Technique for determining total bacterial virus counts in complex aqueous systems.

A direct method is described for measuring bacteriophage concentrations in complex aqueous systems. Conditions for sample clarification, phage recognition, and recovery were optimized. In contrast to the plaque assay, this procedure permits quantitation of total numbers of phages independent of bacterial host. Also, the modifications increase the sensitivity of the sedimentation assay, permitting detection of particles at a minimum concentration of 10(4) per ml. Maximal total phage concentrations in the aqueous phase of sewage and activated sludge mixed liquor were 1.3 x 10(6) and 4.3 x 10(7) per ml, respectively.     

大肠杆菌合成的乙型肝炎病毒核心抗原(HBcAg)转化为e抗原(HBeAg)的探索

本文以带有HBcAg基因重组质粒的大肠杆菌转化株Ecoli MM206所合成的HBcAg进行HBcAg转化为HBeAg的探索研究。菌经超声破碎获得的菌裂解液对生理盐水透析两天后,酶联检测发现抗原性部分转化为HBeAg。将菌裂解液或HBcAg精制品用2-巯基乙醇处理,可使抗原性发生进一步转化。但是分子筛层析证明抗原蛋白分子大小没有明显变化。这种制品有可能作为诊断试剂用以检测抗-HBe,而且实验结果表明HBeAg是由HBeAg衍变来的。为要提高HBeAg的稳定性,以碘乙酰胺处理,使还原的抗原蛋白通过羧甲基化反应封闭游离的巯基。经上述处理的HBeAg通过分子筛层析可与大部分细菌杂蛋白分开,制品只有HBeAg活性而测不到HBeAg活性,因而提高了抗原蛋白的稳定性与纯度。     

鲍曼不动杆菌烈性噬菌体的分离与纯化

利用柱层析方法,纯化鲍曼不动杆菌（Acinetobacter baumannii）烈性噬菌体AB1。首先采用聚乙二醇6000沉淀方法,初步分离裂解液中的噬菌体,噬菌体纯度由6.1×1010 pfu/mg提高到37×1010 pfu/mg,噬菌体回收率为58.8%,蛋白质去除率为90.6%;噬菌体粗提样品经Sepharose 4B凝胶过滤层析柱进一步纯化,纯度提高到73×1010 pfu/mg,噬菌体回收率为95.7%,蛋白质去除率为48.1%;收集的噬菌体样品最后经DEAE-52阴离子交换层析柱处理,噬菌体纯度为40×1010 pfu/mg,回收率为50.8%,蛋白去除率15.6%。内毒素分析结果显示,Sepharose 4B凝胶过滤层析纯化的噬菌体样品中,内毒素含量为443.8 EU/mg,而DEAE-52阴离子交换层析纯化的噬菌体样品中,内毒素含量为544.4 EU/mg。实验结果显示,PEG沉淀方法与Sepharose 4B凝胶过滤方法能够有效地提高噬菌体纯度,而DEAE-52阴离子交换层析则不能提高噬菌体的纯度,也无法有效地去除样品中的内毒素。     

Evaluation of a rapid microbial detection method via phage lytic amplification assay coupled with Live/Dead fluorochromic stains

AIMS: To develop a method for rapid detection of bacteria via bacteriophage amplification coupled with exogenous fluorochromic stains. METHODS AND RESULTS: A method for the rapid detection of bacteria was developed which consisted of exposing the sample suspected to contain target cells to host-specific phage. After at least one infection cycle, bacteria known to be infected by the phage (helper cells) were added and the number of nascent phage particles was estimated using the Live/Dead BacLight Bacterial Viability kit. Using Pseudomonas aeruginosa, it was shown that the dead helper cell population following phage infection was proportional to the initial number of target cells present in the original sample. Approximately 1 x 10(1) CFU per ml of P. aeruginosa could be detected within 4 h without the need for enrichment. CONCLUSIONS: The phage lytic amplification assay coupled with exogenous fluorochromic stains was able to detect approx. 1 x 10(1) CFU per ml of the target bacterium within 4 h. SIGNIFICANCE AND IMPACT OF THE STUDY: A method to detect low number of bacterial cells in a sample within 4 h without the need for enrichment was developed.     

Direct detection of lactococcal bacteriophages in cheese whey using DNA probes

Abstract We have designed a new method for the rapid detection of lactococcal phage directly in milk samples. The method was based on a dot blot analysis and did not require a biological assay with sensitive indicator strains. Culture media or whey permeate samples containing phage were spotted directly onto a nylon membrane and the phage were lysed in situ prior to hybridization. For skim milk, whole milk and whey samples, the samples were first treated with 50 mM EDTA, 20% Triton X-100 and heated at 60°C for 5 min, prior to spotting on the membrane. The detection limit was approximately between 10⁵ and 10⁷ pfu/spot. A large number of samples could be processed simultaneously and the results were obtained within 24 h.     

