The identification of Pseudomonas cepacia and its occurrence in clinical material

During the 19 year period ending December 1984, 4840 strains of Gram-negative non-fermentative bacteria were submitted to the National Collection of Type Cultures for identification. Of these, 195 strains (4.0% of the total) were identified as Pseudomonas cepacia which demonstrates both that the species is regularly encountered in clinical material in the UK and that several laboratories have experienced difficulty in identifying the organism. The sources from which the 195 strains were isolated are reported and also the characteristics by which the species may be recognized. The clinical significance of Ps. cepacia is reviewed, and the resistance of this species to disinfectants and antimicrobial agents commonly used to treat pseudomonas infections is discussed to underline the necessity for the precise identification of Ps. cepacia.     

Surface characteristics of Pseudomonas cepacia

Two major surface characteristics of Pseudomonas cepacia were examined in this study: reactivity with lectins and hydrophobicity. The results indicated that the surfaces of P. cepacia strains are heterogeneous with regard to the distribution of lectin receptors. Only lima bean agglutinin was found to strongly agglutinate all strains. The strains were also heterogeneous with regard to hydrophobicity as determined by adhesion to hexadecane. The degree of hydrophobicity, however, was not significantly altered when selected strains were mixed with either fibronectin or bovine serum albumin. In addition, the strains exhibited no apparent affinities for buccal epithelial cells and gave no evidence for an ability to haemagglutinate human red cells.     

Identification and distribution of Pseudomonas stutzeri in clinical material

During the 19 year period ending December 1984, 114 (2.4%) of 4840 strains of Gram-negative non-fermentative bacteria submitted, mainly by laboratories in the UK, to the National Collection of Type Cultures for computer-assisted identification were strains of Pseudomonas stutzeri. These figures suggest that Ps. stutzeri is a relatively uncommon species in clinical material in the UK but that when it does occur laboratories have difficulty in identifying it. The sources from which the strains were isolated and also characteristics of the species by which it may be recognized are reported. The clinical significance of Ps. stutzeri is discussed and also the susceptibility of this species to antimicrobial agents.     

Identification and distribution of Pseudomonas stutzeri in clinical material

During the 19 year period ending December 1984, 114 (2.4%) of 4840 strains of Gram-negative non-fermentative bacteria submitted, mainly by laboratories in the UK, to the National Collection of Type Cultures for computer-assisted identification were strains of Pseudomonas stutzeri. These figures suggest that Ps. stutzeri is a relatively uncommon species in clinical material in the UK but that when it does occur laboratories have difficulty in identifying it. The sources from which the strains were isolated and also characteristics of the species by which it may be recognized are reported. The clinical significance of Ps. stutzeri is discussed and also the susceptibility of this species to antimicrobial agents.     

Rapid differentiation of Pseudomonas pseudomallei from Pseudomonas cepacia

The Minitek disc system was utilized for the differentiation of Pseudomonas pseudomallei, the causative agent of melioidosis, from Ps. cepacia. The system was simple to use, inexpensive, and furnished rapid, clear-cut test results after 4 h. This procedure is suitable for differentiating soil bacteria presumptively identified as Ps. pseudomallei, Ps. cepacia or flavobacteria, and for the rapid confirmation of the presumptive identification of either Ps. pseudomallei or Ps. cepacia obtained by commercial identification-kit systems in the clinical laboratory.     

Bacteriocin, plasmid and pectolytic diversity in Pseudomonas cepacia of clinical and plant origin.

Pseudomonas cepacia strains of plant and clinical origin were compared with the type strains of P. cepacia, P. kingii and P. multivorans. Conventional biochemical tests and antibiotic sensitivity patterns supported the previous proposals of synonymy between P. cepacia, P. kingii and P. multivorans. However, bacteriocin production patterns, onion maceration tests and hydrolysis of low pH pectate agar clearly differentiated strains of clinical and plant origin into two distinct groups; these tests may therefore be helpful in epidemiological studies. In contrast, plant and clinical strains were of equal lethality to mice. Agarose gel electrophoresis indicated the presence of one or more plasmids (molecular weights 9 X 10(6) to 120 X 10(6)) in 15 out of 16 strains of both types examined.     

