黄曲霉毒素对费氏弧菌发光的影响

[目的]探讨黄曲霉毒素对一种发光细菌——费氏弧菌发光的抑制效应.[方法]黄曲霉毒素或产黄曲霉毒素的菌株培养液对费氏弧菌进行处理后,利用多功能酶标仪检测费氏弧菌的发光强度,研究黄曲霉毒素对费氏弧菌发光的影响.[结果]黄曲霉毒素浓度的对数值与费氏弧菌发光的抑制率呈线性关系,依据所得的回归方程可以快速准确地检测不同微生物产毒素的情况:6株不同来源的黄曲霉菌株均能够产毒素,以黄曲霉毒素含量表示的毒素量在14.94 - 46.45mg/L之间,1株米曲霉不产毒素.[结论]费氏弧菌发光强度的改变可以较准确地反映微生物产毒素的能力,尤其是微生物产黄曲霉毒素的能力,为在工农业生产中快速检测黄曲霉毒素提供了新的线索,有望发展成为一种检测黄曲霉毒素的新技术.     

Effects of Magnesium Sulfate on the Luminescence of Vibrio fischeri under Nutrient-Starved Conditions

In this study, we investigated the relationship between MgSO₄ and luminescence in Vibrio fischeri under nutrient-starved conditions. When V. fischeri was cultured in an artificial seawater medium, the luminescence intensity was low relative to that observed under normal growth conditions. It decreased during the initial 14 h, and then increased slightly at 24 h. This regulation of luminescence was not dependent on the quorum-sensing mechanism, because the cell densities had not reached a critical threshold concentration. Under MgSO₄-starved conditions, luminescence was not fully induced at 14 h, and decreased at 24 h. In contrast, induction of luminescence occurred under MgSO₄-supplemented conditions, but MgSO₄ alone was insufficient to induce luminescence, and required NaHCO₃ or KCl. These results suggest that the luminescence of V. fischeri is controlled by an exogenous sulfur source under nutrient-starved conditions. In addition, they indicate that the induction of sulfur-dependent luminescence is regulated by the NaHCO₃ or KCl concentration.     

Evaluating the Acute Toxicity of Estrogen Hormones and Wastewater Effluents Using Vibrio fischeri

Toxicity evaluation of environmental substances such as those in wastewater and contaminated water bodies has become an important part of environmental monitoring of pollution. The study evaluated the toxicity of estrogen hormones and the removal of toxicity in full-scale wastewater treatment plants (WWTPs) using the marine bacterium, Vibrio fischeri, and to determine if there is a correlation between the hormones and the toxicity in the effluents. Three different types of full-scale WWTPs were investigated and presence of estrogens in the treated wastewater was evaluated by enzyme linked immunoassay (ELISA). The toxicity of individual estrogens (E2, EE2, and a mixture of E1, E2, and E3) was investigated as well as influents and treated wastewater. The results revealed that all estrogen hormones had less than 50% inhibitions and fell in the Class II group that exhibits slight acute toxicity. The toxicity of the individual E2 hormone had higher inhibitions when compared to the individual synthetic EE2 and the mixture of the hormones. The toxicity results of the WWTP revealed that biological treatment can reduce the toxicity of the influent to an extent. The findings suggest that the residual estrogen contents as well as toxicity can be reduced in certain WWTPs.     

Experimental design of a simple medium for the bioluminescence of Aliivibrio fischeri and mathematical modelling for growth estimation

Increasing numbers of studies are using Aliivibrio fischeri (A. fischeri), a marine bioluminescent bacterium as a model, however the culture medium used for its growth are complex and expensive. The objectives of this study were: (1) to evaluate the effect of yeast extract, tryptone, and NaCl to select a simple and inexpensive culture medium suitable for A. fischeri growth and bioluminescence induction; and (2) to compare the performance of mathematical models to predict the growth of A. fischeri. A fractional factorial design was performed to evaluate the effect of yeast extract, tryptone, and sodium chloride on the luminescence of A. fischeri. The result showed that sodium chloride is the most important factor, congruent with its inducer role in bioluminescence. The best medium for bioluminescence induction was selected through an optimization plot, this medium is inexpensive, and generates the same luminescence as commercial formulations. The estimation of A. fischeri growth at OD₆₀₀ measurement was statistically analyzed. All evaluated models fitted the data adequately (r²  > 0.96). The nonlinear models Gompertz, Richards and logistic provided a lower variation and a better fit of the growth estimation (r² >0.99), showing that these mathematical models can be used for the accurate growth prediction of A. fischeri.     

