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PubMed1976

Treatment of Pseudomonas aeruginosa infections with pyocines.

The interactions of a contractile, a filamentous and a small pyocine with a sensitive strain of Pseudomonas aeruginosa (no. P14) were examined in vivo. The purification procedure used yielded high-activity pyocine preparations that were not toxic to mice. The inhibitory activity of such preparations, when injected into mice by various routes, was retained for up to 24 h. However, high molecular-weight pyocines given intraperitoneally in the presence of a lethal dose of strain P14 administered by the same route did not prevent the fatal outcome of infection unless they are given before or together with the bacteria. The small pyocine had no protective effect. In burned mice infected with strain P14, topical application of a filamentous pyocine failed to improve the chances of survival. The results suggest that there is little future for pyocine therapy.

Administration, Topical↗
PubMed1975

Defective pyocin particles produced by some mutant strains of Pseudomonas aeruginosa.

Mutants of Pseudomonas aeruginosa, defective in the production of active R-type pyocins, were isolated from pyocinogenic strains and their products were characterized. Polysheath-like structures were found in induced lysates of 29 out of 42 mutants. Two mutants (strain P15-16 and M189) were found to produce special defective particles, which were characterized in detail. The other 11 mutants did not produce significant amounts of any structure visible under an electron microscope. Serum blocking powers were found in lysates from P15-16 and M189 to significant amounts. Defective particle produced by strain P15-16 lacked the sheath component, whereas M189 had morphological defects at the junction between sheath and baseplate, and also in the architecture of baseplate. Both defective particles could adsorb to the surface of bacteria, that were sensitive to pyocin, at the tip of their fibers without killing cells. All M189 particles attached to the bacteria had the extended sheaths. Therefore, attachment to the bacteria by fibers is not sufficient to kill cells, and contraction of sheath must occur after the initial adsorption by fibers for pyocin to express its biological activity. Defective particles of strain P15-16, which was derived from strain P15 (a pyocin R1 producer), could be converted to active forms by an in vitro complementation reaction with extracts from certain mutants originated from strain PAO (a pyocin R2 producer). This result indicated the exchangeability of components between R-type pyocins belonging to the different groups.

Adsorption↗
PubMed1984

Genetic determinant of pyocin AP41 as an insert in the Pseudomonas aeruginosa chromosome.

The genetic determinant for pyocin AP41 , a bacteriocin produced by Pseudomonas aeruginosa PAF, was transferred to P. aeruginosa PAO and analyzed. By conjugation experiments, the pyocin determinant was found to be located on the chromosome, being closely linked to argG at about 45 min on the genetic map. Cloning of the pyocin AP41 gene into the plasmid R68.45 was attempted in vivo by taking advantage of its linkage at argG. R' argG+ plasmids were isolated by interspecific conjugation between P. aeruginosa and Escherichia coli recA argG strains. Some of the R' argG+ plasmids did contain the pyocin AP41 determinant. Genetic and physical analyses of these R' plasmids indicated that the pyocin AP41 determinant was located within a 2.9-kilobase extra segment found at a certain position of the chromosome of various pyocin AP41 producer strains.

Bacteriocins↗
PubMed1993

Regulation of pyocin genes in Pseudomonas aeruginosa by positive (prtN) and negative (prtR) regulatory genes.

Most strains of Pseudomonas aeruginosa produce various types of bacteriocins (pyocins), namely, R-, F-, and S-type pyocins. The production of all types of pyocins was shown to be regulated by positive (prtN) and negative (prtR) regulatory genes. The prtN gene activates the expression of various pyocin genes, probably by the interaction of its product with the DNA sequences conserved in the 5' noncoding regions of the pyocin genes. The prtR gene represses the expression of the prtN gene, and its product, predicted from the nucleotide sequence, has a structure characteristic of phage repressors and seems to be inactivated by the RecA protein activated by DNA damage. A model for the regulation of the pyocin genes is proposed.

Amino Acid Sequence↗
PubMed1991

The structural basis for pyocin resistance in Neisseria gonorrhoeae lipooligosaccharides.

