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.     

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.     

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.     

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.     

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.     

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.     

A globally disseminated M1 subclone of group A streptococci differs from other subclones by 70 kilobases of prophage DNA and capacity for high-frequency intracellular invasion

The M1inv+ subclone of M1 group A streptococci that spread globally in the late 1980s and early 1990s was previously identified by restriction fragment length polymorphism (RFLP), M protein, and SpeA exotoxin sequence analyses. Strains representing this subclone were characterized with regard to carriage of bacteriophage and capacity to invade cultured human epithelial cells. The M1inv+ subclone was found to harbor two entirely different prophages, phage T13 and phage T14, which together supplement its genome with nearly 70 kb of DNA. Phage T14 encodes the SpeA exotoxin and is closely related to the classic converting phage T12. Plaque-forming characteristics and RFLP analyses of phages T13 and T14 were compared to each other and to phage T12. Other subclones of M1, isolated in the 1970s to the early 1980s, lacked both prophages. The M1inv+ subclone was previously reported to be efficiently internalized by human epithelial cells. This potential was confirmed and expanded by comparing a variety of clinical isolates. The capacity for high-frequency invasion of epithelial cells was not transmitted to a laboratory strain of group A streptococci by the above-mentioned bacteriophages.     

Identification and characterization of a newly isolated shiga toxin 2-converting phage from shiga toxin-producing Escherichia coli

Shiga toxins 1 (Stx1) and 2 (Stx2) are encoded by toxin-converting bacteriophages of Stx-producing Escherichia coli (STEC), and so far two Stx1- and one Stx2-converting phages have been isolated from two STEC strains (A. D. O'Brien, J. W. Newlands, S. F. Miller, R. K. Holmes, H. W. Smith, and S. B. Formal, Science 226:694-696, 1984). In this study, we isolated two Stx2-converting phages, designated Stx2Phi-I and Stx2Phi-II, from two clinical strains of STEC associated with the outbreaks in Japan in 1996 and found that Stx2Phi-I resembled 933W, the previously reported Stx2-converting phage, in its infective properties for E. coli K-12 strain C600 while Stx2Phi-II was distinct from them. The sizes of the plaques of Stx2Phi-I and Stx2Phi-II in C600 were different; the former was larger than the latter. The restriction maps of Stx2Phi-I and Stx2Phi-II were not identical; rather, Stx2Phi-II DNA was approximately 3 kb larger than Stx2Phi-I DNA. Furthermore, Stx2Phi-I and Stx2Phi-II showed different phage immunity, with Stx2Phi-I and 933W belonging to the same group. Infection of C600 by Stx2Phi-I or 933W was affected by environmental osmolarity differently from that by Stx2Phi-II. When C600 was grown under conditions of high osmolarity, the infectivity of Stx2Phi-I and 933W was greatly decreased compared with that of Stx2Phi-II. Examination of the plating efficiency of the three phages for the defined mutations in C600 revealed that the efficiency of Stx2Phi-I and 933W for the fadL mutant decreased to less than 10(-7) compared with that for C600 whereas the efficiency of Stx2Phi-II decreased to 0.1% of that for C600. In contrast, while the plating efficiency of Stx2Phi-II for the lamB mutant decreased to a low level (0.05% of that for C600), the efficiencies of Stx2Phi-I and 933W were not changed. This was confirmed by the phage neutralization experiments with isolated outer membrane fractions from C600, fadL mutant, or lamB mutant or the purified His6-tagged FadL and LamB proteins. Based on the data, we concluded that FadL acts as the receptor for Stx2Phi-I and Stx2Phi-II whereas LamB acts as the receptor only for Stx2Phi-II.     

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.     

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.     

