A comparison of 2 methods for determining the hydrophobicity of Streptococcus groups A and B

A simple method for the evaluation of the hydrophobic properties of streptococci, pathogenic for humans, by their adherence to polystyrene and a modified method for measuring their hydrophobic properties by their sorption on hexadecane have been developed. The results obtained in the evaluation of the hydrophobic properties of streptococci by these two methods have been compared and the complete correlation of these results in classifying the cultures as hydrophobic and hydrophilic has been shown. For the first time the differences in the hydrophobic properties of different strains of group B streptococci have been established.     

香菇单孢杂交子代群体灰色关联度和ISSR分析

以香菇Lentinula edodes栽培菌株秋6和K95-1为亲本,通过单孢菌株配对杂交获得21个杂交子,观察杂交子及亲本菌丝体生长情况,进行栽培出菇试验。采用灰色关联度分析法,对9个正常出菇的杂交子从8个性状方面进行综合评价,结果表明,杂交子QK-8和QK-15关联度仅次于亲本秋6,农艺性状和子实体性状表现最好。采用ISSR技术对21个杂交子及亲本进行DNA多态性聚类分析,单孢杂交后代遗传分化十分明显,归于同一类群的杂交子在菌丝生长、子实体形态和农艺性状等方面常表现相似,ISSR分析能为优良杂交子初步筛选提供重要参考。     

Evaluation of candidate probiotic strains for gilthead sea bream larvae (Sparus aurata) using an in vivo approach

AIMS: The aim of this study was to evaluate the effect of six bacterial strains on gilthead sea bream larvae (Sparus aurata). METHODS AND RESULTS: Six bacterial strains isolated from well-performing live food cultures were identified by sequencing fragments of their 16s rDNA genome to the genus level as Cytophaga sp., Roseobacter sp., Ruergeria sp., Paracoccus sp., Aeromonas sp. and Shewanella sp. Survival rates of gilthead sea bream larvae transferred to seawater added these bacterial strains at concentrations of 6 +/- 0.3 x 10(5) bacteria ml(-1) were similar to those of larvae transferred to sterilized seawater and showed an average of 86% at 9 days after hatching, whereas, survival rates of larvae transferred to filtered seawater were lower (P < 0.05), and showed an average of 39%, 9 days after hatching. CONCLUSION: Several bacterial strains isolated from well-performing live food cultures showed a positive effect for sea bream larvae when compared with filtered seawater. SIGNIFICANCE AND IMPACT OF THE STUDY: The approach used in this study could be applied as an in vivo evaluation method of candidate probiotic strains used in the rearing of marine fish larvae.     

高粱材料耐盐性综合评价方法的初步建立与验证

用0、0.2%、0.4%、0.6%、0.8%5个浓度的NaCl溶液对10份高粱材料进行处理,测定了发芽盐害率、芽高盐害率、侧根数盐害率、根长盐害率、1/2叶片萎蔫持续时间和整个叶片萎蔫持续时间6项指标。以发芽期和苗期的各项指标为基础,根据每个指标盐害率制定各指标的得分方法,最后通过各个材料的得分判断其耐盐性,建立了一套高粱材料的耐盐评价标准。以此标准对10份高粱材料的耐盐性进行评价,排列出这10份高粱材料的耐盐次序,将高粱材料划分为4个耐盐等级。从这10份高粱材料中随机挑选5份耐盐性不同的材料通过盆栽试验对其耐盐性进行鉴定,以检验该评价方法的可靠性,结果表明:盆栽试验的鉴定结果与按照耐盐评分标准得出的结果一致,由此确定此评价方法运用于筛选高粱材料的耐盐性是准确、可靠的。同时在这10份高粱材料中筛选出了耐盐性较强的高粱材料河农1号和能饲1号。     

Characterization of Lactobacillus plantarum from wine must by PCR species-specific and RAPD-PCR

AIMS: Physiological and molecular analysis such as PCR species-specific and randomly amplified polymorphic PCR (RAPD-PCR) have been used for typing of Lactobacillus plantarum strains from typical wine must. METHODS AND RESULTS: Phenotypic tests such as API 50CH and evaluation of D-L-lactate production from glucose were used to perform a preliminary characterization of lactobacilli. Furthermore, 18 strains of lactobacilli were analyzed by PCR species-specific oligonucleotides based on short sequences of the recA gene. CONCLUSIONS: Four strains were identified as belonging to the L. plantarum species and were further analysed by RAPD-PCR. The RAPD-PCR profiles were similar in all strains that had positive results for species-specific PCR, suggesting that the four L. plantarum strains were closely related. SIGNIFICANCE AND IMPACT OF THE STUDY: Using PCR species-specific as a preliminary screening test and then RAPD-PCR can be as considered the most reliable method of performing a rapid and correct typing of L. plantarum from wine must.     

