Complex Population Structure and Virulence Differences among Serotype 2 Streptococcus suis Strains Belonging to Sequence Type 28

Streptococcus suis is a major swine pathogen and a zoonotic agent. Serotype 2 strains are the most frequently associated with disease. However, not all serotype 2 lineages are considered virulent. Indeed, sequence type (ST) 28 serotype 2 S. suis strains have been described as a homogeneous group of low virulence. However, ST28 strains are often isolated from diseased swine in some countries, and at least four human ST28 cases have been reported. Here, we used whole-genome sequencing and animal infection models to test the hypothesis that the ST28 lineage comprises strains of different genetic backgrounds and different virulence. We used 50 S. suis ST28 strains isolated in Canada, the United States and Japan from diseased pigs, and one ST28 strain from a human case isolated in Thailand. We report a complex population structure among the 51 ST28 strains. Diversity resulted from variable gene content, recombination events and numerous genome-wide polymorphisms not attributable to recombination. Phylogenetic analysis using core genome single-nucleotide polymorphisms revealed four discrete clades with strong geographic structure, and a fifth clade formed by US, Thai and Japanese strains. When tested in experimental animal models, strains from this latter clade were significantly more virulent than a Canadian ST28 reference strain, and a closely related Canadian strain. Our results highlight the limitations of MLST for both phylogenetic analysis and virulence prediction and raise concerns about the possible emergence of ST28 strains in human clinical cases.     

猪链球菌毒力因子和鉴定方法的研究进展

猪链球菌(Streptococcus suis)是一种重要的人畜共患病病原体,能够引起猪疫病,人类感染该菌可导致脑膜炎、败血症甚至死亡。鉴于其对养猪业的巨大经济影响和对公共卫生事业的威胁,关于猪链球菌的研究日益深入,即对其毒力相关蛋白和鉴定方法的研究进展做一综述。     

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.     

Zebrafish as a Model to Assess the Teratogenic Potential of Nitrite

High nitrate levels in the environment may result in congenital defects or miscarriages in humans. Presumably, this is due to the conversion of nitrate to nitrite by gut and salivary bacteria. However, in other mammalian studies, high nitrite levels do not cause birth defects, although they can lead to poor reproductive outcomes. Thus, the teratogenic potential of nitrite is not clear. It would be useful to have a vertebrate model system to easily assess teratogenic effects of nitrite or any other chemical of interest. Here, we demonstrate the utility of zebrafish (Danio rerio) to screen compounds for toxicity and embryonic defects. Zebrafish embryos are fertilized externally and have rapid development, making them a good model for teratogenic studies. We show that increasing the time of exposure to nitrite negatively affects survival. Increasing the concentration of nitrite also adversely affects survival, whereas nitrate does not. For embryos that survive nitrite exposure, various defects can occur, including pericardial and yolk sac edema, swim bladder noninflation, and craniofacial malformation. Our results indicate that the zebrafish is a convenient system for studying the teratogenic potential of nitrite. This approach can easily be adapted to test other chemicals for their effects on early vertebrate development.     

Identification of a Novel Virulence-Related Gene in Streptococcus suis Type 2 Strains

The trag is a novel infection-related factor identified using in vivo-induced antigen technology (IVIAT) from SS2 expression libraries with swine convalescent sera. PCR-based studies of various S. suis strains collected from different sources revealed that the trag gene was present in all the virulent SS2 strains, but absent in the avirulent T15 strain. PCR and Southern hybridization analyses of a trag-knockout strain created through homologous recombination confirmed the coding sequence of trag replaced by spc ^R cassette in the ?trag mutant. Zebrafish was used to identify the role of trag in SS2 virulence. The reduction of virulence in the trag mutant compared to the wild-type in animal model systems, laid the foundation for further studies.     

