猪链球菌生物被膜形成的耐药机制

猪链球菌病(Streptococcus suis)是一种严重影响各国养猪业发展和人类健康的人兽共患传染病,可以引起败血症、关节炎、脑膜炎等多种疾病,造成巨大的经济损失.猪链球菌生物被膜的形成是导致其致病性和耐药性增加的主要原因.为了预防和治疗猪链球菌病以及解析其耐药的可能机制,深入了解和掌握猪链球菌生物被膜的形成和耐药...     

Neutrophil extracellular Taps play an important role in clearance of Streptococcus suis in vivo

Streptococcus suis infection induces formation of neutrophil extracellular traps (NETs) in vitro; however, the contribution of NETs‐mediated killing to the pathogenesis of S. suis in vivo is yet to be elicited. The findings of the present study indicated that extracellular DNA fiber can be induced in a murine model in response to S. suis infection. A nuclease that destroys their structure was used to evaluate the role of NETs on S. suis infection. Treatment with nuclease resulted in a greater bacteria load and higher serum TNF‐α concentrations in response to S. suis infection, indicating that NETs structure played an essential role in S. suis clearance and inflammation. Furthermore, nuclease treatment resulted in more severe clinical signs during and higher mortality from S. suis infection. These findings indicated that NETs structure contributes to protection against S. suis infection.     

Effect of Licochalcone A on Growth and Properties of Streptococcus suis

Streptococcus suis (S.suis) is an important emerging worldwide pig pathogen and zoonotic agent with rapid evolution of virulence and drug resistance. In this study, we wanted to investigate the effect of licochalcone A on growth and properties of Streptococcus suis. The antimicrobial activity of licochalcone A was tested by growth inhibition assay and the minimal inhibitory concentrations (MICs) also were determined. The effect of licochalcone A on S.suis biofilm formation was characterized by crystal violet staining. The effect of licochalcone A on suilysin secretion was evaluated by titration of hemolytic activity. To understand the antimicrobial effect, gene expression profile of S.suis treated by licochalcone A was analyzed by DNA microarray. Our results demonstrated that licochalcone A showed antimicrobial activity on S.suis with MICs of 4 µg/ml for S.suis serotype 2 strains and 8 µg/ml for S.suis serotype 7 strains. Biofilm formation was inhibited by 30–40% in the presence of licochalcone A (3 µg/ml) and suilysin secretion was also significantly inhibited in the presence of licochalcone A (1.5 µg/ml). The gene expression profile of S.suis in the presence of licochalcone A showed that 132 genes were differentially regulated, and we analyzed the regulated genes in the aspect of the bacterial cell cycle control. Among the deregulated genes, the genes responsible for the mass doubling was increased expression, but the genes responsible for DNA replication and cell division were inhibited the expression. So, we think the regulation of the cell cycle genes might provide a mechanistic understanding of licochalcone A mediated antimicrobial effect against S.suis.     

Impact of the <Emphasis Type="Italic">Staphylococcus epidermidis</Emphasis> LytSR two-component regulatory system on murein hydrolase activity,pyruvate utilization and global transcriptional profile

Background  

Staphylococcus epidermidis has emerged as one of the most important nosocomial pathogens, mainly because of its ability to colonize implanted biomaterials by forming a biofilm. Extensive studies are focused on the molecular mechanisms involved in biofilm formation. The LytSR two-component regulatory system regulates autolysis and biofilm formation in Staphylococcus aureus. However, the role of LytSR played in S. epidermidis remained unknown.     

sarA negatively regulates Staphylococcus epidermidis biofilm formation by modulating expression of 1 MDa extracellular matrix binding protein and autolysis‐dependent release of eDNA

