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PubMed1993

Epidemiologic typing of multiply drug-resistant Pseudomonas aeruginosa isolated from an outbreak in an intensive care unit.

From June to August 1991, there was an outbreak of Pseudomonas aeruginosa infections in an intensive care unit in a general hospital in Sao Paulo, Brazil. We obtained 14 isolates from 14 patients, 11 from tracheal aspirate, and 3 from surgical wound exudates. These strains were typed by serotyping, pyocin typing, and pulsed-field electrophoresis (CHEF) of chromosomal DNA (chrDNA), and the different typing methods were analyzed. These three methods demonstrated seven identical strains. We also performed an extensive antibiogram (33 drugs) in all 14 isolates. The incidence of resistance to aminoglycosides, extended-spectrum beta-lactams, and quinolones was very high among the seven identical isolates; however, the antibiogram profile differed significantly among the isolates. Our results suggest that a unique strain caused several cross-transmitted infections during this period of time, and the emergence of antimicrobial resistance has been occurring before and after the establishment of the epidemic strain by selective drug use. The chrDNA fingerprinting proved to be versatile and precise for epidemiologic investigations of P. aeruginosa infections.

Brazil↗
PubMed1984

[Pseudomonas septicemia after endoscopic interventions on the bile duct system].

In the first half of 1982 there was an increase in septicaemia cases, especially among patients with biliary-tract drainage. The septicaemia incidence rose from 1.25% to 4.4%. The proportion of Pseudomonas septicaemias was especially high: of 20 patients (21 episodes of septicaemia) nine had infections with Pseudomonas aeruginosa alone, three a mixed septicaemia. In ten of these twelve patients there was impaired drainage by a malignant tumour. Three patients with a tumour stenosis died, mainly of the septicaemia. The cause of this increased incidence of Pseudomonas septicaemias lay in contamination of the instruments; it was in part sustained by the Endo-Washer. The connection between the septicaemia after endoscopic-retrograde cholangiography and recontamination of the endoscope was discovered after sero- and pyocin-typing of the Pseudomonas strains. By changing the methods of disinfection the Pseudomonas incidence among endoscopies fell. However, in individual samplings Pseudomonas can still be demonstrated on instruments as well as on the Endo-Washer.

Blood↗
PubMed1994

Epidemiology of chronic Pseudomonas aeruginosa infections in cystic fibrosis.

The epidemiology of chronic colonization of airways with Pseudomonas aeruginosa was monitored in 44 patients with cystic fibrosis (CF) by DraI/SpeI macrorestriction analyses of 489 isolates. Sequential P. aeruginosa isolates (144) that had been collected from 32 CF patients over < or = 2.5 years were investigated, and 12 patients were followed for 8 years after onset of colonization. Forty-eight different genotypes were uncovered from 481 typeable isolates. Ten genotypes were found in > 1 unrelated CF patient. The 6 most frequent clones were identified in 58% of isolates. Ten of the 12 patients monitored for 8 years were harboring their initially acquired P. aeruginosa clone at all times, with subtle shifts of fragment patterns indicating subclonal variation. During colonization, the bacteria gradually lost pyocin and phage typing responses, supporting the view that genotypically discordant P. aeruginosa strains develop a common phenotype.

Adolescent↗
PubMed1989

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.

Alginates↗
PubMed2003

Genome mosaicism is conserved but not unique in Pseudomonas aeruginosa isolates from the airways of young children with cystic fibrosis.

Pseudomonas aeruginosa strains from the chronic lung infections of cystic fibrosis (CF) patients are phenotypically and genotypically diverse. Using strain PAO1 whole genome DNA microarrays, we assessed the genomic variation in P. aeruginosa strains isolated from young children with CF (6 months to 8 years of age) as well as from the environment. Eighty-nine to 97% of the PAO1 open reading frames were detected in 20 strains by microarray analysis, while subsets of 38 gene islands were absent or divergent. No specific pattern of genome mosaicism defined strains associated with CF. Many mosaic regions were distinguished by their low G + C content; their inclusion of phage related or pyocin genes; or by their linkage to a vgr gene or a tRNA gene. Microarray and phenotypic analysis of sequential isolates from individual patients revealed two deletions of greater than 100 kbp formed during evolution in the lung. The gene loss in these sequential isolates raises the possibility that acquisition of pyomelanin production and loss of pyoverdin uptake each may be of adaptive significance. Further characterization of P. aeruginosa diversity within the airways of individual CF patients may reveal common adaptations, perhaps mediated by gene loss, that suggest new opportunities for therapy.