Pyocin resistance in a strain of Neisseria gonorrhoeae has been found to be associated with structural differences in the oligosaccharide moieties of the gonococcal outer membrane lipooligosaccharides (LOS). N. gonorrhoeae strain 1291 had been treated with several pyocins, usually lethal bacteriocins produced by Pseudomonas aeruginosa, and a series of surviving mutants were selected. The LOS of these pyocin-resistant mutants had altered electrophoretic mobilities in sodium dodecyl sulfate-polyacrylamide gels (Dudas, K. C., and Apicella, M. A. (1988) Infect. Immun. 56, 499-504). Structural analyses of the oligosaccharide portions of the wild-type (1291 wt) and five pyocin-resistant strains (1291a-e) by liquid secondary ion mass spectrometry, tandem mass spectrometry, and methylation analysis revealed that four of the mutant strains make oligosaccharides that differ from the wild-type LOS by successive saccharide deletions (1291a,c-e) and, in the oligosaccharide of 1291b, by the addition of a terminal Gal to the 1291c structure. The composition, sequence, and linkages of the terminal tetrasaccharide of the wild-type LOS are the same as the lacto-N-neotetraose terminus of the human paragloboside (Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4Glc-ceramide), and both glycolipids bound the same monoclonal antibodies O6B4/3F11 that recognize this terminal epitope. None of the pyocin-resistant mutants bound this antibody. The 1291b LOS bound a monoclonal antibody that is specific for Gal alpha 1----4Gal beta 1----4Glc-ceramide (Pk glycosphingolipid) and shared a common composition, sequence, and linkages with this latter glycosphingolipid. Organisms that bound the anti-Pk monoclone occurred at the rate of approximately 1/750 among the wild-type parent strain. This structural information supports the conclusion that treatment with pyocin selects for mutants with truncated LOS structures and suggests that the oligosaccharides contained in the LOS of the wild-type strain and 1291b mimic those of human glycosphingolipids.

Carbohydrate Sequence↗
PubMed1985

Purification and characterization of pyocins from Pseudomonas aeruginosa.

Three types of pyocins were found in Pseudomonas aeruginosa strain 986 and named pyocin type P25, P50, and P70. Production of these types was inducible by UV irradiation. Their molar mass was estimated. The pyocins obtained were different from the known pyocins R, S, and F in their chemical and physical properties. No immunological cross reaction was observed among these pyocins.

Amino Acids↗
PubMed1985

In vitro pyocin activity of Pseudomonas aeruginosa strains pretreated with antibiotics.

Fifty-six selected strains of Pseudomonas aeruginosa belonging to 8 different pyocin types (H, I, 15, 6, PTI-1, PTI-2, PTI-3, PTI-4) were treated with subinhibitory concentrations (MIC/2) of either gentamicin or carbenicillin. Both treatments induced changes in pyocin patterns for all types but at different levels. The percentage of strains that retained their pyocin pattern were more or less equal in both treatments. In treated and untreated producers, the growth inhibition ability for 5 different strains of Enterobacteriaceae (Escherichia coli K12, E. coli EB, Proteus vulgaris, Salmonella typhi, Shigella flexneri) was also investigated. In all pyocin patterns the number of producers that inhibit the growth of these strains was lower after treatment with gentamicin or with carbenicillin, a smaller decrease was detected in the latter treatment. It appeared that the subinhibitory concentrations of these antibiotics are capable of protecting the Enterobacteriaceae strains from the action of the pyocins.

Bacteriocins↗
PubMed1990

Purification of the pyocin S2 complex from Pseudomonas aeruginosa PAO1: analysis of DNase activity.

Pyocin S2 purified from mitomycin C-induced lysates of Pseudomonas aeruginosa strain PAO1 has been shown to consist of a complex of two proteins. Further analysis of the purified S2 complex revealed that the 74 kd S2 pyocin demonstrates DNase activity which can be blocked by S2-specific antisera. Chromosomal DNA from pyocin sensitive cells treated with the pyocin S2 complex in vitro did not show any degradation, suggesting that the 10 kd protein inhibits the DNase activity of the S2 protein. These results suggest an alternative mechanism for the toxicity associated with the S2 pyocin.

Antibodies, Monoclonal↗
PubMed1997

Susceptibility of Pseudomonas aeruginosa of various pyocin types to the newly synthesized ampicillin derivative, N-(6,7-difluoroquinolonyl)ampicillin.

Six hundred and thirty-two isolates of Pseudomonas aeruginosa of 17 pyocin types were collected in 1993 in Taiwan. Types 1, 10, 3, 35 and 12 were the most common pyocin types identified in Taiwan with isolation frequencies of 47.3%, 24.4%, 7.6%, 3.6% and 2.2%, respectively. Several pyocin subtypes were determined. All pyocin types (one isolate of each tested) were resistant to ampicillin and nalidixic acid, but sensitive to fluoroquinolone antibiotics, such as norfloxacin and enoxacin, indicating that cross-resistance to quinolone antibiotics of nalidixic acid and fluoroquinolone derivatives has not developed. A new ampicillin derivative of 6,7-difluoroquinolonic acid, N-(6,7-difluoroquinolonyl)-ampicillin (AU-1), was synthesized by coupling ampicillin with 6,7-difluoroquinolonic acid (FP-3). Compound AU-1 was much more active than either ampicillin or FP-3 alone against all pyocin types of P. aeruginosa and induced filamentation in most growing cells.

Ampicillin↗
PubMed1983

Isolation and characterization of a new bacteriophage, KF1, immunologically cross-reactive with F-type pyocins.

