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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↗
PubMed1969

Epidemiological fingerprinting of Pseudomonas aeruginosa by the production of and sensitivity of pyocin and bacteriophage.

A new method has been devised to trace cross-infection by Pseudomonas aeruginosa. Unknown strains growing logarithmically in liquid media were treated with mitomycin C to induce the liberation of pyocin and phage. The lysates were then tested against 27 selected indicator strains, and the zones of clearing were differentiated as to killing by pyocin or lysis by phage. Twenty-four standard pyocin-phage lysates were then applied to each of the unknowns, and the sensitivity pattern was recorded. Thus, an "epidemiological fingerprint" consisting of 51 operational characteristics was established for each isolate. Organisms from the same source had identical or similar fingerprints, but organisms from different origins could easily be distinguished. Pyocin production, pyocin sensitivity, and phage production were found to be stable genetic characters; however, spontaneous mutations in phage sensitivity were frequently encountered. The epidemiological fingerprint has proven to be a sensitive tool in establishing the identity or dissimilarity of unknown strains. This method has been of great value in tracing the epidemiology of P. aeruginosa in the hospital environment. Each of the 157 P. aeruginosa strains tested has been typable by this method.

Bacteriocins↗
PubMed1974

Pyocin typing of Pseudomonas aeruginosa: a simplified method.

A simplified method has been devised for typing Pseudomonas aeruginosa by pyocin production. Pyocins are produced as strains grow overnight in Trypticase soy broth (without glucose) plus 1% potassium nitrate. Because P. aeruginosa can use nitrate instead of oxygen as a terminal electron acceptor, mechanical shaking is not necessary, nor is induction by mitomycin C. Pyocins can now be produced in screw-cap tubes in a water bath or incubator. A total of 250 strains were tested as possible pyocin indicators, which included 60 strains already used in pyocin-typing systems. The final set contained 18 indicators which were chosen because (i) they had clear positive or clear negative reactions, thus eliminating reactions difficult to read, (ii) they had few zones due to bacteriophage lysis, and (iii) they were most sensitive in differentiating clinical isolates of P. aeruginosa. The final typing method was tested in several studies and the results were clear; thus definitive epidemiological conclusions could be made. Because it is simple to perform and easily automated, the new method should have application in many hospitals; however, it should be used only in carefully planned epidemiological studies. The method and its application are described in detail, and some pitfalls are discussed.

Bacteriocins↗
PubMed1967

In vitro and in vivo characterization of pyocin.

Pyocin, a bacteriocin obtained from lysates of ultraviolet-induced cultures of Pseudomonas aeruginosa was characterized in vitro and in vivo after 1,000-fold purification by chemical, column, and differential centrifugation procedures. Electron micrographs of negatively stained pyocin preparations contained rod-shaped particles which resembled the contractile tail protein of the T-even phages of Escherichia coli. Although two separate and distinct pyocin fractions were eluted from diethylaminoethyl cellulose (pH 7.5) during the purification procedure, the particles appeared identical. In addition, the two fractions exhibited a close correlation between their titers and the particle numbers as observed in the electron microscope. The particles were approximately 20 by 90 mmu with a core diameter of 5 mmu and a sheath length of 50 mmu. Neither intact phage nor ghosts were seen in any of the preparations, although ringlets of two different diameters, which appeared to correspond to the diameters of the sheath and inner core, were observed. Other studies indicated that, although crude preparations were stable to freezing and thawing, purified preparations lost all of their activity under similar treatment. However, the addition of 50% glycerol to purified preparations completely protected activity. Conversely, aged normal human or rabbit sera enhanced the antibacterial activity of pyocin approximately fourfold, although serum albumin and hemoglobin had no effect. In vivo studies indicated that purified pyocin was not lethal for mice when injected intraperitoneally in concentrations of 28,000 to 1,400,000 units (5.6 to 276 mug of protein), nor was 7,200 to 36,000 units dermonecrotic for rabbits.

Animals↗
PubMed1979

Pseudomonas aeruginosa bacteremia: relationship of bacterial enzyme production and pyocine types with clinical prognosis in 100 patients.