金针菇自交后代生物学特性的研究

选用6个生产用金针菇菌种(白色品种菌株F10、F4、FM、F21,黄色品种菌株F29、F3)分别进行自交,对其S1代自交群体的菌丝生长速度、产量、原基发生早晚、农艺性状进行综合分析。结果表明,自交导致后代群体的平均生长速度、平均产量降低。黄色自交子代菌株平均生长速度快于白色自交子代菌株。菌丝生长速度与产量不具有相关性,产量与现原基早晚有较强相关性,相同自交系中黄色品种现原基早于白色品种,F3菌株黄色后代现原基早于白色后代。在各菌株自交子代群体中,FM菌株子代具有高产优势,F3菌株子代有短菌龄优势,F10菌株子代有较好的商品表型特征,可根据育种目标选择自交子代群体中的优势菌株加以利用。     

Evaluation of purified recombinant spike fragments for assessment of the presence of serum neutralizing antibodies against a variant strain of porcine epidemic diarrhea virus

Since 2010, variant strains of porcine epidemic diarrhea virus(PEDV) have caused disasters in the pork industry. The spike(S) protein, as the major immunity-eliciting antigen, has previously been used for serological testing and has been found to correlate significantly with the results of the serum neutralization(SN) test. However, further evaluation of this method is needed as new epidemic strains of PEDV emerge. Hence, the main objective of this study was to assess sow sera and determine the correlation between enzyme-linked immunosorbent assay(ELISA) results(involving a newly isolated GDS01 virus-based ELISA and ELISAs based on seven recombinant fragments comprising overlapping S1 and partial S2 sequences) and SN titers. Furthermore, we determined the reliability of the ELISAs based on receiver operating characteristics(ROC) curve analyses. For the most promising ELISA, i.e., the SP4 ELISA, the correlation coefficient(r) and the area under curve(AUC) were determined to be 0.6113 and 0.8538, respectively. In addition, we analyzed the homology of the SP4 sequences obtained from different strains(including vaccine strains) and found that various strains showed a high degree of homology in this region. Thus, we conclude that SP4 is a promising serological testing protein for use in the field.     

Microcolony morphology as a criterion in bacterial diagnosis. The differentiation of Pneumococcus from alpha-hemolytic Streptococcus

The authors have developed a method permitting the microscopic study of the morphology of bacteria and their relative position in microcolonies. Thus, the use of this method has made it possible to distinguish Streptococcus pneumoniae from other bacteria by the morphology of their microcolonies. In the study of 75 streptococcal strains, all strains yielding positive results in two or three tests, similarly to all strains pathogenic for mice, formed microcolonies with granular (pneumococcal) morphology, while all strains yielding negative results have been found to form microcolonies with catenulate ("nonpneumococcal") morphology. The authors suggest that the morphology of microcolonies is a more reliable criterion for the identification of S. pneumoniae than other tests used separately.     

IDENTIFICATION OF NEW TARGET SEQUENCES FOR PCR DETECTION OF VIBRIO PARAHAEMOLYTICUS BY GENOME COMPARISON

A genome comparison method was used to identify specific target sequences for the polymerase chain reaction (PCR) detection of Vibrio parahaemolyticus, and the CDS value of this bacterium was compared with that of 139 other bacterial genomes. It was found that 20 CDS of V. parahaemolyticus were relatively specific according to their E value in BLAST (a new tool for comparing protein and nucleotide sequences), and four of them were selected for the design of PCR primers. There were positive amplification products of these four pairs of primers from nine V. parahaemolyticus strains, whereas there were no amplification products from nine other Vibrionaceae strains and four non -Vibrionaceae strains. An evaluation of detection sensitivities revealed that these four pairs of primers can be used in a PCR assay for the detection of V. parahaemolyticus.