LapG mediates biofilm dispersal in Vibrio fischeri by controlling maintenance of the VCBS-containing adhesin LapV

Efficient symbiotic colonization of the squid Euprymna scolopes by the bacterium Vibrio fischeri depends on bacterial biofilm formation on the surface of the squid’s light organ. Subsequently, the bacteria disperse from the biofilm via an unknown mechanism and enter through pores to reach the interior colonization sites. Here, we identify a homolog of Pseudomonas fluorescens LapG as a dispersal factor that promotes cleavage of a biofilm-promoting adhesin, LapV. Overproduction of LapG inhibited biofilm formation and, unlike the wild-type parent, a ΔlapG mutant formed biofilms in vitro. Although V. fischeri encodes two putative large adhesins, LapI (near lapG on chromosome II) and LapV (on chromosome I), only the latter contributed to biofilm formation. Consistent with the Pseudomonas Lap system model, our data support a role for the predicted c-di-GMP-binding protein LapD in inhibiting LapG-dependent dispersal. Furthermore, we identified a phosphodiesterase, PdeV, whose loss promotes biofilm formation similar to that of the ΔlapG mutant and dependent on both LapD and LapV. Finally, we found a minor defect for a ΔlapD mutant in initiating squid colonization, indicating a role for the Lap system in a relevant environmental niche. Together, these data reveal new factors and provide important insights into biofilm dispersal by V. fischeri.     

Colonization of Euprymna scolopes squid by Vibrio fischeri

Specific bacteria are found in association with animal tissue. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no fitness consequence (commensal), or be beneficial (mutualistic). While much attention has been given to pathogenic interactions, little is known about the processes that dictate the reproducible acquisition of beneficial/commensal bacteria from the environment. The light-organ mutualism between the marine Gram-negative bacterium V. fischeri and the Hawaiian bobtail squid, E. scolopes, represents a highly specific interaction in which one host (E. scolopes) establishes a symbiotic relationship with only one bacterial species (V. fischeri) throughout the course of its lifetime. Bioluminescence produced by V. fischeri during this interaction provides an anti-predatory benefit to E. scolopes during nocturnal activities, while the nutrient-rich host tissue provides V. fischeri with a protected niche. During each host generation, this relationship is recapitulated, thus representing a predictable process that can be assessed in detail at various stages of symbiotic development. In the laboratory, the juvenile squid hatch aposymbiotically (uncolonized), and, if collected within the first 30-60 minutes and transferred to symbiont-free water, cannot be colonized except by the experimental inoculum. This interaction thus provides a useful model system in which to assess the individual steps that lead to specific acquisition of a symbiotic microbe from the environment. Here we describe a method to assess the degree of colonization that occurs when newly hatched aposymbiotic E. scolopes are exposed to (artificial) seawater containing V. fischeri. This simple assay describes inoculation, natural infection, and recovery of the bacterial symbiont from the nascent light organ of E. scolopes. Care is taken to provide a consistent environment for the animals during symbiotic development, especially with regard to water quality and light cues. Methods to characterize the symbiotic population described include (1) measurement of bacterially-derived bioluminescence, and (2) direct colony counting of recovered symbionts.     

Logarithmic Transformation is Essential for Statistical Analysis of Fungicide EC50 Values

Half maximal (50%) effective concentration (EC50) values are widely used to express fungicide potency and sensitivity of plant pathogens. This study explored the necessity of logarithmic transformation for statistical analysis of EC50 values. The results demonstrated that without logarithmic transformation, none of the five sets of epoxiconazole EC50 data (n = 26–33) against Sclerotinia sclerotiorum fitted a normal distribution. But after logarithmic transformation, four of the five datasets became normally distributed. Of the five sets of pyraclostrobin EC50 data (n = 29–32), only one dataset fitted a normal distribution. After logarithmic transformation, four datasets became normally distributed. Logarithmic transformation transformed the heterogeneity of variance across the five sets of epoxiconazole EC50 data to homogeneity but failed to improve the heterogeneity of variance across the five sets of pyraclostrobin EC50 data. For 150 isolates' EC50 values to epoxiconazole and 153 isolates' EC50 values to pyraclostrobin, the intervals of arithmetic means ± standard deviations (SD) covered 85.3% and 90.2% of data points, respectively, whereas the intervals of geometric means (*) multiplied/divided by the multiplicative SD (S*) covered 69.3% and 70.9% of data points, respectively, which approximated the theoretical value of 68.3%. Distribution normality and homogeneity of variance are prerequisites for analysis of variance (anova ) and the two parameters could be improved by logarithmic transformation, therefore, power and efficiency of statistical tests on EC50 data will be greatly enhanced by this kind of transformation.     