Adaptation, Physiological↗
PubMed1999

Characteristics of sheep-rumen isolates of Pseudomonas aeruginosa inhibitory to the growth of Escherichia coli O157.

Screening facultative sheep-rumen bacteria which inhibit growth of Escherichia coli produced 11 strains of Pseudomonas aeruginosa. The isolates showed three different pulsed-field gel electrophoresis patterns and strains from different sheep produced pyocins that varied in strain specificity. Representative strains were resistant to ampicillin, methicillin, erythromycin, fusidic acid and augmentin, but not to tetracycline or nalidixic acid. Tested strains attached in large numbers to cultured rumen epithelial cells, potentially providing a means of survival in this ecosystem.

Animals↗
PubMed1974

Resistance of Pseudomonas aeruginosa to gentamicin and related aminoglycoside antibiotics.

This study was undertaken to investigate biochemical, genetic, and epidemiological aspects of resistance to aminoglycoside antibiotics among 650 consecutive isolates of Pseudomonas aeruginosa from Parkland Memorial Hospital, Dallas, Tex. In 364 strains, minimal inhibitory concentrations were 25 mug/ml or greater for gentamicin (G), tobramycin (T) or kanamycin (K). Four patterns of resistance were noted: (A) G, T, K (four strains), (B) G, K (23 strains), (C) T, K (one strain), and (D) K (336 strains). Gentamicin acetyltransferase (GAT) activities were associated with resistance to gentamicin in strains of groups A and B, whereas kanamycin phosphotransferase activity was found in strains of group D. The GAT from group B strains acetylates both gentamicin and tobramycin. Resistance to gentamicin and susceptibility to tobramycin may reflect the fact that the K(m)'s for tobramycin (25 to 44 mug/ml) of GAT activities in these group B strains are much greater than the K(m)'s for gentamicin (1.9 to 2.7 mug/ml) and exceed the minimal inhibitory concentrations for tobramycin (1.25 to 7.5 mug/ml). GAT from strains of group A was associated with resistance to G, T, and K. Gentamicin acetyltransferases can be distinguished by their specificities for aminoglycoside substrates. The substrate specificity of GAT from group B strains is similar to that reported for GAT(I), but the specificity of GAT from group A strains differs from those described for GAT(I) and GAT(II). Conjugal transfer of gentamicin or tobramycin resistance from our strains of P. aeruginosa to various potential recipient strains was not observed. Pyocin typing showed that many, but not all, of the strains resistant to gentamicin were similar, and retrospective epidemiological investigation revealed that these strains were isolated almost exclusively from patients in the adult and pediatric burn intensive care units and geographically continguous areas of the hospital.

Acetyltransferases↗
PubMed1974

Agricultural plants and soil as a reservoir for Pseudomonas aeruginosa.

Pseudomonas aeruginosa was detected in 24% of the soil samples but in only 0.13% of the vegetable samples from various agricultural areas of California. The distribution of pyocin types of soil and vegetable isolates was similar to that of clinical strains, and three of the soil isolates were resistant to carbenicillin. Pseudomonas aeruginosa multiplied in lettuce and bean under conditions of high temperature and high relative humidity (27 C and 80-95% relative humidity) but declined when the temperature and humidity were lowered (16 C, 55-75% relative humidity). The results suggest that soil is a reservior for P. aeruginosa and that the bacterium has the capacity to colonize plants during favorable conditions of temperature and moisture.

California↗
PubMed1975

Preliminary studies of fluorescent pseudomonads capable of growth at 41 C in swimming pool waters.

During the summer of 1973 cultures of Pseudomonas aeruginosa and other fluorescent pseudomonads capable of growth at 41 C were obtained from swimming pool waters at a training center for the mentally retarded. Isolates were subjected to selected physiological tests, pyocine typing, and immunotyping. High counts of P. aeruginosa or other fluorescent pseudomonads consisted mainly of single predominant types. Both P. aeruginosa strains and unidentified fluorescent Pseudomonas strains predominated in pool waters at different times.