Regulation of pyrimidine biosynthesis in Pseudomonas cepacia

Pyrimidine biosynthesis was investigated in Pseudomonas cepacia ATCC 17759. The presence of the de novo pyrimidine biosynthetic pathway enzyme activities was confirmed in this strain. Following transposon mutagenesis of the wild-type cells, a mutant strain deficient for orotidine 5-monophosphate decarboxylase activity (pyrF) was isolated. Uracil, cytosine or uridine supported the growth of this mutant. Uracil addition to minimal medium cultures of the wild-type strain diminished the levels of the de novo pyrimidine biosynthetic enzyme activities, while pyrimidine limitation of the mutant cells increased those de novo enzyme activities measured. It was concluded that regulation of pyrimidine biosynthesis at the lelel of enzyme synthesis in P. cepacia was present. Aspartate transcarbamoylase activity was found to be regulated in the wild-type cells. Its activity was shown to be controlled in vitro by inorganic pyrophosphate, adenosine 5-triphosphate and uridine 5-phosphate.     

Antibiotic activity and siderophores of Pseudomonas cepacia

The ability of 46 strains of Pseudomonas cepacia to inhibit phytopathogenic fungi and the effect of iron on their antifungal activity were studied. The antifungal effect of the bacteria and the antimicrobial activity of their crude yellow and violet pigments showed a 4-5-fold decrease in the presence of Fe(III). The addition of 100 micrograms/ml of FeCl3 to the medium decreased the biosynthesis of violet and yellow pigments; the complex of the yellow pigment with Fe(III) promoted the growth of the P. cepacia producing strain under iron-deficient conditions. The data obtained suggest a participation of some P. cepacia pigments in iron transport. The resistance of the P. cepacia strains to the synthetic chelating agents hydroxyethylenediphosphonic and diethylenediaminepentaacetic acids was demonstrated, which may indicate a high Fe(III)-binding constant of P. cepacia siderophores.     

Methods of <Emphasis Type="BoldItalic">Candida dubliniensis</Emphasis> identification and its occurrence in human clinical material

Candida dubliniensis was reported as a new species in 1995. This species is often misidentified as Candida albicans. The aims of this work were to determine the occurrence of C. dubliniensis in various clinical materials, to evaluate several ways to identify it and to examine the genetic variability of isolates. Among 7706 isolates originally identified as C. albicans, 237 were identified as C. dubliniensis (3.1%). Most of the C. dubliniensis isolates were obtained from the upper and lower respiratory tract (61.4 and 22.9%). Five phenotypic methods including latex agglutination were used (cultivation on CHROMagar Candida, on Staib agar, at 42 °C and in medium with 6.5% NaCl), but only cultivation on the medium with an increased concentration of NaCl and latex agglutination gave reliable results. Species-specific polymerase chain reaction was used as the confirmation method. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry provided less reliable results. In fact, 78.9% of C. dubliniensis isolates had scores above 1.7. However, the rest of them (21.1%) were also identified as C. dubliniensis even when the scores were lower than 1.7. Divergences among C. dubliniensis strains were evaluated by means of pulsed-field gel electrophoresis. Eighty-six selected C. dubliniensis isolates showed a 69.6% level of similarity. The results of this study expand the knowledge of the incidence, means of identification and genotypic divergence of C. dubliniensis isolates.     

Comparative Zone Electrophoresis of Enzymes of Pseudomonas solanacearum and Pseudomonas cepacia

The technique of starch-gel electrophoresis with specific staining for a series of enzymes was used to compare 21 Pseudomonas strains representing both P. cepacia and P. solanacearum. These experiments produced no evidence for close similarity of the two species. Twelve strains of P. solanacearum were compared by means of data obtained from nine different enzymes, and the data indicate that these strains belong in two biotypes. Except for the assignment of two strains, these groups are the same as the two major groups previously derived from nutritional properties and from deoxyribonucleic acid hybridization experiments. Eleven enzymes were available for comparisons of the P. cepacia strains. Eight of these strains form a homogeneous group, but the last strain, number 249, differs considerably from the other representatives of the species.     

