Determining the prevalence,identity and possible origin of bacterial pathogens in soil

Soil biomes are vast, exceptionally diverse and crucial to the health of ecosystems and societies. Soils also contain an appreciable, but understudied, diversity of opportunistic human pathogens. With climate change and other forms of environmental degradation potentially increasing exposure risks to soilborne pathogens, it is necessary to gain a better understanding of their ecological drivers. Here we use the Galleria mellonella insect virulence model to selectively isolate pathogenic bacteria from soils in Cornwall (UK). We find a high prevalence of pathogenic soil bacteria with two genera, Providencia and Serratia, being especially common. Providencia alcalifaciens, P. rustigianii, Serratia liquefaciens and S. plymuthica strains were studied in more detail using phenotypic virulence and antibiotic resistance assays and whole-genome sequencing. Both genera displayed low levels of antibiotic resistance and antibiotic resistance gene carriage. However, Serratia isolates were found to carry the recently characterized metallo-β-lactamase blaSPR-1 that, although not conferring high levels of resistance in these strains, poses a potential risk of horizontal transfer to other pathogens where it could be fully functional. The Galleria assay can be a useful approach to uncover the distribution and identity of pathogenic bacteria in the environment, as well as uncover resistance genes with an environmental origin.     

大肠埃希氏菌的分型方法及其研究进展

大肠埃希氏菌是一种条件性致病菌,致病性的大肠埃希氏菌具有高度的传染性,会严重危害健康。快速准确地测定大肠埃希氏菌的污染来源对有效缩小疫情影响范围极有帮助,从而避免对人类健康和经济贸易造成重大损失。建立简便高效的分型方法是微生物溯源的关键,常见的大肠埃希氏菌分型方法可分为表型分型和分子分型,这些分型方法各有优劣,具有不同的适用范围。本文详细介绍了大肠埃希氏菌的分型方法,并对国内外大肠埃希氏菌分型的研究进展进行综述,为致病菌溯源方法的选择提供参考依据,对防御并控制致病菌引起的流行病传播具有重要的意义。     

宁波市城区内河肠道病原细菌定量 PCR 分析

了解城市内河中病原菌含量对城市生态安全具有重要意义。应用定量PCR（ qPCR）方法,对宁波市城区6条重要内河水体中的病原菌含量及主要水质指标进行分析。结果表明,祖关山河、苏家河、南北河、后西河、史魏家河和直落河每升水体中含有病原菌基因的拷贝数分别为：3.34×105、1.47×105、4.68×105、1.75×106、9.65×106和1.33×106;且病原菌基因的含量与水体溶解氧负相关,与总氮、氨氮、浊度、总磷、高锰酸钾指数、叶绿素a和电导率呈正相关关系,但与水体透明度、温度和酸碱度无明显关系。定量PCR分析结果可望为评价城市内河水体质量提供重要参考。     

Identification and sequence of an unstable DNA element in the entomopathogenic bacteria Photorhabdus temperata strain K122

AIMS: A search was conducted for a difference in genome composition between phenotypic variants of the insect pathogenic bacteria, Photorhabdus temperata. METHODS: An unstable 300 bp fragment of DNA was identified by amplified fragment length polymorphism (AFLP) analysis, which was not, however, associated with phenotypic variation. RESULTS: During prolonged culturing of the bacteria, one copy of the repeated fragment was deleted and a restriction site linked to one of the copies was lost or gained. The sequence did not show substantial identity to any in the database, but a 16-bp region was identical to part of the marR gene of Escherichia coli. SIGNIFICANCE AND IMPACT OF THE STUDY: The work has implications for the understanding of genetic instability in this and other pathogenic species of bacteria. In addition, the complete unstable element may be useful as a genetic tool in Photorhabdus spp.     