Anti-Bacterial Agents↗
PubMed1977

Bacteriocin typing by leakage of ultraviolet light-absorbing material.

A rapid and reproducible method of bacteriocin typing is described based on leakage of ultraviolet light-absorbing material (UVAM), detectable in supernatants of bacteriocin-sensitive cultures, by means of a spectrophotometer. The prerequisites for reproducible results, with nonsignificant fluctuations in standard error of the mean, are: a set of standardized bacteriocins, produced under defined conditions and of determined strength. These must interact with the unknown bacterial culture in suspension and at a given ratio in order to achieve an optimal multiplicity of interaction. Pyocin and colicin typing by the "scrape and streak" technique of Gillies (J. Hyg. 62:1-10, 1963) was compared with the UVAM leakage method in 275 tests; the two tests were found to be in good agreement for the strains tested.

Bacteria↗
PubMed1978

Biological activities of pyochelins: iron-chelating agents of Pseudomonas aeruginosa.

Strains of Pseudomonas aeruginosa able to grow readily in serum (serum resistant) produce siderophores in large quantity, enabling them to extract iron from transferrins. The term pyochelin has been proposed for this group of compounds. Pyochelin extractable with ethyl acetate and designated pyochelin A appears to be a mixture of catechols and other phenolates. The structures of water-soluble siderophores, designated pyochelin B, have not been determined. Pyochelins enabled growth in serum of strains of serum-sensitive P. aeruginosa and other gram-negative bacilli. Serum-resistant strains of P. aeruginosa tended to be more virulent than equally toxigenic strains of the serum-sensitive group. However, incorporation of pyochelins into the inocula of serum-sensitive strains could reduce, rather than enhance, their virulence. Utilization of pyochelins by serum-sensitive strains of P. aeruginosa rendered some of these organisms resistant to pyocins which were otherwise lethal to them.

Bacteria↗
PubMed1993

Cloning of a gonococcal DNA sequence that complements the lipooligosaccharide defects of Neisseria gonorrhoeae 1291d and 1291e.

An isogenic set of gonococcal lipooligosaccharide (LOS) mutants derived from pyocin treatment of Neisseria gonorrhoeae 1291 was used to identify cloned gonococcal DNA fragments. A gene bank from N. gonorrhoeae 1291c chromosomal DNA was made in pLEE10, a shuttle vector that replicates in the gonococcus and Escherichia coli. A plasmid (pSG30) that could transform the LOS mutants 1291d and 1291e to reactivity with monoclonal antibody 3F11 and to production of an LOS component with migration identical to that of the parent, 1291, was identified. pSG30 contains a 9-kb EcoRI fragment. Curing studies indicate that pSG30 encodes gene products that affect LOS biosynthesis in trans. Subcloning identified a 2.6-kb HincII fragment (pSG38) that retained the ability to modify the LOS of 1291d and 1291e. The DNA regions involved in modification of 1291d and 1291e were named lsi-4 and lsi-5, respectively. The region of pSG38 that was involved in LOS modification was further localized by the construction of exonuclease III deletion plasmids. Transformation of these constructs identified a 750-bp fragment that retains the ability to modify 1291e and a 540-bp fragment which retains the ability to modify 1291d.

Base Sequence↗
PubMed2000

Construction and characterization of Haemophilus ducreyi lipooligosaccharide (LOS) mutants defective in expression of heptosyltransferase III and beta1,4-glucosyltransferase: identification of LOS glycoforms containing lactosamine repeats.

To begin to understand the role of the lipooligosaccharide (LOS) molecule in chancroid infections, we constructed mutants defective in expression of glycosyltransferase genes. Pyocin lysis and immunoscreening was used to identify a LOS mutant of Haemophilus ducreyi 35000. This mutant, HD35000R, produced a LOS molecule that lacked the monoclonal antibody 3F11 epitope and migrated with an increased mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Structural studies indicated that the principal LOS glycoform contains lipid A, Kdo, and two of the three core heptose residues. HD35000R was transformed with a plasmid library of H. ducreyi 35000 DNA, and a clone producing the wild-type LOS was identified. Sequence analysis of the plasmid insert revealed one open reading frame (ORF) that encodes a protein with homology to the WaaQ (heptosyltransferase III) of Escherichia coli. A second ORF had homology to the LgtF (glucosyltransferase) of Neisseria meningitidis. Individual isogenic mutants lacking expression of the putative H. ducreyi heptosyltransferase III, the putative glucosyltransferase, and both glycosyltransferases were constructed and characterized. Each mutant was complemented with the representative wild-type genes in trans to restore expression of parental LOS and confirm the function of each enzyme. Matrix-assisted laser desorption ionization mass spectrometry and SDS-PAGE analysis identified several unique LOS glycoforms containing di-, tri-, and poly-N-acetyllactosamine repeats added to the terminal region of the main LOS branch synthesized by the heptosyltransferase III mutant. These novel H. ducreyi mutants provide important tools for studying the regulation of LOS assembly and biosynthesis.