Pseudomonas aeruginosa strain NIH S produced a bacteriophage, KF1, immunologically cross-reactive with F-type pyocins. Phage KF1 was neutralized by both anti-pyocin F1 and anti-pyocin F3 sera, although the efficiency was very low. About eleven polypeptides were detected by SDS-polyacrylamide gel electrophoresis of the phage. Most of the subunit proteins were different from those of F-type pyocins, but the molecular weights of minor subunit proteins P3 and P6 seemed to be the same as those of band 1 and band 5 of F-type pyocins, respectively. The head of the phage appeared to have an icosahedral structure, approximately 63 nm in diameter, with a long (190 nm, 11 nm wide and about 45 striations) flexuous tail connected to a fiber structure (about 53 nm in length). The density in CsCl and the sedimentation coefficient of the phage were 1.54 g/ml and 392S, respectively. Some other biochemical properties were described. The nucleic acid of the phage was linear, double stranded DNA of molecular weight 4 x 10(7). The density of the DNA in CsCl was 1.719 g/ml, the melting temperature was 95.4 degrees C. The guanine plus cytosine content was calculated to be 60 to 64%.

Bacteriocins↗
PubMed1994

Use of pyocin to select a Haemophilus ducreyi variant defective in lipooligosaccharide biosynthesis.

Haemophilus ducreyi, a cause of genital ulcer disease in developing countries, appears to facilitate the heterosexual transmission of the human immunodeficiency virus in Africa. Despite an increase in studies of this gram-negative human pathogen, little is known about the pathogenesis of chancroid. Our studies have shown that the lipooligosaccharides (LOS) of H. ducreyi may play an important role in ulcer formation. Monoclonal antibody and mass spectrometric analyses identified a terminal trisaccharide present on H. ducreyi LOS that is immunochemically similar to human paragloboside. This epitope is present on the LOS of Neisseria gonorrhoeae, and it may be the site of attachment for pyocin lysis. We have used pyocin, produced by Pseudomonas aeruginosa, to select LOS variants with sequential saccharide deletions from N. gonorrhoeae. On the basis of the similarities between N. gonorrhoeae and H. ducreyi LOS, we employed the same technique to determine if H. ducreyi strains were susceptible to pyocin lysis. In this study, we report the generation of a pyocin N-resistant H. ducreyi strain which synthesizes a truncated version of the parental LOS. Further studies have shown that this H. ducreyi variant has lost the terminal LOS epitope defined by monoclonal antibody 3F11. This report demonstrates that H. ducreyi is sensitive to pyocins and that this technique can be used to generate H. ducreyi LOS variants. Such variants could be used in comparative studies to relate LOS structure to biologic function in the pathogenesis of chancroid.

Carbohydrate Sequence↗
PubMed1982

Dansyl chloride labeling of Pseudomonas aeruginosa treated with pyocin R1: change in permeability of the cell envelope.

Pyocin R1, a bacteriocin of Pseudomonas aeruginosa, caused an increase in binding of fluorescent label, 1-dimethylaminonaphthalene-5-sulfonyl chloride (dansyl chloride), to sensitive cells. In pyocin R1-treated cells, cytoplasmic soluble proteins and crude ribosomes as well as cell envelopes were labeled by dansyl chloride. The amount of bound dye was proportional to the multiplicity of pyocin R1 and reached a maximal level at high multiplicity. In addition, pyocin R1 rapidly caused an increase in fluorescence intensity of the hydrophobic probes N-phenyl-1-naphthylamine, pyrene, and perylene, which were mixed with cells. These results show that pyocin R1 damages locally a cell envelope barrier to hydrophobic solutes and allows dyes to penetrate into the intracellular space across the barrier.

1-Naphthylamine↗
PubMed1993

Functional domains of S-type pyocins deduced from chimeric molecules.

Functional domain structures of pyocins AP41, S1, and S2 were assigned by examining the functions of chimeric pyocins and deletion derivatives. Pyocins AP41, S1, and S2 are essentially composed of three domains, the receptor-binding domain, the translocation domain, and the DNase domain, in that order from the N terminus to the C terminus. The alignment of these domains is distinct from that in E2-group colicins with functions similar to those of these pyocins. Pyocins AP41 and S2 have a fourth domain between the receptor-binding and the translocation domains, which is dispensable for their killing functions.

Amino Acid Sequence↗
PubMed1979

Studies on a receptor for pyocin in a R mutant of Salmonella minnesota.