Genetic markers validate using the natural phenotypic characteristics of shed feathers to identify individual northern goshawks Accipiter gentilis

The recognition of individual animals is essential for many types of ecological research, as it enables estimates of demographic parameters such as population size, survival and reproductive rates. A popular method of visually identifying individuals uses natural variations in spot, stripe or scar markings. Although several studies have assessed the accuracy of these methods in mammals, crustaceans and fish, there have been few attempts to determine whether phenotypic characteristics are accurate when used for birds. Furthermore, even less is known about whether shed or moulted body parts can be reliably used to visually identify individuals. Here we assessed the accuracy of using phenotypic characteristics to identify avian individuals using a double‐marking experiment, whereby nine microsatellite genetic markers and natural markings on shed feathers were used to independently identify northern goshawks Accipiter gentilis. Phenotypic and genetic identification of individuals was consistent in 94.4% (51/54) comparisons. Our results suggest that the phenotypic characteristics of shed feathers can be reliably used as a non‐invasive and relatively inexpensive technique to monitor populations of an elusive species, the northern goshawk, without having to physically re‐capture or re‐sight individuals. We posit that using natural markings on shed feathers will also be a reliable method of identifying individuals in avian species with similar phenotypic characteristics, such as other Accipiter species.     

Merging chemical ecology with bacterial genome mining for secondary metabolite discovery

The integration of chemical ecology and bacterial genome mining can enhance the discovery of structurally diverse natural products in functional contexts. By examining bacterial secondary metabolism in the framework of its ecological niche, insights into the upregulation of orphan biosynthetic pathways and the enhancement of the enzyme substrate supply can be obtained, leading to the discovery of new secondary metabolic pathways that would otherwise be silent or undetected under typical laboratory cultivation conditions. Access to these new natural products (i.e., the chemotypes) facilitates experimental genotype-to-phenotype linkages. Here, we describe certain functional natural products produced by Xenorhabdus and Photorhabdus bacteria with experimentally linked biosynthetic gene clusters as illustrative examples of the synergy between chemical ecology and bacterial genome mining in connecting genotypes to phenotypes through chemotype characterization. These Gammaproteobacteria share a mutualistic relationship with nematodes and a pathogenic relationship with insects and, in select cases, humans. The natural products encoded by these bacteria distinguish their interactions with their animal hosts and other microorganisms in their multipartite symbiotic lifestyles. Though both genera have similar lifestyles, their genetic, chemical, and physiological attributes are distinct. Both undergo phenotypic variation and produce a profuse number of bioactive secondary metabolites. We provide further detail in the context of regulation, production, processing, and function for these genetically encoded small molecules with respect to their roles in mutualism and pathogenicity. These collective insights more widely promote the discovery of atypical orphan biosynthetic pathways encoding novel small molecules in symbiotic systems, which could open up new avenues for investigating and exploiting microbial chemical signaling in host–bacteria interactions.     

The role of scent marking in the social communication of wild golden lion tamarins, Leontopithecus rosalia

The role of scent marking in the social communication of mammals is widely variable. One reason for this variation is that the function of scent marking may vary with different ecological and social conditions. The purpose of this study was to test four nonexclusive hypotheses explaining the role of scent-marking frequency in different ecological and social contexts for wild golden lion tamarins. Relative to ecological contexts, we compared scent-marking frequency during seasons of abundant and scarce food resources. Relative to social contexts, we compared scent-marking frequency when groups were isolated and when groups were in the presence of neighbouring groups. We found that the tamarins used scent marking to mark the location of food resources. Additionally, males used scent marking to communicate intrasexual dominance within their groups, while females did not. Our results also indicate that alpha females increased their scent-marking frequency to communicate to members of other groups, while the presence of members of other groups did not elicit a similar response by alpha males. We did not find evidence for a territorial function of scent marking in golden lion tamarins. Copyright 2003 Published by Elsevier Science Ltd on behalf of The Association for the Study of Animal Behaviour      

Obtaining of fluorescent-labeled nodule bacteria strains of wild legumes for their detection in vive and in vitro