Bacteriophage ecology in a small community sewer system related to their indicative role in sewage pollution of drinking water

In view of various studies looking for the merit of coliphages as indicators of water pollution with viruses originating from faecal material, a small agricultural community (population of approximately 1500 inhabitants of all ages, 2-3 km from Haifa) was selected in order to understand these bacteriophage ecology (F-RNA and somatic coliphages) in its sewer and oxidation pond system. Along the sewer lines, it was possible to isolate constantly both bacteriophage types (F-RNA and somatic coliphages) at 10(2)-10(4) plaque-forming units (pfu) ml(-1). The average numbers of somatic and F-RNA phages isolated from oxidation pond were 10(3)-10(4) pfu ml(-1); however, somatic coliphages were undetectable for several months (April-August). Significant high correlation (0.944 < R(2) < 0.99) was found between increased anionic detergent concentrations and F-RNA coliphage numbers. Infants less than 1 year old excreted both phage types and few only F-RNA coliphages (at high numbers > 10(5) pfu g(-1)) for up to 1 year. The excretion of F-RNA coliphages was highly linked to Escherichia coli F(+) harborage in the intestinal track as found in their faecal content. Finally, three bacterial hosts E. coli F(+), F(-) and CN(13) tested for survivability in sewage filtrate revealed that E. coli F(+) had the highest survivability under these conditions. Presence of somatic and F male-specific phages in sewer lines of a small community are influenced by several factors such as: anionic detergents, nutrients, temperature, source (mainly infants), shedding and survival capability of the host strain. Better understanding of coliphages ecology in sewer systems can enhance our evaluation of these proposed indicator/index microorganisms used in tracking environmental pollution of water, soil and crop contamination with faecal material containing enteric viruses.     

Real-time optical detection of methicillin-resistant Staphylococcus aureus using lytic phage probes

Staphylococcus aureus (S. aureus)-specific bacteriophage was used as a probe for detection of methicillin-resistant S. aureus (MRSA) in aqueous solution using a novel optical method. Biorecognition phage monolayers transferred to glass substrates using Langmuir-Blodgett (LB) technique were exposed individually to MRSA in solution at logarithmic concentrations ranging from 10(6) to 10(9)cfu/ml, and observed for real-time binding using a CytoVivatrade mark optical light microscope system. Results indicate that LB monolayers possessed high levels of elasticity (K), measuring 22 and 29mN/m for 10(9) and 10(11)pfu/ml phage concentrations, respectively. Near-instantaneous MRSA-phage binding produced 33+/-5%, 10+/-1%, 1.1+/-0.1%, and 0.09+/-0.01% coverage of the substrate that directly correlated to a decrease in MRSA concentrations of 10(9), 10(8), 10(7), and 10(6)cfu/ml. The exclusive selectivity of phage monolayers was verified with Salmonella enterica subsp. enterica serovar typhimurium (S. typhimurium) and Bacillus subtilis.     

Disinfecting properties of performic acid against bacteriophage phi X 174 as a model of small envelope--free viruses

Performic acid HCOOH (PFA) is a wide-spectrum disinfectant. It inactivates viruses, bacteria and bacterial spores, mycobacteria as well as microscopic fungi. Its main drawback is its instability, which makes it a logical necessity that it is to be prepared prior to use from its components HCOOH and H2O2. The mixing of 8 ml HCOOH of the concentration 850 ml/l and 17 ml H2O2 of the concentration 300 ml/l in a 100 ml-volume reagent bottle with a ground-in glass stopper gives, after an 1-hour rest at room temperature and after another 1 hour in a refrigerator, a stock solution that contains about 50 ml/l of PFA the actual concentration of which is determined iodometrically. Bacteriophage phi X 174 (host E. coli C) is characterized by cubic ikosahedral-type symmetry of particles free of envelope, has 27 mm in diameter and contains single-strand cyclic DNA; formerly was classed among Parvoviridae. The possibility of plaque assay-based quantitative determination of the number of infectious particles makes if it a feasible model for assessing disinfectant action on small hydrophilic viruses under conditions close to those of practical disinfection procedures. PFA stock solution diluted to 1 X 10(-3) (0.05 ml/l of effective component) inactivates the model virus of a concentration 10(8) pfu/ml aqueous suspension within 5 min so that no virus is detectable; the drop in the number of pfu amounts to 7 log orders of magnitude. In the presence of 400 ml/l of serum, the identical effect is achieved within 5 min by PFA stock solution diluted to 5 X 10(-3). The lowest PFA concentration that reliably inactivates bacteriophage phi X 174 in aqueous suspension is identical with the lowest concentration inactivating Coxsackie B 1 virus in tissue cultures. On textile, glass, plastic, rubber and metal carriers contaminated by swabbing or by a dried drop of bacteriophage suspension containing about 1 X 10(9) pfu/ml, the lowest reliably effective concentrations of PFA range within 0.25-0.025 ml/l, i.e. PFA stock solutions diluted to 5 X 10(-3)-5 X 10(-4), depending on the type of carrier and the type of contamination.     