Biofilm formation is essential for Staphylococcus epidermidis pathogenicity in implant‐associated infections. Nonetheless, large proportions of invasive Staphylococcus epidermidis isolates fail to form a biofilm in vitro. We here tested the hypothesis that this apparent paradox is related to the existence of superimposed regulatory systems suppressing a multicellular biofilm life style in vitro. Transposon mutagenesis of clinical significant but biofilm‐negative S. epidermidis 1585 was used to isolate a biofilm positive mutant carrying a Tn917 insertion in sarA, chief regulator of staphylococcal virulence. Genetic analysis revealed that inactivation of sarA induced biofilm formation via overexpression of the giant 1 MDa extracellular matrix binding protein (Embp), serving as an intercellular adhesin. In addition to Embp, increased extracellular DNA (eDNA) release significantly contributed to biofilm formation in mutant 1585ΔsarA. Increased eDNA amounts indirectly resulted from upregulation of metalloprotease SepA, leading to boosted processing of autolysin AtlE, in turn inducing augmented autolysis and release of eDNA. Hence, this study identifies sarA as a negative regulator of Embp‐ and eDNA‐dependent biofilm formation. Given the importance of SarA as a positive regulator of polysaccharide mediated cell aggregation, the regulator enables S. epidermidis to switch between mechanisms of biofilm formation, ensuring S. epidermidis adaptation to hostile environments.     

Formation of biofilms under phage predation: considerations concerning a biofilm increase

Bacteriophages are emerging as strong candidates for combating bacterial biofilms. However, reports indicating that host populations can, in some cases, respond to phage predation by an increase in biofilm formation are of concern. This study investigates whether phage predation can enhance the formation of biofilm and if so, if this phenomenon is governed by the emergence of phage-resistance or by non-evolutionary mechanisms (eg spatial refuge). Single-species biofilms of three bacterial pathogens (Pseudomonas aeruginosa, Salmonella enterica serotype Typhimurium, and Staphylococcus aureus) were pretreated and post-treated with species-specific phages. Some of the phage treatments resulted in an increase in the levels of biofilm of their host. It is proposed that the phenotypic change brought about by acquiring phage resistance is the main reason for the increase in the level of biofilm of P. aeruginosa. For biofilms of S. aureus and S. enterica Typhimurium, although resistance was detected, increased formation of biofilm appeared to be a result of non-evolutionary mechanisms.     

Potential mechanisms for the effects of tea extracts on the attachment,biofilm formation and cell size of Streptococcus mutans

Tea can inhibit the attachment of Streptococcus mutans to surfaces and subsequent biofilm formation. Five commercial tea extracts were screened for their ability to inhibit attachment and biofilm formation by two strains of S. mutans on glass and hydroxyapatite surfaces. The mechanisms of these effects were investigated using scanning electron microscopy (SEM) and phytochemical screening. The results indicated that extracts of oolong tea most effectively inhibited attachment and extracts of pu-erh tea most effectively inhibited biofilm formation. SEM images showed that the S. mutans cells treated with extracts of oolong tea, or grown in medium containing extracts of pu-erh tea, were coated with tea components and were larger with more rounded shapes. The coatings on the cells consisted of flavonoids, tannins and indolic compounds. The ratio of tannins to simple phenolics in each of the coating samples was ～3:1. This study suggests potential mechanisms by which tea components may inhibit the attachment and subsequent biofilm formation of S. mutans on tooth surfaces, such as modification of cell surface properties and blocking of the activity of proteins and the structures used by the bacteria to interact with surfaces.     

Inhibition and eradication of Salmonella Typhimurium biofilm using P22 bacteriophage,EDTA and nisin

Abstract

P22 phage >10⁵ PFU ml^?1 could be used to inhibit Salmonella Typhimurium biofilm formation by 55–80%. Concentrations of EDTA >1.25?mM and concentrations of nisin >1,200?µg ml^?1 were also highly effective in reducing S. Typhimurium biofilm formation (≥96% and ≥95% reductions were observed, respectively). A synergistic effect was observed when EDTA and nisin were combined whereas P22 phage in combination with nisin had no synergistic impact on biofilm formation. Triple combination of P22 phage, EDTA and nisin could be also used to inhibit biofilm formation (≥93.2%) at a low phage titer (10² PFU ml^?1), and low EDTA (1.25?mM) and nisin (9.375?µg ml^?1) concentrations. A reduction of 70% in the mature biofilm was possible when 10⁷ PFU ml^?1 of P22 phage, 20?mM of EDTA and 150?μg ml^?1 of nisin were used in combination. This study revealed that it could be possible to reduce biofilm formation by S. Typhimurium by the use of P22 phage, EDTA and nisin, either alone or in combination. Although, removal of the mature biofilm was more difficult, the triple combination could be successfully used for mature biofilm of S. Typhimurium.     