A series of expression vectors containing genes of fluorescent proteins TurboGFP and TurboRFP under the phage T5 constitutive promoter regulation, intended for lifetime marking of nodule bacteria is created: a series of vectors based on a broad-host-range replicon BBRI, for marking strains with an expression of reporter gene from a transformed plasmid and a series of vectors based on a plasmid pRL765gfp for marking strains by introduction genes of fluorescent proteins in a bacterial chromosome. It was shown that transformation is the most preferable method of constructions transfer in nodule bacteria cells, as in the presence of mob locus in the vectors necessary for conjugation, exists the possibility of occasional plasmid mobilization and its transition from marked strain cells in other soil bacteria. With application of the created vector constructions we obtained fluorescent tagged strains of Rhizobium sp., Mesorhizobium sp., Ensifer (Sinorhizobium) sp., Bradyrhizobium sp., Phyllobacterium sp., Agrobacterium sp. Also their suitability for experiments in vivo and in vitro is shown.     

Hydrobionts as reservoir hosts for infectious agents of bacterial sapronoses

Data on parasitism of the infectious agents of sapronoses in hydrobionts (protozoans, crustaceans, worms, mollusks, echinoderms, and fishes) are considered from the population-ecological viewpoint. The symbiotic relationships between populations of pathogenic bacteria and protozoans are of the host-parasite type. An ultrastructural analysis demonstrates that phagocytosis is incomplete both in planktonic forms and in biofilms formed by bacteria and protozoa. This provides for long-term survival of infectious agents in the environment. The migration of Yersinia pseudotuberculosis along trophic chains from the lowest to the highest level has been simulated experimentally. The long-term survival of pathogenic bacteria in aquatic animals and the ability of bacteria to migrate along trophic chains, reaching soil animals and humans, provide evidence that comprehensive studies on the routes of circulation of pathogens in natural ecosystems are highly relevant from the ecological and epidemiological viewpoints.     

小菌落突变株：一种利于持续复发性感染的细菌致病形式

小菌落突变株是一种具有独特表型及致病特征且生长缓慢的细菌亚群。在表型上,小菌落突变株表现为生长率低、菌落形态不规则及生化特性异常,这使得临床微生物学家在鉴定时遇到了挑战。在临床上,小菌落突变株比相应的野生株更能持续存在于哺乳动物细胞中,且对抗生素更不敏感。当宿主细胞形成应急的保护性环境时,小菌落突变株会引发隐性或复发性感染。这篇综述涵盖了小菌落突变株相关表型、遗传及临床上的特征,重点描述目前研究最多的葡萄球菌。     

Small colony variants: a pathogenic form of bacteria that facilitates persistent and recurrent infections

Small colony variants constitute a slow-growing subpopulation of bacteria with distinctive phenotypic and pathogenic traits. Phenotypically, small colony variants have a slow growth rate, atypical colony morphology and unusual biochemical characteristics, making them a challenge for clinical microbiologists to identify. Clinically, small colony variants are better able to persist in mammalian cells and are less susceptible to antibiotics than their wild-type counterparts, and can cause latent or recurrent infections on emergence from the protective environment of the host cell. This Review covers the phenotypic, genetic and clinical picture associated with small colony variants, with an emphasis on staphylococci, for which the greatest amount of information is available.     

Detection and kinetics of mucosal pathogenic bacteria binding with polysaccharides

The detection and kinetics of mucosal pathogenic bacteria binding on polysaccharide ligands were studied using a surface plasmon resonance biosensor. The kinetic model applied curve-fitting to the experimental surface plasmon resonance sensorgrams to evaluate the binding interactions. The kinetic parameters for the mucosal pathogenic bacteria (Pseudomonas aeruginosa, Pseudomonasfluorescens, Serratia marcescens) with the alginate ligand were determined from a kinetic model. In addition, the binding interactions of the mucosal pathogenic bacteria with polysaccharide binding pairs (Pseudomonas aeruginosa/alginate, Streptococcus pneumoniae/pneumococcal polysaccharide, Staphylococcus aureus/pectin) were also compared with their kinetic parameters. The rate constants of association for Pseudomonas aeruginosa with the alginate ligand were higher than those for Pseudomonas fluorescens. Serratia marcescens had no detectable interaction with the alginate ligand. The adhesion affinity of Pseudomonas aeruginosa with alginate was higher than that for the other binding pairs. The binding affinities of the pathogenic bacteria with their own polysaccharide were higher than that of Staphylococcus aureus with pectin. Measuring the contact angle was found to be a feasible method for detecting binding interactions between analytes and ligands.     