PRACTICAL APPLICATIONS

An automatic BLAST method was developed in this study, by which species-specific sequences can be screened out rapidly. In this way, new and specific genes of Vibrio parahaemolyticus were identified to be used as target sequences for PCR detection. In terms of acceptable specificity and sensitivity, the four pairs of primers were selected by screening, which can be applied in PCR assays and other molecular methods. These kinds of methods might become commercial detection products in the new future. In addition, this method for searching specific DNA sequences can also be used for the mining specific sequences in other genus and species, such as Salmonella , Staphylococcus , etc.     

2株动物微生态制剂菌的分离鉴定及其生物学特性初步研究

目的从饲料中进行微生物的分离与培养,筛选动物微生态制剂候选菌株。方法利用变性梯度凝胶电泳(DGGE)筛选得到的7株微生物区分为2种菌,经测序确定其为热带假丝酵母和植物乳杆菌;检测了不同pH、温度、胆盐、金属铜和需/厌氧对其生长的影响。结果当pH小于2.5或铜离子高于150 ppm时,植物乳杆菌无法生长,而热带假丝酵母数量略有下降;胆盐和金属离子对2种菌影响较小,42℃培养条件下相对于30℃培养时热带假丝酵母数量下降了6个数量级。结论筛选得到的2株菌具有应用于动物微生态制剂的潜力,为动物微生态制剂候选菌筛选和评价提供了基础数据。     

A new and rapid bioassay for the detection of gliotoxin and related epipolythiodioxopiperazines produced by fungi

Gliotoxin is an immunosuppressive cytotoxin produced by numerous environmental or pathogenic fungal species. For this reason, it is one of the mycotoxins which must be systematically searched for in samples for biological control. In this study, a new, rapid and sensitive method for detecting gliotoxin has been developed. This bioassay is based on the induction of morphological changes in cultured cells (human KB cell line) by gliotoxin. Interpretation of the assay can be carried out after 1 h of incubation, either by direct microscopic observation, or with an automated microplate-reader at 630 nm. The limit of detection is 18-20 ng of gliotoxin in the well, depending on the used observation method. A high degree of specificity of the detection is brought about by the ability of the reducing reactant dithiothreitol to inhibit the biological activities of epipolythiodioxopiperazines (ETPs), such as gliotoxin, by reducing their polysulfide bridge. The bioassay allows a rapid primary screening of samples and a semi-quantitative evaluation of the gliotoxin concentration in extracts. The method has been used to study the gliotoxin production by different fungal strains, allowing to highlight 3 strains of Aspergillus fumigatus producing gliotoxin in various extracts.     

Methods to evaluate nodulation competitiveness between Sinorhizobium meliloti strains using melanin production as a marker

Three methods to evaluate the relative ability of different strains of Sinorhizobium meliloti to occupy nodules formed on alfalfa after co-inoculation were compare in this study. Results obtained using the three methods of evaluation together, provided insight into the relative nodulation competitiveness between two given sinorhizobial strains. A simple visual phenotypic marker, i.e., melanin production was used to distinguish individual strains in a given assay. As such, melanin producing strains were compared with melanin non-producing strains throughout this study. Method 1 required the use of an ELISA plate, took 35 min for the analysis of 40 nodules, and allowed strain identification by melanin production 2 days after nodule harvest. Method 2 required 3 h for the analysis of 40 nodules, used an ELISA plate, growth of bacteria on Petri dishes, and melanin production was analysed after 48 h of cell culture. Finally, method 3 involved the whole nodulated plant root, required less material than the above methods, and results were obtained after 24 h. Only method 2 was useful in determining if both a melanin producing strain and a melanin non-producing strain had occupied an individual nodule. Each of the three methods represented a rapid way of studying strain competition for field studies, using a natural trait as a marker.     

The possible contribution of electron microscopy to the understanding of the mechanism of non-disjunction in man.

Sprout inhibition of onion bulbs can be effectively accomplished by low doses of radiation [2,3]. However, wholesomeness data on irradiated onions, particularly with respect to their mutagenic activity, are still insufficient for evaluation [6]. Therefore we examined the mutagenic activity of irradiated onions in bacterial systems. Because onion bulbs contain a considerable amount of free amino acids, we used indicator strains carrying the marker for mutagenicity other than the amino acid requirement.In this paper we describe the results on irradiated onions. We used tests with solid and liquid media, assaying for the streptomycin (SM) dependence in a strain having a tetracycline (TC)-resistance factor, as well as DNA repair tests using two sets of indicator strains.     