Strains of Pseudomonas aeruginosa were studied from 100 patients with bacteremia. In vitro quantitation of extracellular enzymes (lecithinase, protease, and elastase) and pyocine typing of these isolates were performed. No significant difference was found in the quantity of the enzymes produced or in the pyocine types by isolates obtained from patients dying from bacteremia or surviving this serious infection. Quantitation of the extracellular enzymes and pyocine types of blood isolates were contrasted with similar data obtained from sputum, urine, and skin isolates. One third of all strains produced minimal or no extracellular enzymes regardless of their source. However, the highest enzyme-producing strains were observed in the blood isolates. Although a greater variability of pyocine types was found in blood culture isolates, there was no significant difference between the pyocine types found in blood, urine, sputum, or skin isolates. The lack of correlation between the in vitro quantity of lecithinase, protease, and elastase produced by P. aeruginosa strains isolated from bacteremic patients and the prognosis of these patients supports the possible local rather than systemic significance of these extracellular enzymes in the pathogenesis of P. aeruginosa bacteremia.

Extracellular Space↗
PubMed1977

A fluorescent probe response to the interaction of pyocin R1 with sensitive cells.

Additon of pyocin R1, a bacteriocin of Pseudomonas aeruginosa, to sensitive cells caused a fluorescence increase of 8-anilino-1-naphthalenesulfonate (ANS) in the cell suspension. The reaction was rapid, starting with a short time lag after adsorption of pyocin onto the cells and finishing within several minutes. The fluorescence response was attributed to the interaction of the cell body and ANS, not to that of the medium outside the cells and ANS. The maximal amplitude of fluorescence after pyocin addition was dependent on temperature, and the relation appeared to be biphasic. Similarly, Arrhenius plots of the initial rate of fluorescence change were biphasic. The transition of slopes in both cases occurred in the temperature range between 18 and 19 degrees. These results suggest that ANS interacts with lipids in the cell envelope and that pyocin causes a structural change of the cell envelope leading to increased fluorescence of ANS.

1-Naphthylamine↗
PubMed1978

Effect of pyocin R1 on the glucose metabolism of sensitive cells of Pseudomonas aeruginosa.

The effect of pyocin R1 on the glucose metabolism of sensitive Pseudomonas cells was investigated. Upon treatment with pyocin R1, although the rate of O2 uptake of the sensitive cells for glucose or gluconate was not very much affected at first, the final level of O2 uptake was greatly reduced. When 2-oxogluconate was used as a substrate, O2 uptake was immediately halted by pyocin. By determining the amounts of glucose, gluconate, and 2-oxogluconate before and after the reaction and the amount of O2 consumed, it was concluded that glucose was exclusively metabolized via the following pathway with quantitative accumulation of 2-oxogluconate after pyocin treatment. (Formula: see text). The possible mechanism of this change is discussed.

Bacteriocins↗
PubMed1982

Comparative study on fibers isolated from four R-type pyocins, phage-tail-like bacteriocins of Pseudomonas aeruginosa.

Five R-type pyocins have been reported which are almost identical with one another in their morphology and subunit composition, though distinct in receptor-binding specificity. We isolated fibers from pyocin R2, R3, and R4 by essentially the same procedure as used in our previous isolation of pyocin R1 fiber with unimpaired receptor-binding ability. All the isolated fibers including R1 fiber were indistinguishable from one another, in terms of electron microscopic observation and subunit composition analysis by SDS gel electrophoresis. They consisted mainly of Subunit No. 2 (Mw 71,000) and No. 9 (31,000) proteins. Although Subunit No. 9 protein in every fiber was susceptible to trypsin and afforded a fragment with the same molecular weight (about 19,000) detectable in the SDS gel, Subunit No. 2 protein was cleaved with trypsin only after the fiber had been treated with an organomercurial, 4-(p-sulfophenylazo)-2-mercuriphenol. The cleavage of Subunit No. 2 protein proceeded to give several fragments with molecular weights ranging from 64,000 to 34,000, and the fragmentation patterns were electrophoretically distinct at least among R1 fiber, R3 fiber, and others (R2 and R4 fibers). The results indicate that Subunit No. 2 proteins of these fibers are different from one another in the structure surrounding trypsin-susceptible peptide bonds. Immunological investigations with anti-R1 fiber antibodies provided some additional information on the difference among R-type pyocins at the fiber level.

Amino Acids↗
PubMed1979

Sensitivity of Neisseria gonorrhoeae to partially purified R-type pyocines and a possible approach to epidemiological typing.

