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Evaluation of microbial source tracking methods using mixed fecal sources in aqueous test samples.

Microbiological source tracking (MST) methods are increasingly being used to identify fecal contamination sources in surface waters, but these methods have been subjected to limited comparative testing. In this study, 22 researchers employing 12 different methods were provided sets of identically prepared blind water samples. Each sample contained one to three of five possible fecal sources (human, dog, cattle, seagull or sewage). Researchers were also provided with portions of the fecal material used to inoculate the blind water samples for use as library material. No MST method that was tested predicted the source material in the blind samples perfectly. Host-specific PCR performed best at differentiating between human and non-human sources, but primers are not yet available for differentiating between all of the non-human sources. Virus and F+ coliphage methods reliably identified sewage, but were unable to identify fecal contamination from individual humans. Library-based isolate methods correctly identified the dominant source in most samples, but also had frequent false positives in which fecal sources not in the samples were incorrectly identified as being present. Among the library-based methods, genotypic methods generally performed better than phenotypic methods.

Adenoviridae↗

Use of viral pathogens and indicators to differentiate between human and non-human fecal contamination in a microbial source tracking comparison study.

Assays for the detection and typing of adenoviruses, enteroviruses and F+ specific coliphages were performed on samples created as part of a national microbial source tracking methods comparison study. The samples were created blind to the researchers, and were inoculated with a variety of types of fecal contamination source (human, sewage, dog, seagull and cow) and mixtures of sources. Viral tracer and pathogen assays demonstrated a general ability to discriminate human from non-human fecal contamination. For example, samples inoculated with sewage were correctly identified as containing human fecal contamination because they contained human adenovirus or human enterovirus. In samples containing fecal material from individual humans, human pathogen analysis yielded negative results probably because the stool samples were taken from healthy individuals. False positive rates for the virus-based methods (0-8%) were among the lowest observed during the methods comparison study. It is suggested that virus-based source tracking methods are useful for identification of sewage contamination, and that these methods may also be useful as an indication of the public health risk associated with viral pathogens. Overall, virus-based source tracking methods are an important approach to include in the microbial source tracking 'toolbox'.

Adenoviridae↗

Isolation and characterization of the smallest bacteriophage P4 derivatives packaged into P4-size head in bacteriophage P2-P4 system.

Bacteriophage P4, a satellite phage of coliphage P2, is a very useful experimental tool for the study of viral capsid assembly and cos-cleavage. For an in vitro cos-cleavage reaction study of the P2-P4 system, new shortened and selectable markers containing P4 derivative plasmids were designed as a substrate molecules. They were constructed by swapping the non-essential segment of P4 DNA for either the kanamycin resistance (kmr) gene or the ampicillin resistance (apr) gene. The size of the genomes of the resulting markers were 82% (P4 ash8 delRI:: kmr) and 79% (P4 ash8 delRI:: apr) of the wild type P4 genome. To determine the lower limit of genome size that could be packaged into the small P4-size head, these shortened P4 plasmids were converted to phage particles with infection of the helper phage P2. The conversion of plasmid P4 derivatives to bacteriophage particles was verified by the heat stability test and the burst size determination experiment. CsCl buoyant equilibrium density gradient experiments confirmed not only the genome size of the viable phage form of shortened P4 derivatives, but also their packaging into the small P4-size head. P4 ash8 delRI:: apr turned out to be the smallest P4 genome that can be packaged into P4-sized head.

Bacteriophage P2↗

Preliminary investigation of suitable bacteria strains for the detection of bacteriophages in environmental polluted surface waters.