Identification of Nisin-Producing Strains by Nisin-Controlled Gene Expression System

A specific method to identify nisin-producing strains was developed based on Nisin-Controlled gene Expression (NICE) vector pSec:Nuc. The plasmid pSec:Nuc was transformed into non-nisin-producing strain Lactococcus lactis NZ9000, a host commonly used for the NICE system. The generating strain L. lactis NZ9000/pSec:Nuc could sense extracellular inducer nisin and efficiently secrete a reporter protein Nuc, the staphylococcal nuclease (Nuc) into the medium. Instead of using purified nisin, the culture supernatants of nisin-producing strains were also used as inducers. Therefore, the NICE system could be used to identify nisin-producing strains. With this principle, 4 among 56 lactococci strains isolated from raw milk were identified as nisin producers. The results were further confirmed by polymerase chain reaction amplification with their genomic DNA as templates, and nucleotide sequencing revealed that three of them produced nisin A, and the others produced nisin Z. Those results made it possible to isolate and identify nisin-producing strains specifically and rapidly using NICE system.     

A method for evaluating the host range of bacteriophages using phages fluorescently labeled with 5-ethynyl-2′-deoxyuridine (EdU)

The evaluation of bacteriophage (phage) host range is a significant issue in understanding phage and prokaryotic community interactions. However, in conventional methods, such as plaque assay, target host strains must be isolated, although almost all environmental prokaryotes are recalcitrant to cultivation. Here, we introduce a novel phage host range evaluation method using fluorescently labeled phages (the FLP method), which consists of the following four steps: (i) Fluorescently labeled phages are added to a microbial consortium, and host cells are infected and fluorescently labeled. (ii) Fluorescent cells are sorted by fluorescence-activated cell sorting. (iii) 16S rRNA gene sequences retrieved from sorted cells are analyzed, and specific oligonucleotide probes for fluorescence in situ hybridization (FISH) are designed. (iv) Cells labeled with both fluorescently labeled phage and FISH probe are identified as host cells. To verify the feasibility of this method, we used T4 phage and Escherichia coli as a model. We first used nucleic acid stain reagents for phage labeling; however, the reagents also stained non-host cells. Next, we employed the Click-iT EdU (5-ethynyl-2'-deoxyuridine) assay kit from Invitrogen for phage labeling. Using EdU-labeled T4 phage, we could specifically detect E. coli cells in a complex microbial consortium from municipal sewage. We also confirmed that FISH could be applied to the infected E. coli cells. These results suggest that this FLP method using the EdU assay kit is a useful method for evaluating phage host range and may have a potential application for various types of phages, even if their prokaryotic hosts are currently unculturable.     

Circadian rhythms of antibacterial resistance, coagulase and antilysozyme activity of Staphylococcus aureus

AIM: To determine daily dynamics of antibacterial resistance as well as antilysozyme and coagulase activity of S. aureus strains. MATERIALS AND METHODS: On an example of clinical strains of S.aureus isolated from patients with surgical infections daily dynamics of biological characteristics of staphylococci was studied. After 12 hours of incubation strains were tested for coagulase activity by standard method (test tube method), antilysozyme activity by photometric method, and antibacterial resistance by method of serial dilutions in agar. Tests were repeated each 3-hours during a day. RESULTS: Variation of levels of studied biological characteristics of staphylococci during a day was revealed. Structures of coagulase and antilysozyme circadian rhythms had some differences in different S. aureus strains. Alongside with it, similarity in temporal expression of such biological characteristics of staphylococci as antibacterial resistance and antilysozyme activity was noted. CONCLUSION: Obtained data open prospect to use biorhythmological approach in study of biological characteristics of microorganisms during evaluation of their mechanisms of adaptation to changing environmental conditions. Chronobiological approach allows to reveal periods of maximal expression of S. aureus characteristics that could be used for increasing of effectiveness of antibacterial treatment by the choice of optimal time for administration of antibiotic.     

Heterokaryosis between Aspergillus oryzae cyclopiazonic acid-defective strains: method for estimating the risk of inducing toxin production among cyclopiazonic acid-defective industrial strains.