Two Spx Regulators Modulate Stress Tolerance and Virulence in Streptococcus suis Serotype 2

Streptococcus suis serotype 2 is an important zoonotic pathogen causing severe infections in pigs and humans. The pathogenesis of S. suis 2 infections, however, is still poorly understood. Spx proteins are a group of global regulators involved in stress tolerance and virulence. In this study, we characterized two orthologs of the Spx regulator, SpxA1 and SpxA2 in S. suis 2. Two mutant strains (ΔspxA1 and ΔspxA2) lacking the spx genes were constructed. The ΔspxA1 and ΔspxA2 mutants displayed different phenotypes. ΔspxA1 exhibited impaired growth in the presence of hydrogen peroxide, while ΔspxA2 exhibited impaired growth in the presence of SDS and NaCl. Both mutants were defective in medium lacking newborn bovine serum. Using a murine infection model, we demonstrated that the abilities of the mutant strains to colonize the tissues were significantly reduced compared to that of the wild-type strain. The mutant strains also showed a decreased level of survival in pig blood. Microarray analysis revealed a global regulatory role for SpxA1 and SpxA2. Furthermore, we demonstrated for the first time that Spx is involved in triggering the host inflammatory response. Collectively, our data suggest that SpxA1 and SpxA2 are global regulators that are implicated in stress tolerance and virulence in S. suis 2.     

A Real-Time Impedance Based Method to Assess Rhodococcus equi Virulence

Rhodococcus equi is a facultative intracellular pathogen of macrophages and the causative agent of foal pneumonia. R. equi virulence is usually assessed by analyzing intracellular growth in macrophages by enumeration of bacteria following cell lysis, which is time consuming and does not allow for a high throughput analysis. This paper describes the use of an impedance based real-time method to characterize proliferation of R. equi in macrophages, using virulent and attenuated strains lacking the vapA gene or virulence plasmid. Image analysis suggested that the time-dependent cell response profile (TCRP) is governed by cell size and roundness as well as cytoxicity of infecting R. equi strains. The amplitude and inflection point of the resulting TCRP were dependent on the multiplicity of infection as well as virulence of the infecting strain, thus distinguishing between virulent and attenuated strains.     

3D Finite Element Electrical Model of Larval Zebrafish ECG Signals

Assessment of heart function in zebrafish larvae using electrocardiography (ECG) is a potentially useful tool in developing cardiac treatments and the assessment of drug therapies. In order to better understand how a measured ECG waveform is related to the structure of the heart, its position within the larva and the position of the electrodes, a 3D model of a 3 days post fertilisation (dpf) larval zebrafish was developed to simulate cardiac electrical activity and investigate the voltage distribution throughout the body. The geometry consisted of two main components; the zebrafish body was modelled as a homogeneous volume, while the heart was split into five distinct regions (sinoatrial region, atrial wall, atrioventricular band, ventricular wall and heart chambers). Similarly, the electrical model consisted of two parts with the body described by Laplace’s equation and the heart using a bidomain ionic model based upon the Fitzhugh-Nagumo equations. Each region of the heart was differentiated by action potential (AP) parameters and activation wave conduction velocities, which were fitted and scaled based on previously published experimental results. ECG measurements in vivo at different electrode recording positions were then compared to the model results. The model was able to simulate action potentials, wave propagation and all the major features (P wave, R wave, T wave) of the ECG, as well as polarity of the peaks observed at each position. This model was based upon our current understanding of the structure of the normal zebrafish larval heart. Further development would enable us to incorporate features associated with the diseased heart and hence assist in the interpretation of larval zebrafish ECGs in these conditions.     

2型猪链球菌Fbps基因敲除突变体的构建及毒力分析

目的:敲除2型猪链球菌(SS2)强毒株05ZYH33中的Fbps基因,研究该基因敲除对菌株生物活性及毒力的影响,为深入探讨猪链球菌致病机制提供实验基础。方法:从05ZYH33基因组中扩增Fbps基因上、下游同源臂,从pSET1质粒中扩增氯霉素抗性基因Cm,通过重叠PCR的方法将3个片段整合后连接到温敏自杀载体pSET4s上,电转入05ZYH33感受态细胞,通过改变培养温度实现双交换和质粒丢失,最后经抗性筛选获得敲除株05ZYH33ΔFBPS,分析敲除株的生物学性状,以CD1小鼠作为体外感染模型对突变株和野生株进行毒力比较。结果:PCR分析和测序结果均显示Fbps基因敲除成功,动物实验结果显示Fbps基因敲除后05ZYH33的毒力有所下降。结论:与野生株相比,突变株对小鼠的毒力有所降低。     