Common elements regulating gene expression in temperate and lytic bacteriophages of Lactococcus species

A phage-inducible middle promoter (P15A10) from the lytic, lactococcal bacteriophage phi 31, a member of the P335 species, is located in an 888-base pair fragment near the right cohesive end. Sequence analysis revealed extensive homology (> 95%) to the right cohesive ends of two temperate phages of the P335 species, phi r1t and phi LC3. Sequencing upstream and downstream of P15A10 showed that the high degree of homology between phi 31 and phi r1t continued beyond the phage promoter. With the exception of one extra open reading frame in phi 31, the sequences were highly homologous (95 to 98%) between nucleotides 13,448 and 16,320 of the published phi r1t sequence. By use of a beta-galactosidase (beta-Gal) gene under the control of a smaller, more tightly regulated region within the P15A10 promoter, P566-888, it was established that mitomycin C induction of a lactococcal strain harboring the prophage phi r1t induced the P566-888 promoter, as determined from an increase in beta-Gal activity. Hybridization of nine other lactococcal strains with 32P-labeled P566-888 showed that the Lactococcus lactis strains C10, ML8, and NCK203 harbored sequences homologous to that of the phage-inducible promoter. Mitomycin C induced the resident prophages in all these strains and concurrently induced the P566-888 promoter, as determined from an increase in beta-Gal activity. DNA restriction analysis revealed that the prophages in C10, ML8, and NCK203 had identical restriction patterns which were different from that of phi r1t. In addition, DNA sequencing showed that the promoter elements in the three phages were identical to each other and to P566-888 from the lytic phage phi 31. These results point to a conserved mechanism in the regulation of gene expression between the lytic phage phi 31 and at least two temperate bacteriophages and provide further evidence for a link in the evolution of certain temperate phages and lytic phages.     

Characteristics of Staphylococcus aureus strains isolated from clinical and non-clinical human sources in Trinidad: susceptibility to bacteriophages and antimicrobial agents, and toxigenicity

The susceptibility of Staphylococcus aureus strains isolated from human clinical and non-clinical sources in Trinidad to bacteriophages and antimicrobial agents was determined. The ability of the strains to produce enterotoxins and toxic shock syndrome toxin-1 (TSST-1) was also investigated. Of the 554 strains tested, 454 (81.8%) were susceptible to international phage set (IPS) phages with strains isolated from bacteruria (57.1%) and bacteremia (53.3%) having a low sensitivity compared to isolates from aspirates (87.3%) and anterior nares (97.4%). All sources combined, strains were most susceptible to phages belonging to several groups (mixed). Overall, 419 (75.6%) strains were resistant to one or more of nine antimicrobial agents tested. Resistance to penicillin was most prevalent, with 413 (74.5%) strains found to be resistant. Prevalence of resistance to tetracycline, gentamicin, oxacillin, cefuroxime and ciprofloxacin was 5.1%, 2.0%, 0.7%, 0.4% and 0.4%, respectively. Of the 554 strains tested, 307 (55.4%) produced staphylococcal enterotoxins A (SEA), B (SEB), C (SEC) and D (SED) singly or in combination. Strains recovered from high vaginal swabs were least enterotoxigenic (40.0%) as compared to umbilical infection isolates which were most enterotoxigenic (78.9%). TSST-1 was produced by 95 (19.0%) out of 499 strains tested, with isolates from bacteruria found to be most toxigenic (33.3%). It was concluded that the S. aureus strains tested were highly susceptible to bacteriophages and antimicrobial agents (except penicillin) and that enterotoxigenic and TSST-1 producers were widespread and have an aetiologic potential.     

Mammalian heterotrimeric G-protein-like proteins in mycobacteria: implications for cell signalling and survival in eukaryotic host cells

Selectively-infective phage (SIP) is a novel methodology for the in vivo selection of interacting protein-ligand pairs. It consists of two components, (1) a phage particle made non-infective by replacing its N-terminal domains of geneIII protein (gIIIp) with a ligand-binding protein, and (2) an "adapter" molecule in which the ligand is linked to those N-terminal domains of gIIIp which are missing from the phage particle. Infectivity is restored when the displayed protein binds to the ligand and thereby attaches the missing N-terminal domains of gIIIp to the phage particle. Phage propagation is thus strictly dependent on the protein-ligand interaction. We have shown that the insertion of beta-lactamase into different positions of gIIIp, mimicking the insertion of a protein-ligand pair, led to highly infective phage particles. Any phages lacking the first N-terminal domain were not infective at all. In contrast, those lacking only the second N-terminal domain showed low infectivity irrespective of the presence or absence of the F-pilus on the recipient cell, which could be enhanced by addition of calcium. An anti-fluorescein scFv antibody and its antigen fluorescein were examined as a protein-ligand model system for SIP experiments. Adapter molecules, synthesized by chemical coupling of fluorescein to the purified N-terminal domains, were mixed with non-infective anti-fluorescein scFv-displaying phages. Infection events were strictly dependent on fluorescein being coupled to the N-terminal domains and showed a strong dependence on the adapter concentration. Up to 10(6) antigen-specific events could be obtained from 10(10) input phages, compared to only one antigen-independent event. Since no separation of binders and non-binders is necessary, SIP is promising as a rapid procedure to select for high affinity interactions.     