Strains of Neisseria gonorrhoeae from a variety of sources were examined for sensitivity to 11 partially purified R-type pyocines from Pseudomonas aeruginosa. Selective inhibition of gonococci by pyocines of Kageyama groups R1 and R5 was observed. "Matched isolates", those from consorts or different body sites of individual patients, usually had very similar pyocine-sensitivity patterns and identical sensitivities to five antibiotics tested. This study included local isolates, strains from diverse geographic regions, and strains from disseminated gonococcal infections. It also proposed a relationship between pyocine-receptor sites in the lipopolysaccharide of Ps. aeruginosa and N. gonorrhoeae. Topics needing further evaluation are discussed.

Anti-Bacterial Agents↗
PubMed1982

Sensitivity of thermophilic campylobacters to R-type pyocines of Pseudomonas aeruginosa.

Strains of thermophilic campylobacters of human origin were examined for bacteriocine activity and for susceptibility to R-type pyocines of Pseudomonas aeruginosa. None of 50 strains inhibited the growth of any other strain, but 13 of 80 strains (16%) were sensitive to R-type pyocines. Absorption of one of the partially purified pyocine preparations with a sensitive strain resulted in the removal of pyocine activity and a decrease in viable count of the organism by 85%.

Bacteriocins↗
PubMed1972

Purification and characteristics of pyocin B39 II. Physical and chemical properties.

A purified preparation of pyocin B39 was shown to be homogeneous by disc electrophoresis and ultracentrifugation. Pyocin B39 is an antibacterial agent, protein in nature, and is capable of inhibiting the growth of susceptible cells. It caused a susceptible strain of Pseudomonas aeruginosa to release ultraviolet-absorbing material. Pyocin B39 appears to be a protein with a high molecular weight; its sedimentation coefficient is 32.8S. It was destroyed by 7 m urea but was resistant to attack by proteolytic enzymes, such as pepsin and trypsin. From its sedimentation coefficient, a molecular weight of about 2 x 10(6) was calculated. An electron photomicrograph of pyocin B39 revealed it as a rodlike particle, 7.50 pm long and 1.08 pm wide, with striations running at nearly right angles to the axes of the particle.

Bacteriocins↗
PubMed1984

Pyocin R1 inhibits active transport in Pseudomonas aeruginosa and depolarizes membrane potential.

Pyocin R1, a bacteriocin of Pseudomonas aeruginosa, inhibited active transport of proline in the presence of high concentrations of malate and magnesium salt. Pyocin R1 did not affect the respiration of sensitive cells nor induce cell lysis, but it caused a decrease in the intracellular ATP level. In addition, a passive inflow of [14C]thiocyanate anion, a probe of membrane potential, was induced by pyocin R1, showing a depolarization of the cytoplasmic membrane. It is considered that membrane depolarization is a primary action of pyocin R1.

Adenosine Triphosphate↗
PubMed1996

Construction and characterization of pyocin-colicin chimeric proteins.

Chimeric proteins were constructed from pyocin S1 or S2 and colicin E3 or E2, and their characteristics were investigated with special reference to the domain structure. The nuclease domains were interchangeable between two bacteriocins so that a new kind of pyocin, with RNase activity, was created. A bacteriocin which can kill both Pseudomonas aeruginosa and Escherichia coli was also constructed. Investigations with various chimeric proteins indicate that the translocation domain as well as the receptor-binding domain is species specific. Inhibition of lipid synthesis, which is characteristic of pyocins, was also observed with chimeric pyocins carrying the DNase domain of colicin E2 but not with those carrying the RNase domain of E3. Thus, the DNase domain is responsible for the inhibition of lipid synthesis.

Bacterial Proteins↗
PubMed1984

Revised pyocin typing method for Pseudomonas aeruginosa.

In the Gillies and Govan method of pyocin typing for Pseudomonas aeruginosa a cross-streaking technique was used, and 105 main types and 25 subtypes were identified by the patterns of inhibition observed on 13 indicator strains. Disadvantages of the technique included the need to remove test strain growth before application of the indicator strains, the 48-h period needed to obtain a result, and the inability to reliably type mucoid P. aeruginosa. Recent studies have enabled us to overcome these disadvantages and significantly improve the speed and application of pyocin typing. Our revised technique utilizes the same 13 indicator strains which are already used internationally. Test strains were rapidly applied to the surface of agar plates with a multiple inoculator. After incubation for 6 h and exposure to chloroform, the indicator strains were applied in agar overlays without prior removal of the test strain growth. After 18 h of incubation, the pyocin type was recognized by inhibition of particular indicator strains. Additionally, the activity of particulate (R and F) and nonparticulate (S) pyocins could be distinguished on the basis of inhibition zone size, which thus allowed further discrimination. The revised technique allows typing within 24 h, increases the number of identifiable types, and can be used to type mucoid strains.