龋齿DNA疫苗(PGJAP)中污染大肠杆菌噬菌体的检测方法的建立

龋齿DNA疫苗工程菌采用的大肠杆菌DH-5α在生产过程中极易污染大肠杆菌噬菌体,所以应对原始菌种、主菌种和工作菌种及大量生产时的发酵液作大肠杆菌噬菌体检测。用大肠杆菌噬菌体VCSM13为标准噬菌体株,对大肠杆菌C3000和DH-5α分别作噬菌斑检测和pfu值计算,验证并确定以VCSM13作为标准噬菌体株,C3000作为检测菌株,对龋齿DNA疫苗原始菌种、主菌种(第一代)、工作菌种(2007001)和其发酵液(200703)分别作噬菌体检测,并建立了检测大肠杆菌噬菌体的直接噬菌斑法。结果显示VCSM13在DH-5α的噬菌斑计数为76,pfu/ml为7.6×1013,C3000的噬菌斑计数为81,pfu/ml为8.1×1013,龋齿DNA疫苗的原始菌种、主菌种、工作菌种和发酵液,噬菌斑计数全部为0。Pfu也为0。阳性对照为74,pfu/ml是7.4×1013,阴性对照为0。通过对阳性对照样本作增殖法试验及挑斑接种验证后,证明此法操作简单,灵敏度高。     

Some Morphological Types of Bacteriophages in Bovine Rumen Contents

Six morphological types of bacteriophage were found in bovine rumen contents. Minimal total phage count was 5 x 10(7) per ml of rumen fluid.     

HBcAg表达菌株的筛选、初步鉴定与DNA顺序分析

为提高HBcAg(乙型肝炎核心抗原)在大肠杆菌体内表达水平,将有HBcAg的基因片段用核酸外切酶从两端消化,插入载体质粒pUR222的EcoRI酶切位点,转化Ecoli BMH71—18,用菌落原位酶联免疫法由275个转化子筛选到7株阳性克隆,用ELISA比较了它们的表达水平,表达水平最高者为M2066菌株,P/N=2.0时菌体裂解液的稀泽度为1:33000’比表达最低者M2098高8,000倍,比第一代菌株M206高15,000倍,DNA顺序分析结果表明与mRNA起始密码上游的发卡结构去除有关。用SDS-PAGE和聚乙烯簿膜复印法检测菌体裂解液中HBcAg的分子量为21000,42000及63000,呈单体和聚合物形式存在,比由病人肝脏和血液中提取的HBcAg(19000)分子量大,为融合蛋白。经琼脂免疫双扩散与ELISA阻断试验未发现与β半乳糖苷酶有免疫学交叉反应。用ELIDA法,M2066-HBcAg与肝-HBcAg同时检测40份血清标本的抗-HBc,二者符合率95％。     

Suitability of yeast- and Escherichia coli-expressed hepatitis B virus core antigen derivatives for detection of anti-HBc antibodies in human sera

Antibody to hepatitis B virus core antigen (anti-HBc) is one of the most important serological markers during hepatitis B virus (HBV) infection. The quality of the hepatitis B virus core antigen (HBcAg; diagnostic antigen) is crucial to the accuracy of anti-HBc detection. In an attempt to explore the suitability of recombinant HBcAg (rHBcAg) for diagnostic purposes, HBcAg was expressed in Escherichia coli (E. coli) and Pichia pastoris (P. pastoris) and evaluated for the detection of anti-HBc. The expression level of the recombinant protein satisfied the criteria for large-scale biologic production. P. pastoris- and E. coli-derived rHBcAg were purified with gel filtration followed by sucrose gradient (reagents A and C) or with a monoclonal anti-HBc antibody binding (reagents B and D) and were utilized to detect anti-HBc in competitive inhibition enzyme-linked immunosorbent assay (ELISA) format. The ELISA using P. pastoris-derived rHBcAg had a higher specificity and sensitivity than that using E.coli-derived rHBcAg to detect the anti-HBc standard panel. Serum specimens were collected from HBV-infected patients and healthy individuals (voluntary blood donors). Anti-HBc was detected in those specimens using P. pastoris- and E. coli-derived rHBcAg. The positive rate of anti-HBc detection in HBV-infected patients' sera was 100% with reagents A and B, 96.4% with reagent C, and 93.6% with reagent D. The negative rate in healthy control sera was 100% with reagents A and B, 97.0% with reagent C, and 99.7% with reagent D. These data indicate that P. pastoris-derived rHBcAg is superior to E.coli-derived rHBcAg for the detection of anti-HBc using the diagnostic ELISA.     

