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.

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.

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.

Risk of cross-colonization and infection by Pseudomonas aeruginosa in a holiday camp for cystic fibrosis patients.

The risk of cross-colonization and subsequent infection by Pseudomonas aeruginosa in holiday camps for cystic fibrosis patients was studied in 91 children by culturing sputum at their arrival, at their departure, 2 months later, and at regular intervals thereafter. The isolated strains were subjected to serotyping, phage typing, pyocin typing, and genotyping by random amplified polymorphic DNA fingerprinting-PCR. It was concluded from random amplified polymorphic DNA fingerprinting-PCR typing that the Pseudomonas flora was not constant in most children. Some children harbored one genotype, whereas some harbored two or more different genotypes simultaneously. Most culture-positive children easily acquired a strain of another genotype which replaced the former one or coexisted with the original one. The incidence of sputum conversion was 7.7% in previously negative children; the incidence of permanent colonization and infection was 1.9%. This risk was comparable with that observed in the community. We conclude that the risk of cross-infection is trivial compared with the obvious joy and social benefit derived from a holiday camp.

[Prevalence of serotype 0:12 among strains of P. aeruginosa causing nosocomial infection in Spain (1980-1991)]. / Prevalencia del serotipo 0:12 de cepas de P. aeruginosa productoras de infección nosocomial en España (1980-1991).

BACKGROUND: The aim of the present study was to characterize P. aeruginosa strains causing nosocomial infection in Spain between 1980-1990 with special emphasis on the incidence of serotype 0:12 strains. METHODS: 11,411 strains of P. aeruginosa from hospital-acquired infections were studied and epidemiologically characterized by phage-typing, serotyping and sensitivity to antimicrobial agents. The strains of the 0:12 serotype were analyzed by isoenzyme analysis. RESULTS: Although the major serotypes throughout the period studied were: 0:1, 0:6 and 0:11, the existence of serotype 0:12 strains (6%) were detected which had produced nosocomial outbreaks in surrounding countries. This serotype is homogeneous in that the epidemiologic markers and patterns of sensitivity to antibiotics and the multienzyme analysis demonstrate uniformity in the electrophoretic patterns of all the strains studied. CONCLUSIONS: The 0:12 serotype is in Spain indistinguishable by phage typing and studies of antibiotic sensitivity. It may be considered as being of clonal origin and is probably equal to that existing elsewhere in Europe.

[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.

[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.

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.

[Epidemiologic markers in the study of Pseudomonas aeruginosa from a pediatric intensive care unit]. / Marcadores epidemiológicos en el estudio de Pseudomonas aeruginosa procedentes de una unidad de cuidados intensivos pediátrica.

A study was made on 198 Pseudomonas aeruginosa strains isolated at a pediatric intensive care unit from January to August 1988, using pyocin typing and antibiotic typing as markers. The most frequent circulating pyocin types were 31.83 and atypical 23578. The germ showed high resistance in vitro to the antimicrobial drugs used and the resistance patterns were distributed in 19 different antibiotic types.

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.

[Variation in phage typing and pyocin typing of Pseudomonas aeruginosa]. / Varijabilnost fagotipizacije i piocinske tipizacije Pseudomonas aeruginosa.

The most frequently used microbiological methods in detecting hospital infection reservoirs caused by bacterial species Pseudomonas aeruginosa (P. a.) are serotyping, phagotyping and pyocin typing. Isolated were 86 strains of P. a. from various material of 38 patients hospitalized in University Clinical Centre in Ljubljana during 1986-7. Efforts have been made to find out the variability of results obtained by phago- and pyocin typing of successively isolated P.a. strains from the same patient. Changes found in these samples have been significant.

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.

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.

Prevalence and profiles of plasmids in Pseudomonas aeruginosa.

The value of plasmid profile determination as an epidemiological tool in Pseudomonas aeruginosa infections was investigated by determining the prevalence of plasmids in 450 Pseudomonas aeruginosa strains and comparing the technique with other epidemiological tools. Since only 13.9% of these strains harbored plasmids and the majority of these plasmids were antibiotic resistant, the technique appeared to be less appropriate as an epidemiological tool in this organism than other techniques. Comparison of results obtained from plasmid profile determinations with those from antibiotyping, serotyping and pyocin typing in 50 non-epidemic strains showed the technique gave highly reproducible results and was sensitive; its ability to discriminate could be improved by additionally performing conjugation assays and hydrolysis of plasmidic DNA with restriction enzymes. It is concluded that plasmid profiles provide important epidemiological information on Pseudomonas aeruginosa infections when performed in conjunction with either serotyping or, more importantly, pyocin typing.

Marked increase of Pseudomonas aeruginosa serotype 012 in Belgium since 1982.

Routine typing was performed on a total of 7089 Pseudomonas aeruginosa strains isolated in 16 Belgian hospitals in the period from 1977 to 1986. The annual number of strains received ranged from 318 to 1346. The incidence of serotype O:12 was less than 2% until 1981 when it rose to 4%, steadily increasing to become the predominant serotype in 1984 (22%), 1985 (18%) and 1986 (22%). Since 1980 the O:12 isolates have exhibited characteristic patterns on pyocin and phage typing, 89% of O:12 isolates belonging to pyocin types 1, 39, 43, 45 or 105, whereas only 51% of isolates of other serotypes belonged to those pyocin types. Ninety-three per cent of serotype O:12 isolates belonged to phage types 68/119x, 68 or 119x, or were non-typable, whereas only 24.37% of other serotypes isolates exhibited these phage patterns. These distinctive patterns of pyocin and phage types suggest a high degree of homogeneity within the O:12 strains isolated in recent years in Belgium. Multi-centre or country-wide survey of Pseudomonas aeruginosa strains isolated in hospitals using epidemiological markers may be of value in identifying a sudden increase in epidemic strains.

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.

Serological and pyocin typing and antibiotic sensitivity of Pseudomonas aeruginosa strains.

94 strains of Pseudomonas aeruginosa, isolated from hospitalized patients, were typed with serological and pyocinic methods, also testing their sensitivity to 6 antibiotics. The serological method allowed for the typing of almost all the strains (98.9%), (the 0-11 serotype was the most frequent -23.4%-) vs. 80.6% by the pyocinic method. Among those tested, ceftazidime was the most active antibiotic, against the P. aeruginosa strains examined.