Bacteriocins↗
PubMed1986

Improved, computer-generated system for pyocin typing of Pseudomonas aeruginosa.

We applied numerical clustering algorithms to the selection of a new indicator strain set for the pyocin typing of Pseudomonas aeruginosa. The new indicator set is composed of selected indicator strains from the sets described in 1966 by Gillies and Govan (J. Pathol. Bacteriol. 91:339-345) and in 1974 by Jones, Zakanycz, Thomas, and Farmer (Appl. Microbiol. 27:400-406) and is designated the G-F set. This indicator set consists of 14 indicator strains which typed 99.5% of 114 test cultures, has a high degree of discrimination (10 patterns encompass 50% of the test strains), and provides 62.3% reproducibility of the same typing pattern in duplicate tests done on different days. The G-F set of indicator strains provides slightly higher percentages of typable cultures than either of the other two sets, has greater discriminatory capability, and is more reproducible than they are. We recommend that the G-F set of indicator strains be used instead of the two other sets for pyocin typing of P. aeruginosa. We also tested a recently described overlay procedure for pyocin testing of P. aeruginosa and found it to be superior to previous methods in that it is easier to perform, it provides answers in only 24 h instead of 48 h, and it can be used to type mucoid strains (which previous techniques could not readily do). Thus, the application of numerical clustering algorithms and use of a revised typing procedure have produced an improved system for pyocin typing of P. aeruginosa. Similar procedures may be applicable to other typing systems.

Algorithms↗
PubMed1976

Epidemiology of Pseudomonas aeruginosa infections: determination by pyocin typing.

The epidemiology of hospital infections due to Pseudomonas aeruginosa was investigated by pyocin typing. The typing method, which determined the pyocin activity of clinical isolates of P. aeruginosa on 27 indicator strains, was 43.7% reproducible, but elimination of 9 indicator strains doubled the reproducibility and yielded more readable pyocin inhibition zones. Seventy-eight of 1,084 isolates (7.2%) were untypable. In the second part of the study, P. aeruginosa was isolated from 110 patients (5.4% of all admissions) in a 3-month period and typed with the revised method. Twenty pyocin types were identified, 10 of which were obtained from five or more patients. P. aeruginosa was isolated from 45 of 353 environmental samples, including water fountains, ice machines, bar soaps, and germicide solutions for toilet brushes. Twenty percent of the environmental samples were untypable but, among typable strains, the five most common environmental strains were the same as the strains most frequently isolated from patients. The organism was frequently isolated from noses (39%), throats (39%), and stools (29%) of patients with P. aeruginosa infections or colonizations in urine, sputum, surgical wounds, or skin lesions. Six of eight patients had P. aeruginosa in their tracheostomy wounds. Autoinfections by strains already acquired on carrier sites may be significant.

Bacteriocins↗
PubMedDate not supplied

Pyocine typing of Pseudomonas aeruginosa strains isolated in Ahmadu Bello University Teaching Hospital, Zaria.

Pseudomonas aeruginosa strains isolated from clinical and environmental sources in Ahmadu Bello University Teaching Hospital, Zaria, were pyocine typed using the indicator strains and the standardized method of Gillies & Govan. Out of 302 clinical isolates 9.6% were untypable and 3.6% were unclassifiable. Among typable isolates fourteen pyocine types were identified, types 1, 3, 11, 5, and 10 being the commonest. Among ninety-six environmental isolates types 1, 3 and 10 were most common, 18.7% were untypable and 3.1% were unclassifiable. Sink traps were environmental sites most frequently contaminated with Ps. aeruginosa. Out of ten pyocine types identified in environmental strains, eight were the same as those isolated from patients. Seven subtypes were found among the most commonly encountered pyocine type 1 isolates. The procedure is a simple and reliable method to study the epidemiology of infections due to Ps. aeruginosa.

Bacteriocins↗