Different host-specific bacteriophages were monitored in several water systems together with the usual faecal contamination indicator bacteria (thermotolerant coliforms). The host bacteria used for the plaque assay were E. coli K12, E. coli K12 Hfr, E. coli CHfr and S. typhimurium LT2F+. S. typhimurium LT2F+ showed significantly lower phage counts than the three E. coli strains. The phages specific for E. coli K12 (somatic phages) were in high correlation (r = 0.86, r = 0.91; P less than 0.001) with other coliphages, but not with phages specific for S. typ. LT2F+ (r = 0.33; P less than 0.05). There was an high degree of correlation among phages of E. coli K12 Hfr, E. coli C Hfr and S. typhimurium LT2F+ (pilus F-carrying bacteria) (r = 0.96, r = 0.73, r = 0.75; P less than 0.001). Good correlation was seen between faecal coliforms and male-specific phages but not among faecal coliforms and E. coli K12-specific phages (r = 0.30; P less than 0.05). The relative suitability of host strains when using phages in the evaluation of faecal and viral pollution of aquatic environments is discussed.

Coliphages↗

[Transposition of the composite synthetic transposon TnV (Tn5-Rep(pSC101)) is accompanied by the formation of the mini-plasmid pTnV, containing defective Is50-elements].

Using thermoelimination (at 42 degrees C) of the thermoinducible coliphage P1tsCmr omega::TnV (TnV is a Tn5 derivative which contains the replication origin (Rep) of plasmid pSC101), more than 110 KmrCms Escherichia coli K12 clones were selected. It was supposed that the KmrCms phenotype could result only from insertion of TnV (Kmr) into E. coli chromosome and the loss of phage (Cms). It was found that the majority of KmrCms clones (35-90%) contained miniplasmids. Their molecular sizes did not exceed the TnV size (6.1 kb). The formation of miniplasmids called pTnV was observed both in RecA+ cells (C600) and in RecA- (HB101), more often in the latters. Interestingly, that miniplasmids of only several molecular sizes were detected: from 6.1 kb (pTnV60) to 4.35 kb (pTnV43). A restriction analysis showed that DNA of the majority of pTnV plasmids had varying deletions (0.3-1.3 kb) of mainly IS50L element which together with IS50R flank TnV. Very low transposition frequencies (approx. 10(-8) Kmr transconjugants per transferred R388) of all pTnV types (including pTnV60 plasmids containing probably microdeletions of the joining "outside" IS50's ends) suggest that pTnV plasmids are not intermediates in TnV transposition. Possibly the circularized TnV derivatives (pTnV's) are side products of the transposition resulting from the abortive attempts of an excised and autonomous transposon molecule to insert into itself. In the present paper the possible mechanisms of the origin of limited pTnV type numbers are also discussed.

Coliphages↗

[The virological supervision of drinking water quality--a review].

Drinking-water must be free of pathogenic organisms; this challenge refers to pathogenic viruses too. In order to control virological aspects of water quality realistic and practicable monitoring procedures, esp. practical microbial assays, must be performed. They should include in all cases standard plate counts and counts of total or faecal coliform bacteria. Counts of coliphages in combination with standard plate counts and counts of coliform bacteria offer a practical and reliable indicator system for evaluating the virological safety of treated drinking-water supplies in the case of drinking-water reclaimed from contaminated raw water, esp. surface water.

Coliphages↗

[Effect of anionic detergents sodium dodecyl sulfate and alkylbenzyl sulfonate on adsorption and transport behavior of several enterotropic viruses in the soil under model conditions].

The adsorption of poliovirus 3, coxsackievirus B1, and coliphage f2 on soil from an irrigation field loaded for years with waste water and sandy soil from an aquifer was investigated under the influence of two anionic detergents, sodium dodecylsulphate (SDS) and alkylbenzolsulphonate (ABS). The investigation was carried out through batch and column experiments under simulated conditions. The concentrations of both detergents found generally in surface water (0.2-10 mg/l) had no effect on the adsorption of tested viruses. The concentration of 100 mg/l was the lowest intensity that led to an impaired adsorption of viruses on the sediments. The desorbing effect of SDS was relatively higher than ABS. Both detergents gave rise to a desorption and migration of viruses. Especially in soil from an irrigation field the effect was stronger at pH 7.2 than at pH 5.1. Despite low concentrations of detergents in surface water, such procedures as bank filtration, waste water irrigation for the enrichment of groundwater, and the use of sewage sludge on agricultural fields or forest grounds can cause an accumulation of detergents or their metabolites which impairs the binding of viruses to sediments. Therefore, the danger occurrence of a groundwater contamination with enteroviruses should be considered in those sectors.