美洲大蠊成虫肠道抗细菌共生放线菌的筛选及鉴定

【目的】鉴于野外美洲大蠊Periplaneta americana对恶劣环境适应性强,本研究旨在从云南省大理州的野外美洲大蠊成虫肠道中分离、筛选出抗细菌活性放线菌,为抗生素开发提供菌种资源。【方法】采用涂布平板法和平板划线法对美洲大蠊成虫肠道放线菌进行分离;以金黄色葡萄球菌Staphylococcus aureus、耐甲氧西林金黄色葡萄球菌、铜绿假单胞菌Pseudomonas aeruginosa、粪肠球菌Enterococcus faecalis、大肠杆菌Escherichia coli和鼠伤寒沙门氏菌Salmonella typhimurium共6种人体病原细菌为指示菌株,采用牛津杯法对分离自这些放线菌的次生代谢产物进行抗菌活性测定;通过形态学特征和16S rRNA基因序列分析,对具有广谱和明显抗菌活性的放线菌进行鉴定,并经16SrRNA基因序列的BlAST同源性比对及系统发育分析确定它们的分类地位。【结果】从美洲大蠊成虫肠道共分离获得41株放线菌。抗菌活性测定结果表明,34株(82.9%)放线菌对至少1种指示病原细菌具有抑制作用,其中有7株对3种以上病原细菌具有抑制作用,9株表现...     

Metagenomic profile of the bacterial communities associated with Ixodes ricinus ticks

Assessment of the microbial diversity residing in arthropod vectors of medical importance is crucial for monitoring endemic infections, for surveillance of newly emerging zoonotic pathogens, and for unraveling the associated bacteria within its host. The tick Ixodes ricinus is recognized as the primary European vector of disease-causing bacteria in humans. Despite I. ricinus being of great public health relevance, its microbial communities remain largely unexplored to date. Here we evaluate the pathogen-load and the microbiome in single adult I. ricinus by using 454- and Illumina-based metagenomic approaches. Genomic DNA-derived sequences were taxonomically profiled using a computational approach based on the BWA algorithm, allowing for the identification of known tick-borne pathogens at the strain level and the putative tick core microbiome. Additionally, we assessed and compared the bacterial taxonomic profile in nymphal and adult I. ricinus pools collected from two distinct geographic regions in Northern Italy by means of V6-16S rRNA amplicon pyrosequencing and community based ecological analysis. A total of 108 genera belonging to representatives of all bacterial phyla were detected and a rapid qualitative assessment for pathogenic bacteria, such as Borrelia, Rickettsia and Candidatus Neoehrlichia, and for other bacteria with mutualistic relationship or undetermined function, such as Wolbachia and Rickettsiella, was possible. Interestingly, the ecological analysis revealed that the bacterial community structure differed between the examined geographic regions and tick life stages. This finding suggests that the environmental context (abiotic and biotic factors) and host-selection behaviors affect their microbiome.Our data provide the most complete picture to date of the bacterial communities present within I. ricinus under natural conditions by using high-throughput sequencing technologies. This study further demonstrates a novel detection strategy for the microbiomes of arthropod vectors in the context of epidemiological and ecological studies.     

