An interactive microcomputer program for the computation of most probable number

An interactive computer program for the rapid computation of Most Probable Numbers (MPN) with 95% confidence intervals and goodness-of-fit, is presented. The program, written in ‘MICROSOFT BASIC’ employs an iterative algorithm based on a modification of the Newton-Raphson method and accomodates any number of replicates up to ten dilutions. It is applicable to most microcomputers with little or no modification. Since the computed MPN, confidence interval,a nd goodness-of-fit tests are displayed simultaneously, MPN tables are no longer required.     

Application of the most probable number method to estimations of entomopathogenic nematode populations in soil

The calculation of most probable numbers (mpn) was used for the estimation of numbers of infective entomopathogenic nematodes in soil. The mpn concept was first introduced in bacteriology as a means of estimating numbers of organisms in a substrate without a direct count, in cases where direct enumeration could not be applied. In the work reported here, soil samples infested with infective juveniles (IJs) of Heterorhabditis megidis (isolate HF85) were diluted with uninfested soil and the diluted soils were baited with mealworm larvae (Tenebrio molitor). The mpn of infective units of IJs in the undiluted soil was calculated. The mpn calculation was found to be applicable to entomopathogenic nematodes in soil under particular conditions. It could be successfully applied to data for the response of soil units but not to the data based on the response of individual insects, as the latter did not confirm to a Poisson series. The calculated mpn represented between 2.9 and 7.1% of the initial inoculum of IJs. It was suggested that IJs might act as a group in infecting an insect host. Using the data for Tenebrio mortality on parasitisation, the mpn based on quantal response of the mealworms would therefore not give the true density of IJs in the soil sample but the effective density, or the quantity of infective units. Although the biological significance of the infective unit needs further clarification, mpn was found to be a useful parameter for use in comparative experiments.     

Use of the most probable number technique to quantify soil-borne plant pathogens

Bioassays using serial soil dilutions and most probable number (MPN) estimations have been used by various authors to quantify inoculum of soil-borne plant pathogens. The requirements of a reliable bioassay are discussed; they include a good choice of dilution series and reproducible growing conditions. Sources of computer programs for analysis of the data are listed. The importance of testing the fit to the mathematical model used is illustrated and emphasised. Factors affecting the size and stability of the standard errors and of the inherent bias of the most probable number estimate are discussed. Equations are presented for calculating the expected standard errors, approximate confidence limits and least significant differences for different dilution factors and numbers of replicates. The benefits of using uneven replication are illustrated. Mathematical considerations show that the technique should enable differences of an order of magnitude to be detected and MPNs should be quoted with a maximum of two significant figures. Dilution ratios as large as 10 should be avoided. Statistical and biological difficulties, especially in standardising growing conditions when soil moisture is critical, indicate that results should normally be regarded as relative, rather than absolute, measurements of inoculum.     

最大或然数法在光合细菌计数中的应用及效果研究

1引言光合细菌(photosynthetic bacteria,PSB)是能进行不放氧光合作用的一大类细菌的总称,属水圈微生物,广泛分布于地球生物圈,无论江、河、湖、海,水田、旱地都有存在[3].光合细菌在水体自净、调节微生态平衡、促进动植物生长、增加产量和提高产品质量、防病、固氮等方面具有重要作用[3,22].近年来,光合细菌菌剂在水产养殖、家畜养殖、污水处理及植物生产上的应用日益广泛[3,13,19,27,28,31],相继推出多种产品,包括单菌菌剂、复合菌剂,剂型上有液体菌剂、浓缩菌剂和固体菌剂(粉剂)等[20].为保证产品质量和应用效果,有必要对光合细菌菌剂…     

Most probable number methodology for quantifying dilute concentrations and fluxes of Salmonella in surface waters

