Genome fingerprinting as a typing method used on polyagglutinable Pseudomonas aeruginosa isolates from cystic fibrosis patients.

Phenotypical changes occur in the surface of Pseudomonas aeruginosa during the chronic lung infection of cystic fibrosis patients. It is difficult with the classical typing methods, such as serotyping, phage typing and pyocin typing, to decide if a patient has been colonized with a new strain or whether it is the same strain which has reappeared, for instance after chemotherapy in the lungs. This investigation was carried out to evaluate genome fingerprinting as a typing method and to see how it correlated with classical methods and with DNA probe typing. Forty Pseudomonas aeruginosa isolates, 34 polyagglutinable and six monoagglutinable, from 14 cystic fibrosis patients were analysed using genome fingerprinting. The bacterial chromosomes were digested with the restriction endonucleases Dra 1 and Xbal, and separated by field inversion gel electrophoresis. The results were compared with those of a previous work (Ojeniyi et al. 1990) concerning typing with a DNA probe, serotyping using both polyclonal and monoclonal sera, phage typing, pyocin typing and reverse phage typing. The results of genome fingerprinting and DNA probe typing showed the best correlation, followed by pyocin typing. The correlation between the results of genome typing and the other typing methods was low. The discriminatory effect of genome fingerprinting was higher than that of DNA probe typing, and genome fingerprinting was found to be the best single method for epidemiological investigations of polyagglutinable isolates from cystic fibrosis patients.

A simple photometric method for determination of the activity of pyrocin R1.

A simple photometric method for rapid and accurate determination of the activity of pyocin R1, a bacteriocin produced by Pseudomonas aeruginosa strain P15, has been developed. This method is based on the turbidity-decrease observed when the bacteriocin is added to a suspension of sensitive bacteria P. aeruginosa strain P11. Optimum conditions for the turbidity-decreasing activity of pyocin R1 are in 0.01 M Tris-HCl buffer containing 0.2 M NaCl (pH 7.5) at 37 degrees C. A good correlation was found between the dose of pyocin R1 and the rate of the turbidity-decrease (with a correlation coefficient of more than 0.98). The amount of pyocin R1 required for this assay is nearly the same as that used for the conventional colony-counts method. The assay for one sample takes less than 3 min, whereas an overnight wait is necessary for the conventional method. This method is shown to be very suitable for following the time course of activity change observed when pyocin R1 is treated with various chemicals, including receptor substances obtained from sensitive cells. The turbidity-decrease assay was also found to be applicable to the determination of activities of other R-type pyocins.

Isolation of mucoid strains of Pseudomonas aeruginosa from non-cystic-fibrosis patients and characterisation of the structure of their secreted alginate.

When the incubation period of primary isolation plates was extended to 48 h, mucoid strains of Pseudomonas aeruginosa were found in specimens from various infected sites in patients who did not have cystic fibrosis. The 17 mucoid isolates were characterised in terms of mucoid type, pyocin type, and their sensitivity or resistance to seven beta-lactam and two aminoglycoside antibiotics. The carbohydrate, uronic acid (alginate) and protein content of the water-soluble extracellular material of 15 strains was determined. This material was fractionated by ion-exchange chromatography, and the presence of alginate confirmed by the chemical assay of uronic acids and their quantitation by gas-liquid chromatography. Uronic acids were absent from a non-mucoid revertant of one strain. The strains produced alginate with a high content of mannuronic acid and substituted with O-acetyl groups. By proton nuclear magnetic resonance (1H-nmr) analysis the alginate from three strains was shown to lack polyguluronate blocks in its structure. These properties are also found in the alginate of mucoid P. aeruginosa strains from patients with cystic fibrosis.

A preliminary study of fingerprinting of Pseudomonas aeruginosa by whole cell protein analysis by SDS-PAGE.

Forty two strains of Pseudomonas aeruginosa isolated from bronchoalveolar lavage fluid from intubated patients admitted to the Intensive care unit in AIIMS between December 1993 to June 1994 were included in the study. After obtaining typical biochemical profile, antimicrobial susceptibility was performed against ceftazidime, amikacin, gentamicin, ampicillin, cefotaxime and ciprofloxacin. Pyocin typing of these 42 strains was performed by scrape and streak method using 22 indicator strains. Forty strains could be typed showing excellent discrimination but on repeated testing the group designation changed indicating that the system had low reproducibility. SDS-PAGE of whole cell protein profile indicated the presence of 45 protein bands of different molecular weights, individual isolates had 37 to 42 protein band ranging in molecular weight from 340 kDa to 14.3 kDa. On the basis of Dice index of similarity the strains could be grouped into 20 types. Since all strains could be typed, the system has adequate typability. Similar results were obtained on repeated testing indicating good reproducibility.

