Dextropropoxyphene deaths in Denmark from the health authority point of view

Using electron microscopy and DNA-DNA-hybridization, 113 virulent and temperate bacteriophages specific for P. aeruginosa have been assigned to 23 species. In most cases, especially in virulent phages, both particle morphology and DNA homology types were in good correlation and their use was sufficient for clear-cut definition of phage species. No virulent phages of different species had any DNA homology. DNA homology was detected between temperate phages of several species. Temperate phages formed two large groups of two and seven species, respectively. The first group included all transposable bacteriophages. The extent of interspecies DNA homology of phages belonging to each group was not more than 10-15% (except for 25% for phages D 3 and KF 1). No DNA homology was between phages of different groups. The possible origin and function of homologous sequences (genetic modules, linkers, occasional insertional sequences) are discussed. One of the phages (phi C 15) may be considered as the result of recombination between phages belonging to two different species, 295 and SM.     

Bacteriophage diversity in the North Sea

In recent years interest in bacteriophages in aquatic environments has increased. Electron microscopy studies have revealed high numbers of phage particles (10(4) to 10(7) particles per ml) in the marine environment. However, the ecological role of these bacteriophages is still unknown, and the role of the phages in the control of bacterioplankton by lysis and the potential for gene transfer are disputed. Even the basic questions of the genetic relationships of the phages and the diversity of phage-host systems in aquatic environments have not been answered. We investigated the diversity of 22 phage-host systems after 85 phages were collected at one station near a German island, Helgoland, located in the North Sea. The relationships among the phages were determined by electron microscopy, DNA-DNA hybridization, and host range studies. On the basis of morphology, 11 phages were assigned to the virus family Myoviridae, 7 phages were assigned to the family Siphoviridae, and 4 phages were assigned to the family Podoviridae. DNA-DNA hybridization confirmed that there was no DNA homology between phages belonging to different families. We found that the 22 marine bacteriophages belonged to 13 different species. The host bacteria were differentiated by morphological and physiological tests and by 16S ribosomal DNA sequencing. All of the bacteria were gram negative, facultatively anaerobic, motile, and coccoid. The 16S rRNA sequences of the bacteria exhibited high levels of similarity (98 to 99%) with the sequences of organisms belonging to the genus Pseudoalteromonas, which belongs to the gamma subdivision of the class Proteobacteria.     

Evaluation of seven experimental phages for inclusion in the international phage set for the epidemiological typing of Listeria monocytogenes

The purpose of our study was to evaluate the inclusion of seven experimental phages into the international phage set for subtyping Listeria monocytogenes. The seven additional phages included the broad-host-range virulent Myoviridae phage A511 (M. J. Loessner, Appl. Environ. Microbiol. 57:1912-1918, 1991), three temperate phages from the Danish subsystem for typing serotype 1/2 strains (12682, 6223, and 5775) (P. Gerner-Smidt, V.T. Rosdahl, and W. Frederiksen, APMIS 101:160-167, 1993), and three temperate phages isolated by this laboratory in France (9425, 1313, and 197). A panel of 395 Listeria monocytogenes isolates (including 180 that were non-phage typeable by the international set) were used in the study for a comparison of the lytic spectra of the various bacteriophages. These results showed that the inclusion of five of the experimental phages contributed greatly to the overall typeability and discriminatory power of the system, especially for strains within serogroup 1/2.     

Abundance in sewage of bacteriophages that infect Escherichia coli O157:H7 and that carry the Shiga toxin 2 gene

Shiga toxin-converting bacteriophages are involved in the pathogenicity of some enteric bacteria, such as Escherichia coli O157:H7, but data on the occurrence and distribution of such phages as free particles in nature were not available. An experimental approach has been developed to detect the presence of the Shiga toxin 2 (Stx 2)-encoding bacteriophages in sewage. The Stx 2 gene was amplified by PCR from phages concentrated from 10-ml samples of sewage. Moreover, the phages carrying the Stx 2 gene were detected in supernatants from bacteriophage enrichment cultures by using an Stx 2-negative E. coli O157:H7 strain infected with phages purified from volumes of sewage as small as 0.02 ml. Additionally, the A subunit of Stx 2 was detected in the supernatants of the bacteriophage enrichment cultures, which also showed cytotoxic activity for Vero cells. By enrichment of phages concentrated from different volumes of sewage and applying the most-probable-number technique, it was estimated that the number of phages infectious for E. coli O157:H7 and carrying the Stx 2 gene was in the range of 1 to 10 per ml of sewage from two different origins. These values were approximately 1% of all phages infecting E. coli O157:H7.     