Aims: To better understand and manage the fate and transport of Salmonella in agricultural watersheds, we developed a culture‐based, five tube–four dilution most probable number (MPN) method for enumerating dilute densities of Salmonella in environmental waters. Methods and Results: The MPN method was a combination of a filtration technique for large sample volumes of environmental water, standard selective media for Salmonella and a TaqMan confirmation step. This method has determined the density of Salmonella in 20‐l samples of pond inflow and outflow streams as low as 0·1 MPN l^?1 and a low 95% confidence level 0·015 MPN l^?1. Salmonella densities ranged from not detectable to 0·55 MPN l^?1 for pond inflow samples and from not detectable to 3·4 MPN l^?1 for pond outflow samples. Salmonella densities of pond inflow samples were associated with densities of Escherichia coli and faecal enterococci that indicated stream contamination with faeces and with nondetectable pond outflow densities of the faecal indicator bacteria. The MPN methodology was extended to flux determinations by integrating with volumetric measurements of pond inflow (mean flux of 2·5 l s^?1) and outflow (mean flux of 5·6 l s^?1). Fluxes of Salmonella ranged from 100 to greater than 10⁴ MPN h^?1. Conclusions: This is a culture‐based method that can detect small numbers of Salmonella in environmental waters of watersheds containing animal husbandry and wildlife. Significance and Impact of the Study: Applying this method to environmental waters will improve our understanding of the transport and fate of Salmonella in agricultural watersheds, and can be the basis of valuable collections of environmental Salmonella.     

Development of real-time PCR assay for detection and quantification of Sinorhizobium meliloti in soil and plant tissue

Aims:  Sinorhizobium meliloti is a nitrogen-fixing alpha-proteobacterium present in soil and symbiotically associated with root nodules of leguminous plants. To date, estimation of bacterial titres in soil is achieved by most-probable-number assays based on the number of nodules on the roots of test plants. Here, we report the development of two real-time PCR (qPCR) assays to detect the presence of S. meliloti in soil and plant tissues by targeting, in a species-specific fashion, the chromosomal gene rpo E1 and the pSymA gene nod C.
Methods and Results:  rpo E1 and nod C primer pairs were tested on DNA extracted from soil samples unspiked and spiked with known titres of S. meliloti and from plant root samples nodulated with S. meliloti . Results obtained were well in agreement with viable titres of S. meliloti cells estimated in the same samples.
Conclusions:  The developed qPCR assays appear to be enough sensitive, precise and species-specific to be used as a complementary tool for S. meliloti titre estimation.
Significance and Impact of the Study:  These two novel markers offer the possibility of quick and reliable estimation of S. meliloti titres in soil and plant roots contributing new tools to explore S. meliloti biology and ecology including viable but nonculturable fraction.     

Comparative metagenomic,phylogenetic and physiological analyses of soil microbial communities across nitrogen gradients

Terrestrial ecosystems are receiving elevated inputs of nitrogen (N) from anthropogenic sources and understanding how these increases in N availability affect soil microbial communities is critical for predicting the associated effects on belowground ecosystems. We used a suite of approaches to analyze the structure and functional characteristics of soil microbial communities from replicated plots in two long-term N fertilization experiments located in contrasting systems. Pyrosequencing-based analyses of 16S rRNA genes revealed no significant effects of N fertilization on bacterial diversity, but significant effects on community composition at both sites; copiotrophic taxa (including members of the Proteobacteria and Bacteroidetes phyla) typically increased in relative abundance in the high N plots, with oligotrophic taxa (mainly Acidobacteria) exhibiting the opposite pattern. Consistent with the phylogenetic shifts under N fertilization, shotgun metagenomic sequencing revealed increases in the relative abundances of genes associated with DNA/RNA replication, electron transport and protein metabolism, increases that could be resolved even with the shallow shotgun metagenomic sequencing conducted here (average of 75 000 reads per sample). We also observed shifts in the catabolic capabilities of the communities across the N gradients that were significantly correlated with the phylogenetic and metagenomic responses, indicating possible linkages between the structure and functioning of soil microbial communities. Overall, our results suggest that N fertilization may, directly or indirectly, induce a shift in the predominant microbial life-history strategies, favoring a more active, copiotrophic microbial community, a pattern that parallels the often observed replacement of K-selected with r-selected plant species with elevated N.     