Adsorption↗

In vitro requirements for N4 RNA polymerase II-specific initiation.

The synthesis of coliphage N4 middle RNAs requires the activity of three phage-coded proteins, P7, P4, and P17, of molecular weights 40,000, 30,000, and 15,000, respectively. P4 and P7 constitute a heterodimeric RNA polymerase, N4 RNA polymerase II, which cannot transcribe or bind to native N4 DNA. Denatured templates are transcribed but without specificity. We present the development of an in vitro transcription system, composed of purified N4 RNA polymerase II and solubilized P17, which accurately utilizes in vivo sites of transcription initiation. Possible mechanisms for P17 action are discussed.

Base Sequence↗

Purification and characterization of bacteriophage N4-induced DNA polymerase.

Coliphage N4 replication is independent of most host DNA replication functions except for the 5'----3' exonuclease activity of polA, DNA ligase, DNA gyrase, and ribonucleotide reductase (Guinta, D., Stambouly, J., Falco, S. C., Rist, J. K., and Rothman-Denes, L. B. (1986) Virology 150, 33-44). It is therefore expected that N4 codes for most of the functions required for replication of its genome. In this paper we report the purification of the N4-coded DNA polymerase from N4-infected cell extracts by following its activity on a gapped template and in an in vitro complementation system for N4 DNA replication (Rist, J. K., Pearle, M., Sugino, A., and Rothman-Denes, L. B. (1986) J. Biol. Chem. 261, 10506-10510). The enzyme is composed of one polypeptide, Mr 87,000. It is most active on templates containing short gaps synthesizing DNA with high fidelity in a quasi-processive manner. A strong 3'----5' exonuclease activity is associated with the DNA polymerase polypeptide. No 5'----3' exonuclease or strand-displacing activities were detected.

Coliphages↗

M13 procoat inserts into liposomes in the absence of other membrane proteins.

Procoat, the precursor form of the major coat protein of coliphage M13, assembles into the Escherichia coli inner membrane and is cleaved to mature coat protein by leader peptidase. This assembly process has previously been reconstituted using lipids and purified leader peptidase in a cell-free protein synthesis reaction (Watts, C., Silver, P., and Wickner, W. (1981) Cell 25, 347-353; Ohno-Iwashita, Y., and Wickner, W. (1983) J. Biol. Chem. 258, 1895-1900). We now report that procoat can also cross a liposomal membrane composed of only purified phospholipids; leader peptidase is not needed to catalyze insertion. When procoat is synthesized in vitro in the presence of liposomes with encapsulated chymotrypsin, the procoat inserts spontaneously through the membrane and is degraded. The protease was shown by several criteria to be in the lumen of the liposomes. These results demonstrate that the precursor form of an E. coli integral membrane protein can cross a membrane without the aid of leader peptidase or any other membrane proteins.

Capsid↗

Possible role for thymine glycol in the selective inhibition of DNA synthesis on oxidized DNA templates.

Single-stranded DNA of coliphage M13mp8 was treated with the oxidizing agent, KMnO4, under conditions that selectively form cis-5,6-dihydro-5,6-dihydroxythymine (thymine glycol). Treatment of DNA with 0.7 and 1.4 mM KMnO4 introduced approximately 200 and 400 thymine glycol residues, respectively, per genome. When these DNAs were used to transform Escherichia coli, it was observed that phage survival was reduced in a dose-dependent manner. In studies designed to investigate the effect of DNA oxidation products on replication in vitro, a complementary 15-mer oligodeoxynucleotide was annealed to the oxidized template and extended with the Klenow fragment of DNA polymerase I from E. coli. It was observed that lesions in oxidized DNA strongly inhibited DNA elongation and that DNA synthesis was stopped opposite thymine residues. This is taken as suggestive evidence that the thymine glycol is inhibitory to DNA replication.

