measuring crystal violet absorbance, using destaining solution. Cellular growth rates and final cell densities did not correlate with biofilm formation, indicating that differences in biofilm formation under the same environmental conditions were not due to growth rate differences. The mean biofilm production of lineage I strains was significantly greater than that observed for lineage II and lineage III strains. The results from the standardized microtiter plate biofilm assay were also compared to biofilm formation on PVC and stainless steel as assayed by quantitative epifluorescence microscopy. Results showed similar trends for the microscopic and microtiter plate assays, indicating that the PVC microtiter plate assay can be used as a rapid, simple method to screen for differences in biofilm production between strains or growth conditions prior to performing labor-intensive microscopic analyses.     

Limitation of adhesion and growth of Listeria monocytogenes on stainless steel surfaces by Staphylococcus sciuri biofilms

The adhesion and subsequent development of Listeria monocytogenes on stainless steel was studied in the absence and in the presence of a Staphylococcus sciuri biofilm. In the three growth media studied, the percentage of adherent cells was reduced to nearly the same extent by the presence of 1-day biofilms of Staph. sciuri for the two strains of L. monocytogenes studied. One-day biofilms of Staph. sciuri exhibited the same exopolysaccharide content per square centimetre, although they colonized from 3.5 to 35% of the stainless steel depending on the growth media. This suggests that extracellular substances rather than cell-to-cell interactions were involved in the decreased adhesion. After 3 days of culture, Staphylococcus biofilms prevented the adherent L. monocytogenes population from increasing within the biofilm, leading to an average logarithmic cfu difference of 0.9-2.7 between the pure and mixed culture. A competition for nutrients by Staph. sciuri was observed in one of the three media. A role for extracellular polysaccharides produced by the Staphylococcus biofilm in preventing the adhesion of L. monocytogenes and in modifying the balance existing between its planktonic and biofilm phase is hypothesized. A higher proportion of L. monocytogenes cells was observed in the planktonic phase in mixed cultures, suggesting that the extracellular substances produced by Staph sciuri biofilms and involved in the decreased adhesion of L. monocytogenes could modify the balance existing between planktonic and biofilm populations. In addition, co-cultures of L. monocytogenes and Staph. sciuri in broth showed competition for nutrients for Staph. sciuri in one of the three media.