云南土壤宏基因组文库中替加环素耐药基因的筛选与分析

[背景]随着抗生素的广泛应用以及滥用,出现了多重耐药的"超级细菌",并且在临床上已出现对治疗"超级细菌"感染的最后一线抗生素——替加环素耐药的细菌.[目的]对土壤环境中存在的替加环素耐药基因进行筛选调查,探讨替加环素耐药机制,为临床抗生素治疗提供借鉴.[方法]利用功能宏基因组学技术对土壤宏基因组文库进行替加环素耐药阳性...     

The most probable number of blocks for the partitions of the set of codons could have determined the number of standard amino acids

Given a genetic code formed by 64 codons, we calculate the number of partitions of the set of encoding amino acid codons. When there are 0-3 stop codons, the results indicate that the most probable number of partitions is 19 and/or 20. Then, assuming that in the early evolution the genetic code could have had random variations, we suggest that the most probable number of partitions of the set of encoding amino acid codons determined the actual number 20 of standard amino acids.     

Phylogenetic and metagenomic analysis of Verrucomicrobia in former agricultural grassland soil

The bacterial phylum Verrucomicrobia has a widespread distribution, and is known to be one of the most common and diverse phyla in soil habitats. However, members of this phylum have typically been recalcitrant to cultivation methods, hampering the study of this presumably important bacterial group. In this study, we examine the phylogenetic diversity of the Verrucomicrobia in a former agricultural field and gain access to genomic information via a metagenomic approach. We examined Verrucomicrobia -like 16S rRNA gene sequences recovered from general bacterial and phylum-specific libraries, revealing a dominance of subdivisions 1 and 2. A PCR-based screening method was developed to identify inserts containing verrucomicrobial 16S rRNA genes within a large-insert metagenomic library, and on screening of 28 800 clones, four fosmids were identified as containing verrucomicrobial genomic DNA. Full-length sequencing of fosmid inserts and gene annotation identified a total of 98 ORFs, representing a range of functions. No conservation of gene order was observed adjacent to the ribosomal operons. Fosmid inserts were further analyzed for tetranucleotide frequencies to identify remnants of past horizontal gene transfer events. The metagenomic approach utilized proved to be suitable for the recovery of verrucomicrobial genomic DNA, thereby providing a window into the genomes of members of this important, yet poorly characterized, bacterial phylum.     

稻田温室气体排放与土壤微生物菌群的多元回归分析

为揭示多种田间管理措施综合影响下双季稻田温室气体平均排放通量与土壤微生物菌群的多元回归关系,利用静态箱—气相色谱法和稀释培养计数法进行了温室气体排放通量和土壤产气微生物菌群数量的连续观测。两年研究结果显示,稻田甲烷排放通量与土壤微生物总活性和产甲烷菌数量关系密切,甲烷排放通量与二者的关系可分别由指数和二次多项式模型拟合。一元回归分析表明,仅产甲烷菌数量就能单独解释96.9%的稻田甲烷排放通量变异（R2=0.969,P<0.001）,但考虑两种因素以后的二元回归拟合优度高于一元回归（R2=0.975,P<0.001）。氧化亚氮排放通量与土壤硝化细菌和反硝化细菌数量也密切相关（P <0.05）,氧化亚氮排放通量与二者的二元非线性混合回归模型可以解释至少70.4%的稻田氧化亚氮排放通量（R2≥0.704, P <0.001）,其拟合优度也高于一元回归。稻田温室气体排放通量受多种影响因素控制,土壤产气微生物活性和数量是多种因素影响的直接响应,因此二者与温室气体排放存在显著相关,基于田间试验的多元非线性回归分析客观的揭示了温室气体排放通量与环境因子的相关关系。     

Oxidative consumption of nitric oxide by heterotrophic bacteria in soil

Abstract: Uptake rate constants for nitric oxide were measured in a neutral calcic cambisol (KBE) and an acidic luvisol (PBE). The NO uptake was higher under oxic than under anoxic incubation conditions by a factor of about three. Gassing the soils with air containing 10 ppmv NO resulted in the accumulation of nitrate which accounted for 57–94% of the NO consumed. Aerobic heterotrophic bacteria were isolated on glucose-yeast extract medium from soil dilutions corresponding to a most probable number of 10⁸–10⁹ bacteria per gram dry weight soil. One of the isolates (strain PS88, a Pseudomonas sp.) exhibited NO consumption activity that was much higher under oxic than anoxic incubation conditions. When sterile KBE amended with strain PS88 was gassed with air containing 10 ppmv NO, 88% of the consumed NO was recovered as nitrate and nitrite. A screening of various bacteria obtained from culture collections showed a widespread ability for consumption of low NO concentrations. Our results indicate that NO consumption in soil is not only possible by reductive denitrification, but also by oxidation due to aerobic heterotrophic bacteria such as strain PS88.     