Biological properties of Streptococcus bovis bacteriophages isolated from lysogenic cultures and sheep rumen

Biological characteristics of eleven phages for Streptococcus bovis were investigated; seven phage were isolated from ovine rumen and four were virulent mutants of temperate phages of lysogenic cultures. The phages had many properties in common: similar morphology of negative colonies, the identical spectrum of lytic action, related antigens, absolute or high requirement of calcium ions, thermolability, and inactivation by the content of the rumen. Their susceptibility to the inactivating action of acetic acid, urea and temperature was however different. Chloroform and phenol may be used during purification and conservation of the phages.     

Molecular phylogeny of phi29-like phages and their evolutionary relatedness to other protein-primed replicating phages and other phages hosted by gram-positive bacteria

The phi29-like phage genus of Podoviridae family contains phages B103, BS32, GA-1, M2, Nf, phi15, phi29, and PZA that all infect Bacillus subtilis. They have very similar morphology and their genomes consist of linear double-stranded DNA of approximately 20 kb. The nucleotide sequences of individual genomes or their parts determined thus far show that these phages evolved from a common ancestor. A terminal protein (TP) that is covalently bound to the DNA 5'-end primes DNA replication of these phages. The same mechanism of DNA replication is used by the Cp-1 related phages (also members of the Podoviridae family) and by the phage PRD1 (member of the Tectoviridae family). Based on the complete or partial genomic sequence data of these phages it was possible to analyze the evolutionary relationship within the phi29-like phage genus as well as to other protein-primed replicating phages. Noncoding regions containing origins of replication were used in the analysis, as well as amino acid sequences of DNA polymerases, and with the phi29-like phages also amino acid sequences of the terminal proteins and of the gene 17 protein product, an accessory component of bacteriophage DNA replicating machinery. Included in the analysis are also results of a comparison of these phage DNAs with the prophages present in the Bacillus subtilis genome. Based on this complex analysis we define and describe in more detail the evolutionary branches of phi29-like phages, one branch consisting of phages BS32, phi15, phi29, and PZA, the second branch composed of phages B103, M2, and Nf, and the third branch having phage GA-1 as its sole member. In addition, amino acid sequences of holins, proteins involved in phage lysis were used to extend the evolutionary study to other phages infecting Gram-positive bacteria. The analysis based on the amino acid sequences of holins showed several weak points in present bacteriophage classification.     

Lysogeny in Pasteurella multocida]

Studied was the possibility of lysis-producing factors (the phenomenon of lysogeny) with 59 strains of Pastuerella multocida isolated in Bulgaria, Cuba, and Czechoslovakia. It was found that eleven of them were lysogenic in terms that a total of 12 bacteriophage strains were isolated from them; one of them yielded 2 phages. Established were three different indicator strains of Pasteurella multocida-97, SHD, and R-II--by means of which 3 different groups of P. multocida phages were isolated. The latter were stabilized and allowed to multiply up to 10(11) phage particles per 1 cc through continuous passaging, and they could be be stored at + 4 degrees C. In accordance with the host strain for multiplying the isolated P. multocida phages were divided into three different groups: phages 3, 4, 5, 6, 22, and Sl fell into group II, and phages 1075 and S-2--to group III.     

Comparative physiology of the replication of RNA-containing bacteriophages MS2 and Q beta

The yield of the phage QB is always less than the yield of the phage MS2, the conditions of cultivation being similar. This is due to the fact that the yield of the phage QB from an infection centre is lower by a factor of 5--10 than the yield of the phage MS2. The yield of the phages may be increased by optimizing conditions of cultivation. The RNA-containg phages MS2 and QB were cultivated in the peptone-yeast growth medium in fermenters with the working volume of 30 and 50 litres. The yields were 2--5-10(13) particles/ml and 1--4-10(12) particles/ml for the phages MS2 and QB, respectively. The specific infectiveness of purified phage preparations was 2--4-10(12) particles/OD260 (MS2) and 2--4-10(11) particles/OD260 (QB).     

Isolation and properties of Escherichia coli mutants which are nonpermissive for the growth of phage lambda

By selecting survivors of lambda phage infection, mutants of Escherichia coli K12 that block reproduction cycle of the phage have been isolated. Fourteen of these phage-tolerant mutants (lam mutants) were chosen and characterized biochemically and genetically. It was shown that these mutants were tolerant to infection by all the lambdoid phages, except for few cases, but they were susceptible to infection by a non-lambdoid temperate phage (phi299), P1 or T phages. The mutants can be divided into at least three groups: (1) A mutant (lam 16) strain that seems to block normal penetration of phage DNA: (2) Three mutant (lam 64, lam 67 and lam 71) strains that block an "early" step(s) of phage growth, including phage DNA synthesis: (3) Six mutant (lam 24, lam 25, lam 26, lam 27, lam 646 and lam 6) strains that block normal functioning of the gene E products and produce unusual head structures. Some lambdoid phages and lambda mutants that overcome the interference by the lam mutations have been obtained, and were used as tools for characterizing the host mutations. Two (lam 12 and lam 13) mutant strains and one (lam 1) mutant were inferred as affecting the expression of "late" genes, and early gene, respectively, by this test.     