Coliphages↗

Carrier-determined tolerance in vitro.

A primary immune response to normal BDF1 spleen cells was obtained in vitro to the T-independent antigen TNP-T4 coliphage. This anti-TNP response was suppressed by exposing the spleen cells for 6 hr to TNP bound either to isologous or heterologous gamma-globulin. The suppression was hapten specific. In contrast, TNP-albumin conjugates did not induce tolerance in vitro.

Animals↗

[Lysogenic conversion induced by phages phi 80. I. A description of the phenomenon and the cloning of the conversion gene].

Escherichia coli cells lysogenic for coliphage phi 80 stop adsorbing the superinfecting phi 80 phage after having been kept under anaerobic conditions for a long time, which conferred on these cells the TonA phenotype. To determine the location of the gene for lysogenic conversion (cor), BamHI fragments of phi 80 DNA were cloned in pBR322 plasmid. The cells transformed with the recombinant plasmid pDK01 = pBR322 + phi 80 BamHI fragment 1 immediately acquire the TonA phenotype. So, the cor gene(s) is contained in the central phi 80 BamHI fragment (fragment 1) which includes gene 13, the b2 region and the att site.

Adsorption↗

Primary structure of the major coat protein of the filamentous bacterial viruses, If1 and Ike.

The primary structures of the major coat proteins from If1 and Ike filmentous coliphages have been determined by automated Edman degradation before and after cyanogen bromide cleavage and by manual sequencing of certain tryptic peptides. Carboxypeptidase A and B digestion was also used to determine the sequence of the COOH termini of these proteins. A comparison of the major coat proteins from these two phages with those from other filamentous phages show that they all share several common features, namely an asymmetric distribution of positively and negatively charged amino acid residues, which are clustered with the COOH-terminal and NH2-terminal regions respectively, and a region of 19 residues which is located in the middle of the polypeptide chain. The consequences of this charge distribution for a possible mechanism of virus maturation are discussed.

Amino Acid Sequence↗

Klebsiella and Enterobacter strains derived from hospital infections. II. Occurrence and characterization of R-, Lac- and Col- plasmids and their clinical-epidemiological significance.

A total of 269 hospital Klebsiella strains and 103 hospital Enterobacter strains showed 34 and 10 different antibiotic resistance patterns, respectively. Among multiple resistant Klebsiella and Enterobacter strains the Ap Sm Cm Tc resistance pattern was the most frequent (K. aerogenes). Antibiotic resistant strains carried R-plasmids in 27.5%. The presence of R-plasmids was demonstrable in 2.9% of single antibiotic resistant, in 12.8% of double antibiotic resistant, and in 71.4% of multiple antibiotic resistant Klebsiella strains. R-plasmid carriage was most frequent in strains of the species K. pneumoniae and K. atlantae. Transfer frequency of R-plasmids of multiple resistant strains was generally higher into Escherichia coli K12 recipient (10-1 to 10-2) than into K. pneumoniae recipient (10-3 to 10-5). Regarding the individual resistance determinants, transfer frequency of Km-Nm resistance was the highest (65.1%). The lac-plasmid was demonstrable in 18 out of the 77 R-plasmid carrying Klebsiella strains. R- and Lac-plasmids of single and multiple resistant Klebsiella strains could be transferred into E. coli and Klebsiella recipients in mice in vivo. As many as 67% showed fi- and 33% showed fi+ character among the demonstrated 112 R-plasmids. Regarding their incompatibility, the studied five plasmids belonged to groups FI, FII and I alpha. Altogether 14 different groups could be distinguished among R-plasmids on the basis of their phage restriction capability on phage lambda and coliphages T, and another group of R-plasmids showing no restriction at all. Fi+ character was demonstrated in 54.5% of R-plasmids showing phage restriction capability and in 10.9% of R-plasmids showing no phage restriction. Nearly twice as many R-plasmids carrier strains were found among non-typable ones as among those typable by phages. Three R-plasmids showed phage-modification among 51 R-plasmids restrictive for phages. In addition to the phage-type of Klebsiella strains, R-plasmids can also be used as an epidemiologic marker.