Widespread distribution of the bacteriocin-producing lactic acid cocci in Miso-paste products

AIMS: A survey was conducted on the ecological distribution of bacteriocin-producing lactic acid bacteria in Miso-pastes, a typical fermented food in Japan. METHODS AND RESULTS Nine Miso-pastes were sampled for isolation of bacteriocin-producers. Almost all isolated enterococcal strains produced bacteriocins but no isolated tetragenococci did so. The bacteriocin-producing isolates were divided into nine groups by phenotypic tests. As the phenotypic characters were highly diverse, these strains could not be identified to species level on the basis of their phenotypes. The nine representative strains from each group were identified by 16S rRNA analysis. These bacteriocin-producers with one exception (Lactococcus sp.) were identified as strains of the Enterococcus faecium 'species group'. The bacteriocins of the nine strains were classified into five types according to their antibacterial spectral patterns and their SDS-PAGE profiles. The bacteriocins inhibited undesirable bacteria in Miso-pastes, such as Bacillus subtilis, but did not inhibit the useful Tetragenococcus halophila. CONCLUSIONS: The bacteriocin-producing lactic acid cocci were widespread at high frequencies in Miso-pastes. They were considered to play an important role in preventing the growth of undesirable bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that bacteriocin-producers act effectively as safe biopreservatives and may contribute to the biopreservation in Miso-pastes.     

基于高通量测序的鱼菜共生池塘与普通池塘微生物群落结构比较

鱼菜共生(Aquaponics)作为一种可持续、循环型、零排放的新型的复合耕作体系, 因具有良好的生态环境效应和食品安全保障, 成为解决农业生态危机的有效途径。研究采用Illumina高通量测序平台对西北地区典型池塘鱼菜共生养殖模式下, 养殖水环境与根系细菌微生物16S rRNA基因(V3—V5区)进行高通量测序。比较群落结构和微生物多样性表明, 细菌总有效序列为56444条, 细菌物种平均注释OTU数目为945条。在鱼菜共生模式下的优势门类为变形菌门(Proteobacteria)、拟杆菌门(Bacteroidetes)、厚壁菌门(Firmicutes)、蓝菌门(Cyanobacteria)。根系微生物群落中占优势地位的分别为拟杆菌门(Bacteroidetes)和变形菌门(Proteobacteria)。OTU聚类分析结果显示6个样品共有OTU数目为165, 其中新疆水生野生动物救护中心鱼菜共生池塘水样(JH.KX)特有OTU数量最高(137), 其空心菜根系水样(JH.R)最低(30), 而乌鲁木齐市米东区长山子镇水源地养殖基地空心菜根系水样(MD.R)和新疆水生野生动物救护中心对照池塘水样(JH.C)数量相同(85)。通过对各OTU进行注释, 发现了根瘤菌、鱼类致病菌、耐寒细菌、降解有机物细菌、降低重金属及毒素危害的细菌。研究为鱼菜共生生态养殖模式下微生物群落结构及其生态调控机理提供了参考依据。     

Rapid differentiation of bacterial species by high resolution melting curve analysis

Molecular based differentiation of various bacterial species is important in phylogenetic studies, diagnostics and epidemiological surveillance, particularly where unusual phenotype makes the classical phenotypic identification of bacteria difficult. Molecular approach based on the sequence of 16S ribosomal RNA gene analysis can achieve fast and reliable identification of bacteria. High resolution melting (HRM) curve analysis has been developed as an attractive novel technique for DNA sequence discrimination but it’s application for bacteria differentiation has not been well studied yet. We have developed HRM assay for differentiation of sixteen pathogenic or opportunistic bacterial species. Amplified partial 16S ribosomal RNA gene region between 968 and 1401 positions (E. coli reference numbering) was subsequently used in high resolution melting curve analysis of PCR products for bacterial species differentiation. Sixteen bacterial species were simultaneously discerned by difference plot of normalized and temperatures shifted melting curves, without need for spiking of DNA, hetero-duplexing experiments or application of several primer pairs. High resolution melting curve analysis of duplex DNA is simple, fast and reliable tool for bacterial species differentiation and may efficiently complement phenotypic identification of bacteria.     