Determination of antibiotic EC50 using a zero‐flow microfluidic chip based growth phenotype assay

Current existing assay systems for evaluating antimicrobial activity suffer from several limitations including excess reagent consumption and inaccurate concentration gradient preparation. Recently, microfluidic systems have been developed to provide miniaturized platforms for antimicrobial susceptibility assays. However, some of current microfluidic based assays require continuous flows of reagents or elaborate preparation steps during concentration preparation. In this study, we introduce a novel microfluidic chip based growth phenotype assay that automatically generates a logarithmic concentration gradient and allows observing the growth of pathogenic bacteria under different concentrations of antibiotics in nanoliter batch culture reactors. We chose pathogen bacterium Pseudomonas aeruginosa as a model strain and evaluated the inhibitory effects of gentamicin and ciprofloxacin. We determined the EC₅₀ values and confirmed the validity of the present system by comparing the EC₅₀ values obtained through conventional test tube method. We demonstrated that the EC₅₀ values acquired from present assay are comparable to those obtained from conventional test tube cultures. The potential application of present assay system for investigating combinatorial effects of antibiotics on multidrug resistant pathogenic bacteria is discussed and it can be further used for systematic evaluation of antifungal or antiviral agents.     

A rapid screening method for the detection of viable spores in powder using bioluminescence.

A rapid diagnosis of a biological threat in a powder sample is important for fi rst responders who have to make decisions on-site. The present culture-based method does not provide timely results, which is a critical barrier for a quick response when a suspicious powder sample is found. The ATP bioluminescence method, combined with a heat shock, was investigated to determine the presence of spores in powder. The results show that only spore-containing powder samples provided a dramatic increase in the bioluminescence signal after the heat shock, which induces germination of the spores. Various conditions were tested to fi nd the most effective and rapid germination procedure. Elevated temperatures (37 degrees C and 50 degrees C) were more effective in germination than room temperature. At 50 degrees C, a double-strength germinant was more effective in germination than the regular strength. The 37 degrees C/15 min procedure induced the germination of spores most effectively, while a 50 degrees C/2 min procedure provided reasonably high signals, so it could make the entire procedure even faster (< 5 min). The detection limit of the bioluminescence method is < 100 spores.     

水产品中创伤弧菌和副溶血弧菌双重PCR检测方法的建立

目的 建立一种同步检测创伤弧菌和副溶血弧菌的双重PCR方法。方法 选择副溶血弧菌tlh基因和创伤弧菌vvhA基因作为靶序列各设计一对引物。用合成的引物对副溶血弧菌和创伤弧菌进行双重PCR扩增,确定特异性和最低检出限。然后用此方法对53株副溶血弧菌和7株创伤弧菌进行检测。结果 确定了双重PCR检测创伤弧菌和副溶血弧菌的最优反应条件,其中退火温度为60 ℃,方法具有较好的特异性。对副溶血弧菌的最低限为1.0×102 CFU/mL,创伤弧菌最低限为4.2×104 CFU/mL。双重PCR对分离株检测符合率达100%。结论 建立的双重PCR方法简便、快速、特异性好,可同时检测副溶血弧菌和创伤弧菌,为水产品中病原菌的基层检测提供解决方案。     

GroEL/GroES chaperone and Lon protease regulate expression of the Vibrio fischeri lux operon in Escherichia coli

Chaperone GroEL/GroES and Lon protease were shown to play a role in regulating the expression of the Vibrio fischeri lux operon cloned in Escherichia coli cells. The E. coli groE mutant carrying a plasmid with the full-length V. fischeri lux regulon showed a decreased bioluminescence. The bioluminescence intensity was high in E. coli cells with mutant lonA and the same plasmid. Bioluminescence induction curves lacked the lag period characteristic of lon ⁺ strains. Regulatory luxR of V. fischeri was cloned in pGEX-KG to produce the hybrid gene GST-luxR. The product of its expression, GST-LuxR, was isolated together with GroEL and Lon upon affinity chromatography on a column with glutathione-agarose, suggesting complexation of LuxR with these proteins. It was assumed that GroEL/GroES is involved in LuxR folding, while Lon protease degrades LuxR before its folding into an active globule or after denaturation.