Bacteriocin Plasmids↗

[The efficacy of bacteriophage preparations in treating inflammatory urologic diseases].

Urinary infection is the most commonly encountered hospital infection. Antibacterial therapy promotes selection and dissemination of polyresistant microorganism strains, development of intestinal dysbacteriosis, reduction of intestinal contamination resistance. Clinical and bacteriological efficacy of urinary infection treatment with bacteriophage preparations (pyocyanic, proteus, staphylococcal, coliphage, combined pyobacteriophage) was studied. Sensitivity of the infective agent phage isolated from urological patients was tested before treatment. The preparations were adapted to recently isolated agents from urological patients to raise phage sensitivity of the strains. A total of 293 strains were studied. Phage sensitivity made up 68.9%. Bacteriophage preparations were used both locally and orally in 46 patients with acute and chronic urogenital inflammation. Bacteriological efficacy amounted to 84%, clinical one to 92%. It is inferred that phagotherapy is effective and safe therapeutic modality in the treatment of urinary infection in monotherapy and in combination with antibiotics.

Bacteriophage Typing↗

Continuous separation of biomolecules by the laterally asymmetric diffusion array with out-of-plane sample injection.

The laterally asymmetric diffusion array, a biomolecule sorting device, was used to continuously separate a mixture of T2 and T7 coliphage DNA molecules into its constituents. A two-dimensional array of obstacles (in the presence of an average flow v) can be used to rectify the Brownian motion of particles (in this case DNA molecules) so that they diffuse preferentially in one direction, and perpendicular to the direction of the applied field (in this case an electric field). This type of device had not yet been used for actual fractionation of biomolecules, due to difficulties in injection of the sample. Here we show that with a new injection strategy a well-defined, narrow and continuous stream of molecules can be injected into the separation channel, thus enabling this separation technique to be used in a working device. We expect this type of device could now be employed for separation of a variety of different biomolecules, ranging from long dsDNA to small proteins.

Bacteriophage T4↗

Elongational flow studies on conformational change in DNA induced by DNA-binding protein HU.

Interaction of DNA-binding protein HU from Bacillus stearothermophilis (HUBst) with coliphage T2 DNA was investigated by observing an elongational flow-induced birefringence, Deltan, of a T2-phage DNA aqueous solution at various HU concentrations. Localized flow birefringence was observed in the pure elongational flow region, and the strain rate dependence of Deltan had a critical strain rate epsilon;(c) for the appearance of flow birefringence at all of the HU concentrations examined, indicating that a coil-stretch transition occurred at epsilon;(c) in each DNA-HU system. For strain rates larger than epsilon;(c), Deltan increased rapidly and then gradually, approaching a plateau value. The value of epsilon;(c) increased with an increase in HU concentration. Analysis based on the relationship between epsilon; (c) and the Rouse-Zimm relaxation time revealed that the increase in epsilon;(c)with increase in HU can be explained by the decrease in the size of the DNA-HU complex. The plateau birefringence value, Deltan(p), decreased at small HU concentrations but did not change at larger HU concentrations. Considering that Deltan(p) is related to the orientational order parameter of segments, it was concluded that there were at least two stages in the process of compaction of DNA induced by HU.

Bacterial Proteins↗