Impaired motor axon regeneration in the C57BL/Ola mouse

Six stable bacteriophages of Vibrio fluvialis were isolated from 44 surface water specimens collected in Thailand and Japan. Twelve different phages types were found among 109 V. fluvialis isolated from feces of diarrheal patients and the environment. Seventy-three percent (80/109) of these 109 isolates were typable with these phages. One phage type, designated as A (1) was predominant and accounted for 43% of the V. fluvialis examined. The six bacteriophages used in this typing scheme were stable for at least during a three-month storage at 4 degrees C. This proposed bacteriophage typing scheme may be of valuable aid in tracing sources and routes of infection in outbreaks of V. fluvialis infection in man.     

Concentration and purification of mycobacteriophages with polyethylene glycol 6000

Experiments were performed to concentrate and purify mycobacteriophages with polyethylene glycol 6000; 6, 9, and 8 per cent polyethylene glycol 6,000 proved to be optimal concentrations for precipitating phages of Mycobacterium phlei, Mycobacterium smegmatis strain butyricum, and mycobacterium smegmatis strain Rabinowitz, respectively. Preparative polyethylene glycol reverse gradients were constructed for further concentration and purification of the precipitated phages. With the method described, large amounts of crude phage lysates can be concentrated and purified rapidly.     

Genetic diversity in temperate bacteriophages of Streptococcus pyogenes: identification of a second attachment site for phages carrying the erythrogenic toxin A gene

Bacteriophage T12, the prototypic bacteriophage of Streptococcus pyogenes carrying the erythrogenic toxin A gene (speA), integrates into the bacterial chromosome at a gene for a serine tRNA (W. M. McShan, Y.-F. Tang, and J. J. Ferretti, Mol. Microbiol. 23:719-728, 1997). This phage is a member of a group of related temperate phages, and we show here that not all speA-carrying phages in this group use the same attachment site for integration into the bacterial chromosome. Additionally, other phages in the group use the same serine tRNA gene attachment site as phage T12 and yet do not carry speA. The evidence suggests that recombination between phage genomes has been an important means of generating diversity and disseminating virulence-associated genes like speA.     

Resurfacing large cheek defects with rotation flaps from the neck

Eighteen common serotypes representative of group E(1), E(2), E(3), and E(4)Salmonella were characterized using a single set of phages.     

Discovery of a novel thrombopoietin mimic agonist peptide

A random phage peptide library was constructed for the filamentous bacteriophage fuse5. The library was made by inserting a degenerate oligonucleotide which encodes 15 variable amino acids into the NH2-terminal region of the phage gene III protein. This library, containing 1x10(9) different phages, was screened with a human immunoglobulin fusion protein containing the extracellular region of human thrombopoietin receptor. Several phages were isolated following four cycles of enrichment and amplification. These phages specifically bound to the fusion protein. One phage peptide acted as an agonist of the thrombopoietin receptor, since it stimulated the proliferation of thrombopoietin-dependent cells and the differentiation of mouse bone marrow cells to megakaryocytes. The amino acid sequence of this peptide is not present in the primary amino acid sequence of thrombopoietin. This discovery may lead to the design of a small-molecular mimic of thrombopoietin.     

Protection systems of dual-chamber pacemakers against atrial arrhythmia

A new transgenic mouse mutagenesis test system has been developed for the efficient detection of point mutations and deletion mutations in vivo. The mice carry lambda EG10 DNA as a transgene. When the rescued phages are infected into Escherichia coli YG6020-expressing Cre recombinase, the phage DNA is converted into plasmid pYG142 carrying the chloramphenicol-resistance gene and the gpt gene of E. coli. The gpt mutants can be positively detected as colonies arising on plates containing chloramphenicol and 6-thioguanine. The EG10 DNA carries a chi site along with the red and gam genes so that the wild-type phages display Spi- (sensitive to P2 interference) phenotype. Mutant phages lacking both red and gam genes can be positively detected as plaques that grow in P2 lysogens of E. coli. These mutant phages are called lambda Spi-. The spontaneous gpt mutation frequencies of five independent transgenic lines were 1.7 to 3.3 x 10(-5) in bone marrow. When the mice were treated with ethylnitrosourea (single i.p. treatments with 150 mg/kg body weight; killed 7 days after the treatments), mutation frequencies were increased four- to sevenfold over the background in bone marrow. The average rescue efficiencies were more than 200,000 chloramphenicol-resistant colonies per 7.5 micrograms bone marrow DNA per packaging reaction. In contrast to gpt mutation frequencies, spontaneous Spi- mutation frequencies were 1.4 x 10(-6) and 1.1 x 10(-6) in bone marrow and sperm, respectively. No spontaneous Spi- mutants have been detected so far in spleen, although 930,000 phages rescued from untreated mice were screened. In gamma-ray-treated animals, however, induction of Spi- mutations was clearly observed in spleen, at frequencies of 1.4 x 10(-5) (5 Gy), 1.2 x 10(-5) (10 Gy), and 2.0 x 10(-5) (5O Gy). These results suggest that the new transgenic mouse "gpt delta" could be useful for the efficient detection of point mutations and deletion mutations in vivo.     