Design, construction and function of a multicopy display vector using fusions to the major coat protein of bacteriophage M13.

Incorporation of numerous copies of a heterologous protein (bovine pancreatic trypsin inhibitor; BPTI) fused to the mature major coat protein (gene VIII product; VIII) of bacteriophage M13 has been demonstrated. Optimization of the promoter, signal peptide and host bacterial strain allowed for the construction of a working vector consisting of the M13 genome, into which was cloned a synthetic gene composed of a lac (or tac) promoter, and sequences encoding the bacterial alkaline phosphatase signal peptide, mature BPTI and the mature coat protein. Processing of the BPTI-VIII fusion protein and its incorporation into the bacteriophage were found to be maximal in a host bacterial strain containing a prlA/secY mutation. Functional protein is displayed on the surface of M13 phage, as judged by specific interactions with antiserum, anhydrotrypsin, and trypsin. Such display vectors can be used for epitope mapping, production of artificial vaccines and the screening of diverse libraries of proteins or peptides having affinity for a chosen ligand. The VIII display phage system has practical advantages over the III display phage system in that many more copies of the fusion protein can be displayed per phage particle and the presence of the VII fusion protein has little or no effect on the infectivity of the resulting bacteriophage.     

Stability and morphology comparisons of self-assembled virus-like particles from wild-type and mutant human hepatitis B virus capsid proteins

Instead of displaying the wild-type selective export of virions containing mature genomes, human hepatitis B virus (HBV) mutant I97L, changing from an isoleucine to a leucine at amino acid 97 of HBV core antigen (HBcAg), lost the high stringency of selectivity in genome maturity during virion export. To understand the structural basis of this so-called "immature secretion" phenomenon, we compared the stability and morphology of self-assembled capsid particles from the wild-type and mutant I97L HBV, in either full-length (HBcAg1-183) or truncated core protein contexts (HBcAg1-149 and HBcAg1-140). Using negative staining and electron microscopy, full-length particles appear as "thick-walled" spherical particles with little interior space, whereas truncated particles appear as "thin-walled" spherical particles with a much larger inner space. We found no significant differences in capsid stability between wild-type and mutant I97L particles under denaturing pH and temperature in either full-length or truncated core protein contexts. In general, HBV capsid particles (HBcAg1-183, HBcAg1-149, and HBcAg1-140) are very robust but will dissociate at pH 2 or 14, at temperatures higher than 75 degrees C, or in 0.1% sodium dodecyl sulfate (SDS). An unexpected upshift banding pattern of the SDS-treated full-length particles during agarose gel electrophoresis is most likely caused by disulfide bonding of the last cysteine of HBcAg. HBV capsids are known to exist in natural infection as dimorphic T=3 or T=4 icosahedral particles. No difference in the ratio between T=3 (78%) and T=4 particles (20.3%) are found between wild-type HBV and mutant I97L in the context of HBcAg1-140. In addition, we found no difference in capsid stability between T=3 and T=4 particles successfully separated by using a novel agarose gel electrophoresis procedure.     

Charakterisierung des Rhodopseudomonas palustris-Bacteriophagen Rp 1

Zusammenfassung RP 1-Phagen sind wenig stabil. Durch Chloroform werden sie rasch inaktiviert, während entsprechende Konzentrationen an Äther ohne Titerverlust toleriert werden; pH-Werte, kleiner als 5 und größer als 8,5 führen zur Inaktivierung, ebenso Temperaturen über 30°C. Die temperaturabhängige Inaktivierungskinetik ist bei 46°C bi-phasisch. Der Phage hat eine Latenzzeit von 90 bis 100 min, eine rise period von 2 Std und eine Wurfgröße von 10–12.Anhand von Ultradünnschnitten konnte gezeigt werden, daß die intracelluläre Phagenentwicklung im peripheren Cytoplasma abläuft. Durch Fluorescenzfärbung und Einbau von ³H-Thymidin konnte 2-DNS als Phagennucleinsäure bestimmt werden.