Amino Sugars↗
PubMed1994

Role of phosphoglucomutase in lipooligosaccharide biosynthesis in Neisseria gonorrhoeae.

A region of pSG30 that complements the pyocin-derived gonococcal lipooligosaccharide (LOS) mutants 1291d and 1291e was characterized by DNA sequence analysis and an open reading frame of 1,380 bases was identified that is 89% similar and 56% identical over 452 amino acids to the algC gene product from Pseudomonas aeruginosa that encodes phosphomannomutase. Enzymatic analysis of gonococcal crude protein extracts demonstrated that pSG30 encodes phosphoglucomutase (PGM) and phosphomannomutase activity. This activity is absent in 1291d and 1291e but is restored upon introduction of pSG30. PGM encoded by pSG34, a subclone of pSG30, was able to complement Escherichia coli PGM1, a strain deficient in PGM, as determined by bacteriophage C21 plaque formation. A revertant of 1291d that binds monoclonal antibody 2-1-L8 (specific for a 3.6-kDa LOS component) was isolated. The construction of a site-specific deletion of this region in the chromosome of 1291 confirms the role of this open reading frame in LOS biosynthesis.

Amino Acid Sequence↗
PubMed1996

Cloning, complementation, and characterization of an rfaE homolog from Neisseria gonorrhoeae.

Neisseria gonorrhoeae WS1 is a spontaneous pyocin (a bacteriocin produced by Pseudomonas aeruginosa)-resistant mutant of N. gonorrhoeae FA19 that produces a truncated lipooligosaccharide (LOS) and is non-transformable. The LOS-specific mutation in WS1 was moved into a transformable background by transforming FA19 with chromosomal DNA from WS1 (generating strain JWS-1). A clone (pJCL2) capable of restoring JWS-1 to wild-type LOS expression, as detected by its acquisition of reactivity with monoclonal antibodies and by its complemented sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile, was isolated. Sequential unidirectional deletion and DNA sequence analysis of pJCL2 identified an open reading frame, designated lsi-7, that could complement the defect in JWS-1. Homology searches against various databases indicated that lsi-7 bad homology with several Escherichia coli genes involved in the phosphorylation of sugars. lsi-7 is adjacent to the lsi-6 gene, another gene involved in LOS biosynthesis. Complementation studies using Salmonella typhimurium lipopolysaccharide mutants showed lsi-6 and lsi-7 to be gonococcal homologs of S. typhimurium rfaD and rfaE, respectively. Reverse transcriptase PCR analysis demonstrated that lsi-6 and lsi-7 are part of the same transcriptional unit.

Bacterial Proteins↗
PubMed1975

Epidemiology of Pseudomonas aeruginosa in a general hospital: a four-year study.

A retrospective study was done to determine the epidemiology of infection and/or colonization due to Pseudomonas aeruginosa in a Brazilian general hospital. In 1966, 1968, and 1969, there were only two instances where probable cross-contamination was shown; the remaining isolates were unrelated. In late 1971 the hospital experienced a marked increase in P. aeruginosa isolation. Contaminated dextrose solutions used in the infant feeding were the apparent cause of the problem which occurred in the premature and special care nurseries. A contaminated oxygen humidifying bottle was the source of a different outbreak in surgery. There was also evidence in four instances that cross-infection and/or contamination had occurred. Pyocin and serological typing revealed that many strains were involved and led to a clear understanding of the complex epidemiological relationships among all the strains.

Adult↗
PubMed1988

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.

Bacteriophage Typing↗
PubMed1995

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.

Adolescent↗