Selection and properties of totally phage-resistant mutant Pseudomonas putida PpG1

The efficiency of using bacteria in open systems to degrade different anthropogenic toxic pollutants can depend strongly on the interaction between these bacteria and natural bacteriophages. The possibility of selecting bacterial Pseudomonas putida mutants resistant to all bacteriophages of this species known so far was tested (in our work, these mutants were designated totally phage-resistant mutants). In a model experiment, changes in the composition of a population upon prolonged growth of bacteria in the presence of one of the virulent phages were examined. On the basis of the results obtained, it is postulated that: (1) Mutants differing in resistance to various phages accumulate in a population; relative numbers of different mutants can undergo alterations over the course of time; mutants selected in the presence of a given virulent phage do not often manifest complete resistance to this phage. (2) It is possible to isolate totally phage-resistant mutants of P. putida PpG1. These mutants carry up to three different mutations simultaneously; however, these mutants regain sensitivity to many phages upon pseudoreversion occurrence.     

The biological properties of Serratia marcescens bacteriophages]

Sixteen phages active against bacteria of the genus Serratia have been divided into 4 groups on the basis of the study of their biological properties. As a result, 5 typing phages have been selected, which permitted the typing of 77.3% of S. marcescens cultures under study, divided into 13 phage types.     

Identification of peptides, selected by phage display technology, that inhibit von Willebrand factor binding to collagen

A repeated selection of phages from a cyclic hexapeptide phage display library resulted in an enrichment of phages that bound to the monoclonal antibody (MoAb) 82D6A3 (an anti-von Willebrand Factor [vWF] antibody that inhibits binding of vWF to collagen). Two clones were selected that bound both to MoAb 82D6A3 and to rat tail collagen type I in a specific and dose-dependent manner. The two phage clones were further used in a two-direction competition experiment with vWF. vWF was able to displace phages from collagen in a dose-dependent manner with an IC50 of 35 micrograms/mL and phages were able to inhibit vWF binding to collagen. With the use of specific primers, the sequence of the cysteine-flanked hexapeptide inserts could be deduced. The two phage clones carried an almost identical sequence, CVWLWEQC and CVWLWENC, with a substitution of an N for a Q at position 6 of the hexapeptide. Sequence comparison with the known vWF sequence showed the presence of a comparable sequence at position 1129-1136 (VWTLPDQC), located between the collagen-binding A3-domain and the D4-domain. The two cyclic peptides, the putative corresponding vWF peptide, and a peptide with a scrambled cyclic sequence were synthesized. The two cyclic peptides inhibited vWF binding to rat tail collagen type I in a dose-dependent manner, whereas the linear vWF peptide and the scrambled cyclic peptide were inactive. For half maximal inhibition, 100 +/- 12.7 micromol/L and 34.8 +/- 8.59 micromol/L (mean +/- SEM, n = 3) of the N- and the Q-peptide, respectively, were needed. The two cyclic peptides were also able to inhibit vWF binding to calfskin and human collagen type I, but effective concentrations were some 5 to 10 times higher.     

Isolation, purification and ultrastructure of bacteriophages from natural mosquito breeding places in Egypt

Two bacteriophages were isolated from field collected samples representing two different mosquito breeding places. The phage AB-1 (isolated from Abheit village, Faiyoum Governorate "seepage water") and the phage GA-2 (isolated from El-Gabal El-Asfer Qualyobia Governorate "sewage drain water") were purified. Both bacteriophages were ultrastructurally described with respect to their morphology, dimensions, phases of bacterial attack and lysogeny. No major differences were observed between both isolated phages in relation to specificity, however; they were isolated from two different types of breeding places and two different geographic areas as well. This study may assume a wide host range of the isolated phages and reflect how bacterial insecticides used for mosquito larval control could be inhibited by such bacteriophage.     

Evidence for frequent lysogeny in lactobacilli: temperate bacteriophages within the subgenus Streptobacterium

A total of 17 of 21 Lactobacillus strains of the subgenus Streptobacterium were lysogenic. Two different temperate phages isolated from such lysogens are very similar to Lactobacillus casei phage PL-1. The narrow host range of bacteriophage PL-1 appears to be caused by homoimmunity.