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Characterization of the Rhodopseudomonas palustris-bacteriophage Rp 1

Summary Isolation and enrichment of the Rp1-bacteriophage of Rhodopseudomonas palustris, strain 1 e5, resulted in a yield of 10 to 20% of the total amount of phage particles in the lysate and a titer of about 10⁹ pfu/ml. The high lost of phage particles may be due to an unspecific adsorption to the membranes of the bacterial cell. The bacteriophage Rp 1 is quickly inactivated at a pH lower than 5 and higher than 8.5 and a temperature higher than 30°C. The inactivation curve at 46°C shows two phases. The phage is rapidly inactivated by chloroform but not by ether. The one-step-growth curve shows a latency period of 90 to 100 min, a rise period of 2 h, and a burst size of 10 to 12 pfu/cell. The development of Rp1 was demonstrated to be in the peripheral region of the cytoplasm. By fluorescence staining and incorporation of ³H-thymidine the nucleic acid of Rp 1 was found to be a double-stranded DNA. The phage is tentatively put into the group C of Bradley.

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Abkürzungen Rps. Rhodopseudomonas - pfu plaque forming units     

Identification of Salmonella with the O-1 Bacteriophage

The O-1 bacteriophage test of Cherry et al. (1954) for the presumptive identification of salmonellae in the diagnostic laboratory was investigated. A phage lysate with a titer of 10(12) plaque-forming units per ml was found to be optimal. This preparation lysed 98.2% of Salmonella strains tested, while maintaining its high specificity for salmonellae. Gram-negative organisms other than salmonellae were resistant to the O-1 phage; however, 5.9% of Escherichia coli strains tested were susceptible. The O-1 phage test is a simple, rapid, inexpensive, sensitive, and specific procedure for the identification of salmonellae in the diagnostic laboratory. A presumptive identification is obtained 1 day earlier than with conventional biochemical tests.     

Recombinant scFv antibodies against E protein and N protein of severe acute respiratory syndrome virus

Severe acute respiratory syndrome (SARS) brought aglobal outbreak in spring of 2003 [1–3], and more andmore attention has been paid on it when a new caseresurfaced in Singapore last September [4]. By the endof May in 2003, WHO reported a cumulative total of 8202infected cases with 725 deaths from 28 countries.Because of the high transmission and morality rate ofSARS, scientists in many countries have made theirefforts in studying SARS coronavirus (SARS-CoV)[5, 6]. Several genomes of…     

Automated 10-channel capillary chip immunodetector for biological agents detection

The automated 10-channel capillary chip immunodetector (10K-IDWG) is a prototype, which has been developed for automatically operated biological agents (BA) point detection. The current technology uses a chemiluminescence capillary immunoassay (EIA) technique in combination with integrated microfluidics and allows the highly sensitive and rapid detection and preliminary identification of multiple BA in aqueous solutions in the laboratory. The chemiluminescence capillary EIA are performed within a disposable capillary chip containing 10 fused-silica capillaries arranged in parallel coated with selected capture antibodies. A multianode-photomultiplier array is used to detect chemiluminescence intensity in each capillary. Reservoirs for reagents and buffers and a waste disposal reservoir are integrated. This paper describes the technology of the 10K-IDWG and its evaluation with three different BA, the toxin staphylococcal enterotoxin B (SEB), the bacterial analyte Escherichia coli (E. coli) O157:H7 as a model for bacterial pathogens, and the bacteriophage M13 as a model for virus pathogens. The 10K-IDWG is able to detect the above mentioned three BA in an aqueous sample within 29 min (single analyte-detection and multiplexing). Limits of detection (LOD) are 0.1 ng/ml for SEB, 10(4)cfu/ml for E. coli O157:H7, and 5x10(5) pfu/ml for M13. Cross reactivities between the three assays were not observed.     

Application of phage-displayed single chain antibodies in western blot

A phage display single chain fragment variable library constructed on pⅢ protein of M13 filamentous phage was screened using B-lymphocyte stimulator and FP248 as selective molecules. After four rounds of panning, there was a remarkable enrichment in the titer of bound phages. Twenty phage clones were selected from the last round and screened by means of phage-ELISA. With the antibody phages as primary antibodies in Western blot, we developed a method for detecting the specific antigen. The dilutions of antibody phages depend on the affinity between antibody-displayed phage particles and antigens.