Reconsideration of the derivation of Most Probable Numbers,their standard deviations,confidence bounds and rarity values

Aims: The purpose of this work was to derive a simple Excel spreadsheet and a set of standard tables of most probable number (MPN) values that can be applied by users of International Standard Methods to obtain the same output values for MPN, SD of the MPN, 95% confidence limits and test validity. With respect to the latter, it is considered that the Blodgett concept of ‘rarity’ is more valuable than the frequently used approach of improbability (vide de Man). Methods and Results: The paper describes the statistical procedures used in the work and the reasons for introducing a new set of conceptual and practical approaches to the determination of MPNs and their parameters. Examples of MPNs derived using these procedures are provided. The Excel spreadsheet can be downloaded from http://www.wiwiss.fu‐berlin.de/institute/iso/mitarbeiter/wilrich/index.html . Conclusions: The application of the revised approach to the determination of MPN parameters permits those who wish to use tabulated values, and those who require access to a simple spreadsheet to determine values for nonstandard test protocols, to obtain the same output values for any specific set of multiple test results. The concept of ‘rarity’ is a more easily understood parameter to describe test result combinations that are not statistically valid. Provision of the SD of the log MPN value permits derivation of uncertainty parameters that have not previously been possible. Significance and Impact of the Study: A consistent approach for the derivation of MPNs and their parameters is essential for coherence between International Standard Methods. It is intended that future microbiology standard methods will be based on the procedures described in this paper.     

Toward the validation of a new method (MUNIX) for motor unit number assessment

Introduction: This prospectively designed study analyzed the correlation of a new, non-invasive neurophysiological method (Motor Unit Number Index – MUNIX) with two established Motor Unit Number Estimation (MUNE) methods. Methods: MUNIX and incremental stimulation MUNE (IS-MUNE) were done in the abductor digiti minimi muscle (ADM), while MUNIX and spike-triggered averaging MUNE (STA-MUNE) were tested in the trapezius muscle. Twenty healthy subjects and 17 patients with amyotrophic lateral sclerosis (ALS) were examined. Results: MUNIX and MUNE values correlated significantly (ADM: n = 108; Spearman-Rho; r = 0.88; p < 0.01; trapezius muscle: n = 49; Spearman-Rho; r = 0.46; p < 0.01). Discussion: MUNIX indeed reflects the number of motor units in a muscle, and may sensibly be recorded from the trapezius muscle. With MUNIX being both much more patient friendly and much more rapid to assess than MUNE, the results support the use of MUNIX when motor unit number assessment is desired.     

Structure of the Mucosal and Stool Microbiome in Lynch Syndrome

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宏基因组及培养组学技术在粪菌移植中的应用

粪菌移植技术（fecal microbiota transplantation,FMT）是利用健康人群的粪便或经过处理的粪便中的微生物来治疗消化、代谢等系统诸多疾病的一项古老而又新兴的技术。宏基因组、培养组等肠道微生物组前沿研究技术的飞速发展,为粪菌移植治疗疾病提供了强有力的研究和临床实践武器。宏基因组技术可全面揭示健康及疾病状态下肠道微生态的组成及功能变化,而培养组学技术则可以用来分离和鉴定人类肠道中的诸多在常规培养条件下未可培养菌,两项技术联用不仅可以使我们更为深入地理解粪菌移植在临床实践中的因果规律,还将有力推动粪菌移植技术在未来的应用与发展。基于此,本文综述了宏基因组及培养组学技术在粪菌移植中的应用及未来发展趋势。