猪链球菌2型srtF基因敲除突变株的构建及其毒力

利用同源重组基因敲除方法构建猪链球菌2型强毒株05ZYH33 srtF同源突变体。组合PCR、交叉酶切、RT-PCR结果均显示srtF突变体构建成功。突变株与强毒株的菌落形态、生长速率以及对小鼠的致病力均无显著性差异。小鼠竞争实验结果提示,突变株在心脏的定殖及感染能力显著减弱。成功构建的srtF突变株为进一步研究srtF的生物学功能奠定了基础。     

2型猪链球菌gene0969基因敲除突变体的构建及毒力分析

目的:构建2型猪链球菌强毒株05ZYH33毒力岛89K上的Ⅳ型分泌系统组分gene0969敲除突变体,初步分析其活性,为进一步研究猪链球菌假想毒力因子在致病中的作用提供实验基础。方法:以05ZYH33基因组为模板,PCR扩增gene0969基因的上下游片段;以穿梭质粒pSET1为模板,PCR扩增氯霉素抗性基因Cm;采用重叠PCR方法搭建3个片段,并克隆到自杀载体pSET4s上,构建基因敲除载体,通过同源重组构建gene0969突变体,再用小鼠感染模型对突变株和野生株的毒力进行比较。结果:获得了gene0969基因敲除突变体,并发现其毒力与野生型相比有下降趋势。结论:2型猪链球菌假想毒力因子gene0969可能与毒力有关,其作用和机制值得进一步分析。     

Larval Zebrafish Proteome Regulation in Response to an Environmental Challenge

Adaptation to the environment during development influences the life‐long survival of an animal. While brain‐wide proteomic changes are expected to underlie such experience‐driven physiological and behavioral flexibility, a comprehensive overview of the nature and extent of the proteomic regulation following an environmental challenge during development is currently lacking. In this study, the brain proteome of larval zebrafish is identified and it is determined how it is altered by an exposure to a natural and physical environmental challenge, namely prolonged exposure to strong water currents. A comprehensive larval zebrafish brain proteome is presented here. Furthermore, 57 proteins that are regulated by the exposure to an environmental challenge are identified, which cover multiple functions including neuronal plasticity, the stress response, axonal growth and guidance, spatial learning, and energy metabolism. These represent candidate proteins that may play crucial roles for the adaption to an environmental challenge during development.     

溶原性噬菌体在猪链球菌2型分离株中的检出和鉴定

[目的]为了研究噬菌体整合酶基因在猪链球菌2型(Streptococcus suis type 2,SS2)中的分布情况.[方法]根据噬菌体整合酶基因设计引物,建立了PCR方法,并对扩增产物进行测序.[结果]结果显示,25株SS2致病菌株均扩增出目的片段,非毒力株T15、5株其它血清型猪链球菌及兰氏C群猪源链球菌未扩增出目的片段.经丝裂霉素C诱导后,SS2致病菌株出现完全的细胞溶解,而非毒力株T15未出现溶解.SS2致病株HA9801和ZY05719诱导均产生溶原性噬菌体,分别命名为SS2-HA和SS2-ZY,电镜观察,二者均头部呈正六边形,无尾部,其核酸类型为dsDNA,可鉴定为复层噬菌体科(Tectiviridae)的成员.噬菌体SS2-HA和SS2-ZY整合酶基因序列与已报道的SS2噬菌体整合酶基因序列高度同源,显示SS2噬菌体整合酶具有较高的特异性.[结论]从SS2致病株中检出溶原性噬菌体和噬菌体整合酶基因,且噬菌体整合酶基因与SS2溶菌酶释放蛋白(mrp)等7种毒力相关基因有相关性,表明SS2的溶原性噬菌体可能与其致病性有关.     