2018年我国19株猪链球菌4型分离株的病原学特征及分析

[背景]近年来,猪链球菌4型(Streptococcus suis serotype 4,SS4)分离率逐渐上升,但是有关SS4的系统研究报道匮乏.[目的]研究19株SS4临床分离株的病原学特征.[方法]以2株猪链球菌2型(Streptococcus suis serotype 2,SS2)强毒株为参考菌株,对19株S...     

Aeromonas hydrophila biofilm,exoprotease, and quorum sensing responses to co-cultivation with diverse foodborne pathogens and food spoilage bacteria on crab surfaces

Abstract

The effects of dual species interactions on biofilm formation by Aeromonas hydrophila in the presence of Pseudomonas aeruginosa, Pseudomonas fluorescens, Pectobacterium carotovorum, Salmonella Typhimurium, and Listeria monocytogenes were examined. High-performance liquid chromatography and liquid-chromatography-mass spectrometry were performed to identify N-acyl homoserine lactone (AHL) molecules secreted by monocultures and dual cultures grown in crab broth. Field emission scanning electron microscopy was performed to observe attachment and biofilm formation. P. aeruginosa and P. fluorescens inhibited biofilm formation by A. hydrophila on the crab surface, without affecting their own biofilm-forming abilities. Dual biofilms of S. Typhimurium, L. monocytogenes, or P. carotovorum did not affect A. hydrophila biofilm formation. Exoprotease, AHL, and AI-2 levels were significantly reduced in dual cultures of P. aeruginosa and P. fluorescens with A. hydrophila, supporting the relationship between quorum sensing and biofilm formation. Dual-species biofilms were studied in their natural environment and in the laboratory.     

Microbiological,biochemical, physicochemical surface properties and biofilm forming ability of Brettanomyces bruxellensis

Purpose

Brettanomyces bruxellensis is a serious source of concern for winemakers. The production of volatile phenols by the yeast species confers to wine unpleasant sensory characteristics which are unacceptable by the consumers and inevitably provoke economic loss for the wine industry. This ubiquitous yeast is able to adapt to all winemaking steps and to withstand various environmental conditions. Moreover, the ability of B. bruxellensis to adhere and colonize inert materials can be the cause of the yeast persistence in the cellars and thus recurrent wine spoilage. We therefore investigated the surface properties, biofilm formation capacity, and the factors which may affect the attachment of the yeast cells to surfaces with eight strains representative of the genetic diversity of the species.

Methods

The eight strains of B. bruxellensis were isolated from different geographical and industrial fermentation origins. The cells were grown in synthetic YPD medium containing 1% (w/v) yeast extract (Difco Laboratories, Detroit), 2% (w/v) bacto peptone (Difco), and 1% (w/v) glucose. Surface physicochemical properties as electrophoretic mobility and adhesion to hydrocarbon of the cells were studied. The ability of the strains to form biofilm was quantified using a colorimetric microtiter 96-well polystyrene plate. Biochemical characteristics were examined by colorimetric methods as well as by chemical analysis.

Result

Our results show that the biofilm formation ability is strain-dependent and suggest a possible link between the physicochemical properties of the studied strains and their corresponding genetic group.

Conclusion

The capacity to detect and identify the strains of the spoilage yeast based on their biofilm formation abilities may help to develop more efficient cleaning procedures and preventing methods.

     

Cloning and partial characterization of the gene encoding the putative elongation factor Ts of<Emphasis Type="Italic"> Streptococcus suis</Emphasis> serotype 2

Streptococcus suis infection has a substantial impact on the swine industry. In addition, S. suis serotype 2 is recognized as a zoonotic agent. In this paper, we report the cloning and complete sequence of the gene coding for the putative elongation factor Ts (tsf-like) of S. suis. The putative tsf gene seems to be transcribed from a promoter located within the cloned DNA fragment, as its expression is not dependent on insertional orientation within the plasmid. One copy of the tsf gene was detected in the chromosome of S. suis by Southern blot analysis. Interestingly, the elongation factor Ts expressed by all reference strains of all S. suis serotypes were antigenically similar, as determined by Western blot.     