红鳍东方鲀病原鱼肠道弧菌的生物学特性研究

对引起红鳍东方鲀发病死亡的病原细菌进行了分离和主要生物学特性研究,包括病原性、形态特征、理化特性、16S rRNA基因序列及其系统发育学分析、胞外酶及溶血素活性、K抗原及耐药性等.结果表明,引起红鳍东方纯发病死亡的病原细菌为弧菌属(-Vibrio Pacini 1854)的鱼肠道弧菌(V.ichthyoenteri Ishimaru,et al.1996),2株代表菌株16S rRNA基凶序列(GenBank登录号分别为:EF611424和EF635304)与GenBank数据库中鱼肠道弧菌的同源性在98%-100%,且在构建的MP系统发生树中与鱼肠道弧菌聚为一个分支.分离菌不具有淀粉酶、蛋白酶、脂肪酶、DNA酶、脲酶、明胶酶和卵磷脂酶活性,且在含7%家兔脱纤血液营养琼脂培养基上不溶血.不具有K抗原.人工感染试验中分离菌对红鳍东方纯表现出明显的致病性.药敏试验结果显示,4株分离菌对供试37种抗菌药物中的苯唑青霉素和杆菌肽2种耐药.     

Bacteria of the Burkholderia cepacia complex: specific features of diagnostics,genome organization and metabolism

Modern data, related with the identification and typing of the complex B. cepacia bacteria, are analyzed in the article by using the poly-phase taxonomic approach. An optimal scheme for identifying and typing the complex B. cepacia bacteria, involving the microbiological and molecular-biological methods of laboratory diagnostics, is presented. The key and assumed factors of pathogenicity of the discussed bacteria are described. The possible phylogenetic relations of the complex B. cepacia bacteria with phytopathgens as well as with pathogenic bacteria of species Burkholderia, Pseudomonas, Escherichia, B. mallei, B. pdeudomallei, P. seruginosa and E. coli are described. A possible role of genome alterations and mutations in the genome of the complex B. cepacia bacteria (with the latter genome having unusual properties, i.e. a big size, and a considerable quantity of insertion sequences) in creating the conditions for the "pulsing" evolution "jerks", i.e. for a rapid change-over from saprophytism in the soil to a pathogenic causative agent of a viral-and-bacteriological infection. Such mechanism can be regarded as a rapid and radical adaptation of a microorganism under the conditions of changing ecological niches.     

婴幼儿腹泻病原学及其快速诊断

目的探讨小儿腹泻标本病原学特点及流行病学特征,寻求小儿感染性腹泻的快速诊断方法。方法对我院2003年10月至2005年9月门诊及住院的1160例2个月-5岁腹泻患儿的腹泻标本进行细菌学检查及pH检测。结果(1)1160例患儿腹泻标本病原学阳性605例(52.16%),检出病原20种621株,外源性肠道病原菌、内源性肠道条件致病菌以及轮状病毒3种病原构成比依次为35.75%、32.69%、31.56%。主要病原:(1)轮状病毒196株(31.56%),志贺菌118株(19.00%),克雷伯菌86株(13.8%),枸橼酸杆菌67株(10.79%),致泻性大肠埃希菌60株(9.66%)。(2)1160例患儿腹泻标本6个月-2岁患儿占881例(69.91%)。轮状病毒腹泻多发于秋冬季、大便为水样、多见于2岁以内婴幼儿,志贺菌以夏秋季与脓血便检出率较高。(3)病原菌分离阳性腹泻标本pH>7占明显优势,轮状病毒阳性标本、病原检查阴性标本、健康体检对照标本pH<6占明显优势,前者pH与后三者pH统计学比较,差异有高度显著性(P<0.01)。结论引起小儿腹泻的病原种类繁多,轮状病毒居首位,内源性肠道条件致病菌在腹泻标本中的构成比已接近外源性肠道致病菌而成为腹泻的重要病原菌,大便pH可作为急性腹泻病早期简单、快速、准确、价廉的初步诊断及合理应用抗生素的重要指标。