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.

[Pyocin typing of Pseudomonas aeruginosa strains isolated from various sources]. / Cesitli kaynaklardan soyutlanan Pseudomonas aeruginosa suslarinin piyosin tiplendirmesi.

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.

Characterization of five novel Pseudomonas aeruginosa cell-surface signalling systems.

Cell-surface signalling is a sophisticated regulatory mechanism used by Gram-negative bacteria to sense signals from outside the cell and transmit them into the cytoplasm. This regulatory system consists of an outer membrane-localized TonB-dependent receptor (TonB-dependent transducer), a cytoplasmic membrane-localized antisigma factor and an extracytoplasmic function (ECF) sigma factor. Pseudomonas aeruginosa contains 13 potential surface signalling systems of which only six have been studied in detail. In this work we have identified the regulons of five novel P. aeruginosa signalling systems. For that, the ECF sigmas PA0149, PA1912, PA2050, PA2093 and PA4896 have been overexpressed and their target gene candidates have been identified using DNA microarray, proteomic analysis, and/or lacZ reporter construct. All five ECF sigma factors control the production of one TonB-dependent transducer. Interestingly, two sigma factors, PA2050 and PA2093, regulate the synthesis of a second transducer. Furthermore, we show that although all these sigma factors seem to control putative (metal) transport systems, one of them also regulates the expression of P. aeruginosa pyocins. Finally, we also show that the PA1912-PA1911-PA1910 (designated FemI-FemR-FemA in this work) signalling system responds to the presence of the Mycobacterium siderophores mycobactin and carboxymycobactin and is involved in the utilization of these heterologous siderophores.

Specific cleavage at fibers of a bacteriophage-tail-like bacteriocin, pyocin R1, by successive treatment with organomercurial compounds and trypsin.

When pretreated with the organomercurial compound that reversibly blocks the adhesion of pyocin R1 to sensitive cells, this bacteriocin became susceptible to trypsin. Trypsinization resulted in the exclusive fragmentation of subunit protein no. 2 among 20 different subunit components and the disappearance of distal knobs from the fibers as well as in irreversible inactivation.

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.

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.

[Modified procedure for pyocin typing of Pseudomonas aeruginosa (author's transl)]. / Modifiziertes Verfahren zur Pyocintypisierung von Pseudomonas aeurginosa.

The Procedure for pyocin typing of Pseudomonas aeruginosa isolations as proposed by GILLIES and GOVAN has been modified in the following aspects: strains are arranged in a symmetrical way, with a central producer zone and radial indicator streaks, optimal size of inocula for both producer (300 x 10(6)/ml) and indicators (50 X 10(6)/ml) was determined, unsupplemented Tryptic Soy Agar is used giving identical results to those when TSA is supplemented with 5% horse blood. The typing results obtained with this simplified and standardized procedure were identical with those from the cross streak method.

Epidemiological typing of Pseudomonas aeruginosa.

Current knowledge of the typing of Pseudomonas aeruginosa and new methods for characterizing strains are reviewed. A combination of serotyping as a primary screen with pyocin typing for finer discrimination between isolates gives valid epidemiological data, is within the scope of most clinical laboratories and is to be recommended. Phage typing and H typing do provide good discrimination but are not reproducible in practice because a reaction-difference rule has to be applied to phage patterns, and diphasic variation reduces the accuracy of the identification of flagellar antigens. A problem remains with cystic fibrosis isolates, which show considerable heterogeneity in surface properties. For these strains pyocin production typing by the revised method of Govan is indicated. Newer techniques such as isoenzyme profile and DNA probes of the chromosome require independent evaluation and due to their technical difficulty may not be adopted in a routine context for some years to come.

[Studies on the serotyping and pyocintyping of Pseudomonas aeruginosa].

Pseudomonas aeruginosa (118 strains) from Xian were typed by 12 groups O-serum and revised pyocin typing method Results indicated that VI, I, III were major serotypes, VI type were mainly from trauma infected, I type mainly from respiration system infected. Pyocintype were mainly I and UT types. Pyocintype I were mainly 1/c and 1/x subtypes. 85.7% of 1/c subtypes were serotype VI, 84.4% of 1/x subtypes were serotype I.