Lipopolysaccharide (LPS) isolated from the pyocin sensitive R form strain of Salmonella minnesota F6 (chemotype Rd1) inhibited the activity of bacteriophage tail-like pyocin P1 whereas no inhibition occurred with LPS prepared from the pyocin resistant S form of S. minnesota. Subunits of lipopolysaccharide obtained by treatment with sodium deoxycholate and the polysaccharide fraction of the lipopolysaccharide obtained by acid hydrolysis were shown to be still active whereas lipid A fraction had no pyocin neutralizing activity. The (KDO)3-hepI-hepII unit, which terminates the lipopolysaccharide of S. minnesota F6 was, therefore, suggested to determine the specificity of the pyocin P1 receptor.

Bacteriocins↗
PubMed1992

[Pyocin typing of Pseudomonas aeruginosa strains isolated from various sources].

In our study, 103 strains of P.aeruginosa from various clinical specimens were typed by pyocin typing method with 13 indicator strains. As a result of pyocin typing, 18 different pyocin type were detected from 81 typable strains (78.6%), 22 of P.aeruginosa strains (21.3%) could not be typed because of the lack of their pyocin activity. On the other hand, 2 of typable P.aeruginosa strains were found to produce inhibition patterns that differed from standard inhibition patterns according to Govan and Gillies Method. Because of this reason, these two strains could not be classified. As a result of our study, it was observed that, strains that belong to pyocin type-1 were in majority. It was detected that, TSA Medium supplemented with 5% sheep blood can be used easily instead of TSA Medium, supplemented with 5% horse blood.

Culture Media↗
PubMedDate not supplied

Pseudomonas aeruginosa. IV. IV. Pyocine typing of strains isolated from the blue fox (Alopex lagopus), mink (Mustela vison), and dog (Canis familiaris) and from their environment.

Investigation of a total of 354 Ps. aeruginosa strains by "active" pyocine typing, using Govan & Gillies' indicator strains (21, 24) by which it may be possible to demonstrate the sources and routes of pseudomonas infection revealed that: (1) of the isolated pyocine-producing types 85.5% could be fitted into Govan & Gillies' classification, (2) 11.1% of all strains tested did not produce pyocine, (3) p3 was the only pyocine type isolated from an enzootic of fatal, haemorrhagic pneumonia in experimental minks debilitated by change of environment and inoculation with plasmacytosis (Aleutian disease), (4) types 1--5, 8, 10, 17, 29, and 37 could be isolated from the blue foxes and their farm environment, and (5) types 1--3, 5, 6, 9, 10, 15, 17, 28, 31, and 37 were identified in hospitalized dogs and their hospital environment. Pyocine type p1 was predominant in all groups except that of experimental minks and samples from the room in which they were kept. This type, plus type p3, made up more than half the typed strains of Ps. aeruginosa. The sources of infection comprised: (1) Carriers among the blue foxes and their winter watering troughs. During periods without frost the troughs were not cleaned, but were left containing more or less water contaminated with urine, faeces, hairs, and dust--a well-suited habitat for Ps. aeruginosa. (2) In the case of the experimental minks the infection may have been derived from a dog with pseudomoniasis in the hospital, brought to the experimental room in the course of feeding and cleaning. (3) The infection in the Small Animal Clinic seems to be maintained by frequent admission of patients for treatment, especially of chronic otitis externa/media caused by widely resistant strains of Ps. aeruginosa.

Animals↗
PubMed1981

Phagovar determination of Pseudomonas aeruginosa and a comparison of the results with mitomycin C induced pyocin production.

One hundred Pseudomonas aeruginosa isolates recovered from intensive care areas of a university hospital and other clinical sites were characterized on the basis of their susceptibility to a set of twenty one phages. These strains were previously mitomycin C induced for their pyocin production and their serovars were also determined. All isolates identified on the basis of belonging to the same O-serovars and pyocin patterns, belonged to oe phagovar group. Strains which were non-identifiable with commercial antisera, had to be typed by both phagovar assay and mitomycin C induced pyocin production. Neither phagovar assay nor pyocin production alone gave enough individual characteristics of any one isolate, as to allow proper identification. Patterns with the same configurations in both phagovar and pyocin groups were detected among strains within entirely different O-serovar groups.

Bacteriocins↗
PubMed1978

Isolation, purification and characterization of a pyocin.

Seventeen strains of P. aeruginosa were tested for pyocin production using the broth method. Mitomycin C was used for the induction of pyocin in TSB shaked cultures incubated at 32 degrees. The highest pyocin activity was produced by strain 14. TSB shaked cultures in fluting flasks were found to be superior to Erlenmeyer flasks for pyocin production. Pyocin 14 was purified by chemical and chromatographic methods. It was found to be protein in nature and free from carbohydrates. It is stable between pH 5 and 9 and is partially inactivated when kept for one month at 4 degrees. Freezing and thawing, lyophilization and heating at 65 degrees for 5 min completely destroyed its activity. The addition of 4 mg/ml bovine albumin or 2 mg/ml gelatin caused about 60% increase in activity. However, treatment with 10 mM EDTA caused a 98% drop in its activity.

Bacteriocins↗