Population Structure and Antimicrobial Resistance Profiles of Streptococcus suis Serotype 2 Sequence Type 25 Strains

Strains of serotype 2 Streptococcus suis are responsible for swine and human infections. Different serotype 2 genetic backgrounds have been defined using multilocus sequence typing (MLST). However, little is known about the genetic diversity within each MLST sequence type (ST). Here, we used whole-genome sequencing to test the hypothesis that S. suis serotype 2 strains of the ST25 lineage are genetically heterogeneous. We evaluated 51 serotype 2 ST25 S. suis strains isolated from diseased pigs and humans in Canada, the United States of America, and Thailand. Whole-genome sequencing revealed numerous large-scale rearrangements in the ST25 genome, compared to the genomes of ST1 and ST28 S. suis strains, which result, among other changes, in disruption of a pilus island locus. We report that recombination and lateral gene transfer contribute to ST25 genetic diversity. Phylogenetic analysis identified two main and distinct Thai and North American clades grouping most strains investigated. These clades also possessed distinct patterns of antimicrobial resistance genes, which correlated with acquisition of different integrative and conjugative elements (ICEs). Some of these ICEs were found to be integrated at a recombination hot spot, previously identified as the site of integration of the 89K pathogenicity island in serotype 2 ST7 S. suis strains. Our results highlight the limitations of MLST for phylogenetic analysis of S. suis, and the importance of lateral gene transfer and recombination as drivers of diversity in this swine pathogen and zoonotic agent.     

Exploring Beneficial/Virulence Properties of Two Dairy-Related Strains of Streptococcus infantarius subsp. infantarius

The genus Streptococcus includes various species, remarkably different in their behavior, applications, virulence, and safety. Taxonomically Streptococcus infantarius subsp. infantarius belonging to the Streptococcus bovis group, which includes several pathogen species, however, has been found as predominant species in some African dairy products that are widely consumed and considered to be safe. Streptococcus infantarius subsp. infantarius’ safety may be questioned due to the association of this species with clinical cases. In this study, isolates from dairy origin were selected based on their bacteriocinogenic potential and differentiated by their RAPD-PCR profiles. Two strains were identified by 16S rRNA sequencing as St. infantarius subsp. infantarius and investigated regarding their potential beneficial properties and factors related to virulence and safety. A series of in vitro tests included properties related to survival in the gastrointestinal tract and beneficial intestinal activities. Production of bacteriocin/s, detection of related genes, and partial characterization of expressed antimicrobial protein were evaluated. Genes related to folate biosynthesis were detected in both studied strains. Evaluation of physiological tests related to strains virulence, adhesion, and resistance to antibiotics and detections of virulence and biogenic amines production-related genes were also investigated. Taking in consideration all the aspects of the specific nature of St. infantarius subsp. infantarius K1–4 and K5–1 (beneficial properties and virulence characteristics), both strains cannot be considered safe for human or other animals application, even though they have been isolated from dairy products. This study is highlighting the importance of evaluation for presence of potential virulence factors in newly characterized strains in order to be confident in their safety.

     

趋流行为下斑马鱼幼鱼的全脑成像

目的 趋流,意即在水中调整身体方向并逆流而上的能力,是一种在大多数鱼类与两栖类动物中存在的保守行为。虽然关于趋流的研究已有一段很长的历史,并且近年来斑马鱼幼鱼趋流行为的理论机制也被提出,但是分布式的神经环路是如何整合多感知信息、做出决策、并实现行为控制仍然是个未知数。对自由运动的斑马鱼进行全脑神经活动成像为理解这一困难的问题提供了特殊的机会。方法 本文开发了一种微流控装置精确控制环境水流并激发斑马鱼的趋流行为。将该微流控芯片与扩增视野光场显微镜以及追踪系统整合,从而记录自由行为下斑马鱼全脑的神经活动。结果 在整合的微流控装置中稳定观察到了斑马鱼在水流中保持自身位置不变、逆流而上等刻板的趋流行为现象。与此同时,实现了对斑马鱼幼鱼趋流行为过程中的全脑钙活动记录。本文初步发现了几个脑区的神经活动与趋流行为相关。结论 本研究第一次展示了在斑马鱼幼鱼趋流行为的同时记录全脑神经活动的技术。接下来对神经活动和行为数据的分析与建模将有助于更好地理解一种重要自然行为背后的感觉运动转换机制。