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.     

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.     

Interaction of Pseudomonas aeruginosa plasmids and mu-like phages: the suppression of the early stages of cell infection by phage D3112 in the presence of plasmid RPL11

The growth of Mu-like, D3112, B39 and B3 bacteriophages of Pseudomonas aeruginosa on bacterial strains containing R plasmids was studied. Plasmids RPL11, Rms148 and Rms163 were shown to interfere with phage growth: 1) D3112 and B39 phages do not produce plaques on a lawn of PAO1 (Rms148) giving e.o.p. less than 10(-9); 2) RPL11 plasmid restricts phage D3112 growth (e.o.p. less than 10(-9), the growth of phage B3 being also restricted by this plasmid, though in considerably less extent; 3) phage B39 makes small and very turbid plaques on a lawn of PAO1 (Rms163) with e.o.p. 0,13, while c mutants form clear plaques on this lawn and grow with e.o.p. 1,0. The interference of plasmid RPL11 with phage D3112 growth was examined in detail. The plasmid did not affect phage D3112 adsorption and no restriction of phage DNA in R+ cells was found. However, phage genes controlling establishment of lysogeny and the lytic cycle were not expressed after infection. It was observed though, that if a cell contains both prophage D3112 and plasmid RPL11, no interference with repressor synthesis or phage development takes place after induction of prophage. The results obtained allow to conclude that: 1) RPL11 plasmid interference with phage D3112 growth is caused by the plasmid effect on one of the early stages in the development preceding phage DNA integration; 2) the process of primary integration after infection and that of reintegration of DNA after prophage induction are likely to differ.     

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.     

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.     

Epidemiology of toxic shock syndrome toxin-1 production in Staphylococcus aureus strains isolated in Denmark between 1959 and 1990

A total of 436 Staphylococcus aureus bacteremia strains isolated between 1959 and 1990 were tested for the production of toxic shock syndrome toxin-1 (TSST-1) by a semiquantitative reversed passive latex agglutination test. TSST-1 production was found in 147/260 (57%) of phage group I strains, excluding the "80" complex, and in 17/176 (10%) of non-group I strains. Strains of the 52, 52A, 80, 81 complex ("80" complex), constituting a subgroup of group I, did not have the same high frequency of TSST-1 production as the rest of group I strains (4% versus 57%). The "80" complex has almost disappeared in Denmark. TSST-1 production was found with the same high frequency among group I strains from the beginning (1959) and throughout the observation period. The TSST-1 production was associated with the phages 29 and/or 52, which in turn lysed 95% of group I strains. The TSST-1 production was quantitatively greater in the phage group I strains than in the non-group I strains. TSST-1 production of the bacteremia strains was not correlated to the clinical parameters: mortality, age, gender, bacterial focus, underlying diseases, or whether the infection was hospital or community acquired.     

Phage and serological types of bacteria infecting patients in anesthesiology and intensive therapy (1988-1990)

1736 of biological materials, being taken from 264 patients, were investigated since 1988 to 1990. 1410 kinds of microorganisms were cultured from 999 biological materials, in which the growth of bacterial flora was noticed. Following species were isolated most frequently: Pseudomonas aeruginosa 15.39%, Proteus mirabilis 12.91%, Klebsiella pneumoniae 10.43% and Staphylococcus aureus 10.43%. The most frequent serological type according to Fisher's scheme was Pseudomonas aeruginosa--immunotype T 3.7 and according to Habs scheme--immunotype P 16. Strains of Staphylococcus aureus were most frequently sensitive to phages the group II. In case of Klebsiella sp. bacilli, the most predominant strains were not typed either by basic or extended phage sets.