[Pyocine typing of Pseudomonas aeruginosa strains from nosocomial outbreaks - after the elimination of possible sources of mistakes by heat-treatment (author's transl)]. / Pyocintypisierung von Pseudomonas aeruginosa-Stämmen aus nosokomialen Infekten - nach Beseitigung von Fehlermöglichkeiten durch eine Hitzebehandlung.

In 25% of the freshly cultivated Pseudomonas aeruginosa strains (isolated from nosocomial infections) a change of the pyocine type determined according to the method of Gillies and Govan results from passages on selective or non-selective media or from several days' storage at room temperature (Tab. 1 and 2). This inconstancy of the pyocine type can be avoided by exposure of the strains to 56 degrees C for 5-75 min. After this heat treatment the agarose-gel-electrophoresis of the strains shows the loss of all non-conjugative plasmids smaller than 25 kbp (Fig. 2). By typing more than 1,000 strains from nosocomial Pyocyaneus outbreaks we could demonstrate the reliability of the pyocine typing after having cured the strains by heat. Further we propose a binary numbering system for the pyocine types using the results of the 8 indicator strains (Tab. 3).

Characterization of a bacteriophage related to R-type pyocins.

A bacteriophage with a contractile tail which shows very similar features to R-type pyocins was isolated and characterized. This phage, named PS17,was purified by DEAE-cellulose chromatography and CsCl density gradient centrifugation. It was a DNA-containing phage, and the density of the purified particles in CsCl was found to be 1.468. DNA from this phage had a density of 1.720 in CsCl, indicating its guanine plus cytosine content to be 61.2%. The head was polyhedral, 69 nm in diameter, and the tail was 150 nm in length. This phage was neutralized by antiserum preparations against five R-type pyocins, and the antiserum against this phage was active in neutralizing R-type pyocins. The properties of this phage, PS17, were compared with another similar phage, PS3, which was previously reported.

[Verification of nosocomial infection chains with Pseudomonas aeruginosa (author's transl)]. / Zur Erfassung nosokomialer Infektketten mit Pseudomonas aeruginosa.

With all 3 typing methods (sero, lyso, pyocin type) errors can arise. The pyocin test following heat curing can be expected to give the least erroneous results. This was demonstrated by a test of 200 non-selected strains which were grown within 4 weeks. The possibility exists that a single patient discharges several different strains simultaneously or in succession. These results must be considered when efforts are made to detect infection chains in a hospital.

[Four new pyocine types of "Pseudomonas aeruginosa": epidemiological significance (author's transl)]. / Marqueurs épidémiologiques de Pseudomonas aeruginosa: isolement de quatre nouveaux pyocinotypes.

The pyocin-typing of 448 Ps. aeruginosa from several Hospitals in Lisbonne has been done with the Gillies and Govan method; 69% Portuguese strains are in pyocinotypes n degrees 1, 3, 5 and 7; 16 % are in 24 other pyocinotypes. Four new pyocinotypes were found in a group of 34 atypical strains. The relationship between serotype and pyocinotype was investigated and the most frequent serotypes in Portugal (0:11; 0:6; 0:3) has been subdivided into numerous pyocinotypes with epidemiological significance.

Genome fingerprinting of Pseudomonas aeruginosa indicates colonization of cystic fibrosis siblings with closely related strains.

The epidemiology of Pseudomonas aeruginosa infection at a cystic fibrosis (CF) center was monitored over a 3-year period. A total of 835 isolates from 72 unrelated patients and 22 siblings with CF were analyzed by genome fingerprinting and serotyping, bacteriophage typing, and pyocin typing. For genome fingerprinting, bacterial chromosomes were digested with one of the restriction endonucleases SpeI, DraI, XbaI, SspI, and NheI, which cut only rarely, and subsequently separated by field inversion gel electrophoresis. The physical genome analysis allowed us to classify P. aeruginosa strains in terms of DNA relatedness. Related strains differed by fewer than six DraI bands in the fingerprint, whereas unrelated strains differed by more than 20 DraI bands. All unrelated CF patients were colonized with different strains. The absence of a nosocomial spread of organisms at the CF center was attributed to the strict hygiene measures observed at the hospital. CF siblings were harboring either identical or closely related strains; transmission within the family is thought to be the most likely cause.