In Silico Design,Synthesis, and In Vitro Evaluation of Novel Amphipathic Short Linear Peptides Against Clinically Relevant Bacterial Biofilms

Microbial biofilms are organized communities of cells that are associated with a wide spectrum of resistant and chronic infections that lead to the treatment failure. Accordingly, there is an urgent demand to create novel effective therapeutic drugs that can inhibit biofilm formation with new mechanisms of action to surmount the current escalating resistance. In this study, in silico hybrid model was utilized to develop three novel short linear peptides (4, 5, and 6) with potential biofilm inhibiting activities (scores?>?1.0). The peptides were composed of cationic and hydrophobic residues. They were synthesized using solid-phase strategy. Synthesized peptides were purified and characterized by reverse-phase high-performance liquid chromatography and matrix-assisted laser desorption/ionization spectroscopy, respectively. They were evaluated using in vitro assay as potential inhibitors of clinically relevant Gram-positive and Gram-negative biofilms. Peptide (4) with five positive charges at physiological pH (4 cationic moieties and W:R?=?1:4) showed activity against biofilms of Gram-positive strains (Staphylococcus epidermidis and Listeria monocytogenes). On the other hand, peptide (5) with six positive charges (5 cationic moieties and W:R?=?2:2) demonstrated activity against Gram-positive (S. epidermidis) and Gram-negative (Escherichia coli) biofilms. Interestingly, peptide (6), with seven positive charges (6 cationic moieties and W:R?=?2:5) revealed higher and broader spectrum of activity against biofilms of Gram-positive (S. epidermidis, S. aureus, L. monocytogenes) and Gram-negative (E. coli).

     

Various biofilm matrices of the emerging pathogen Staphylococcus lugdunensis: exopolysaccharides,proteins, eDNA and their correlation with biofilm mass

Abstract

Staphylococcus lugdunensis is an emerging high-virulent pathogen causative of hospital-acquired infections. Biofilm formation is a complex pathogenic process that leads to well-established bacterial communities. There is a paucity of data on the composition of the biofilm matrix among S. lugdunensis strains. Here, twenty-two S. lugdunensis clinical isolates, mainly from orthopaedic infections but also from other clinical sources, were sub-grouped by ribotyping and dendrogram analysis. Biofilms were analysed by fluorimetric methods based on FITC-Wheat Germ Agglutinin, SYPRO Ruby and TOTO-1 dyes to detect exopolysaccharides, proteins and extracellular DNA (eDNA), respectively. Biofilm morphology was investigated under confocal laser scanning microscopy (CLSM). Isolates displayed intriguing diversities in biofilm mass and matrix composition. The content of exopolysaccharides was found to be to be strongly associated with the biofilm mass (R² = 0.882), while the content of proteins turned out to be weakly (R² = 0.465) and that of eDNA very weakly associated (R² = 0.202) to the biofilm mass.     

Identification and Experimental Verification of Protective Antigens Against <Emphasis Type="Italic">Streptococcus suis</Emphasis> Serotype 2 Based on Genome Sequence Analysis

Streptococcus suis serotype 2 (S. suis 2) is an important zoonotic pathogen that can cause severe disease and even death in both humans and swine. No effective vaccine is clinically available. In this study, a reverse vaccinology method was first applied to identify protective antigens against S. suis 2. As a consequence, 153 genes encoding vaccine candidates were selected from the whole genome sequence by means of bioinformatics analysis, from which 10 genes were selected based on experimental evidences arising from the study of related bacteria such as Streptococcus pneumoniae, group B streptococcus, S. suis and so on. Of 10 target genes, 8 were successfully expressed in Escherichia coli Rosetta, and expressed proteins were purified and used as the immunogens for evaluating vaccine efficacy in a mouse infection model. The results have confirmed that RTX family exoprotein A (RfeA), epidermal surface antigen (ESA), immunoglobulin G (IgG)-binding protein (IBP), and suilysin (SLY) can induce a protective response of the vaccinated animals against S. suis 2, whereas RfeA, ESA, and IBP mainly induce humoral-mediated immunity, and SLY elicits a combined pattern of both humoral- and cellular-mediated immunity. Although immunoprotection of SLY against S. suis 2 was reported previously, RfeA, ESA, and IBP were explored first in this study.