Aspergillus oryzae strains are used extensively in the food industry. Some of these strains excrete alpha-cyclopiazonic acid (CPA), a mycotoxin which may provoke toxicoses in rats. Physicochemical methods may reveal the presence of this toxin, but they are inadequate to screen CPA-nonproducing (CPA-) strains. CPA production is revealed by either bacterial growth inhibition or alkalinization of the culture medium. This first biological property was used to devise a time-saving screening method to isolate mutants affected in their ability to produce CPA. The second method was used as a further test. After N-methyl-N'-nitro-N-nitrosoguanidine treatment, we isolated CPA- mutants from CPA producer strains (CPA+) and CPA+ mutants from CPA- strains. The mutants unable to produce CPA may be used in the food industry to reduce or eliminate the risk of intoxication in humans. Heterokaryon formation between different mutant strains was carried out to evaluate the risks of obtaining CPA from a mixture of mutants modified in their ability to synthesize this toxin. Pairings between two CPA+ strains always gave rise to CPA+ heterokaryons. Pairings between CPA+ and CPA- strains led, most often, to CPA+ heterokaryons. This could be directly correlated to the more frequent genotype (CPA+) in the heterokaryon. CPA hypoproducer and hyperproducer heterokaryons were obtained. Pairings between CPA- strains always gave rise to CPA- heterokaryons. These results suggest that the risks of producing this toxin from two CPA- individuals are not high.     

Heterokaryosis between Aspergillus oryzae cyclopiazonic acid-defective strains: method for estimating the risk of inducing toxin production among cyclopiazonic acid-defective industrial strains

Aspergillus oryzae strains are used extensively in the food industry. Some of these strains excrete alpha-cyclopiazonic acid (CPA), a mycotoxin which may provoke toxicoses in rats. Physicochemical methods may reveal the presence of this toxin, but they are inadequate to screen CPA-nonproducing (CPA-) strains. CPA production is revealed by either bacterial growth inhibition or alkalinization of the culture medium. This first biological property was used to devise a time-saving screening method to isolate mutants affected in their ability to produce CPA. The second method was used as a further test. After N-methyl-N'-nitro-N-nitrosoguanidine treatment, we isolated CPA- mutants from CPA producer strains (CPA+) and CPA+ mutants from CPA- strains. The mutants unable to produce CPA may be used in the food industry to reduce or eliminate the risk of intoxication in humans. Heterokaryon formation between different mutant strains was carried out to evaluate the risks of obtaining CPA from a mixture of mutants modified in their ability to synthesize this toxin. Pairings between two CPA+ strains always gave rise to CPA+ heterokaryons. Pairings between CPA+ and CPA- strains led, most often, to CPA+ heterokaryons. This could be directly correlated to the more frequent genotype (CPA+) in the heterokaryon. CPA hypoproducer and hyperproducer heterokaryons were obtained. Pairings between CPA- strains always gave rise to CPA- heterokaryons. These results suggest that the risks of producing this toxin from two CPA- individuals are not high.     

Pyrolysis patterns of 5 close Corynebacterium species analyzed by artificial neural networks

In the present study, an artificial neural network was trained with the Stuttgart Neural Networks Simulator, in order to identify Corynebacterium species by analyzing their pyrolysis patterns. An earlier study described the combination of pyrolysis, gas chromatography and atomic emission detection we used on whole cell bacteria. Carbon, sulfur and nitrogen were detected in the pyrolysis compounds. Pyrolysis patterns were obtained from 52 Corynebacterium strains belonging to 5 close species. These data were previously analyzed by Euclidean distances calculation followed by Unweighted Pair Group Method of Averages, a clustering method. With this early method, strains from 3 of the 5 species (C. xerosis, C. freneyi and C. amycolatum) were correctly characterized even if the 29 strains of C. amycolatum were grouped into 2 subgroups. Strains from the 2 remaining species (C. minutissimum and C. striatum) cannot be separated. To build an artificial neural network, able to discriminate the 5 previous species, the pyrolysis data of 42 selected strains were used as learning set and the 10 remaining strains as testing set. The chosen learning algorithm was Back-Propagation with Momentum. Parameters used to train a correct network are described here, and the results analyzed. The obtained artificial neural network has the following cone-shaped structure: 144 nodes in input, 25 and 9 nodes in 2 successive hidden layers, and then 5 outputs. It could classify all the strains in their species group. This network completes a chemotaxonomic method for Corynebacterium identification.     

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...