Degradation of diphenylether by Pseudomonas cepacia

The microbial degradation of hard coal implies the cleavage of diaryl ether linkages in the coal macromolecule. We investigated the biodegradation of diphenylether as a model compound representing this substructure of coal. A bacterial strain isolated from soil and identified as Pseudomonas cepacia, was able to grow with diphenylether as sole source of carbon. During microbial growth, three metabolites were detected in the culture supernatant by high pressure liquid chromatography. As product of ring hydroxylation and subsequent rearomatization, 2,3-dihydroxydiphenylether was identified by UV, mass and nuclear magnetic resonance spectrometry and gas chromatography analyses. The cleavage of the ether linkage led to the formation of phenol and 2-pyrone-6-carboxylic acid, the latter being not further degraded by Pseudomonas cepacia. The possible cleavage mechanism of the ether linkage is discussed.Non-standard abbreviations DPE diphenylether - PCA 2-pyrone-6-carboxylic acid - GC gas chromatography - MS mass spectrometry - HPLC high pressure liquid chromatography     

Physiological aspects of disinfection resistance in Pseudomonas cepacia

A Pseudomonas cepacia population was isolated which had reduced susceptibility to iodine and maintained resistance when subcultured several times in phosphate buffer. This population was also resistant to iodine after growth in a minimal medium containing glycerol but not glucose. Addition of cAMP to glucose-grown cells caused increased resistance to iodine. Iodine-resistant cultures also demonstrated reduced susceptibility to chlorination but not to heat or metals (Cu/Ag). The results indicate that halogen resistance can be expressed in varying degrees, dependent on the carbon source, and cAMP may promote this expression. Thus, a catabolite repression-like mechanism may cause resistant cultures grown in some media to become more sensitive to halogens.     

Hydroxyamino acid utilization and alpha-ketobutyrate toxicity in Pseudomonas cepacia

Growth of Pseudomonas cepacia 249 on D-threonine required a mutation to permit D-hydroxyamino acid deaminase formation and L-valine to overcome alpha-ketobutyrate toxicity. Strain 249 lacked a second D-hydroxyamino acid deaminase formed by other strains.     

Molecular cloning of salicylate hydroxylase genes from Pseudomonas cepacia and Pseudomonas putida

The sal gene encoding Pseudomonas cepacia salicylate hydroxylase was cloned and the sal encoding Pseudomonas putida salicylate hydroxylase was subcloned into plasmid vector pRO2317 to generate recombinant plasmids pTK3 and pTK1, respectively. Both cloned genes were expressed in the host Pseudomonas aeruginosa PAO1. The parental strain can utilize catechol, a product of the salicylate hydroxylase-catalyzed reaction, but not salicylate as the sole carbon source for growth due to a natural deficiency of salicylate hydroxylase. The pTK1- or pTK3-transformed P. aeruginosa PAO1, however, can be grown on salicylate as the sole carbon source and exhibited activities for the cloned salicylate hydroxylase in crude cell lysates. In wild-type P. cepacia as well as in pTK1- or pTK3-transformed P. aeruginosa PAO1, the presence of glucose in addition to salicylate in media resulted in lower efficiencies of sal expression P. cepacia apparently can degrade salicylate via the meta cleavage pathway which, unlike the plasmid-encoded pathway in P. putida, appears to be encoded on chromosome. As revealed by DNA cross hybridizations, the P. cepacia hsd and ht genes showed significant homology with the corresponding plasmid-borne genes of P. putida but the P. cepacia sal was not homologous to the P. putida sal. Furthermore, polyclonal antibodies developed against purified P. cepacia salicylate hydroxylase inactivated the cloned P. cepacia salicylate hydroxylase but not the cloned P. putida salicylate hydroxylase in P. aeruginosa PAO1. It appears that P. cepacia and P. putida salicylate hydroxylases, being structurally distinct, were probably derived through convergent evolution.     

A generalized transducing phage of Pseudomonas cepacia

A generalized transducing phage, named CP75, was derived from a lysogenic strain of Pseudomonas cepacia. The frequency of transduction per phage particle ranged from 1.0 X 10(-6) to 2.0 X 10(-6) for a given marker. About half of the 105 P. cepacia strains tested were sensitive to the phage. The molecular size of the CP75 genome was approximately 52 kb.     

Biotransformation of Benzyl Alcohol by Pseudomonas cepacia

To cDNAs encoding class I chitinases of rice were expressed in Escherichia coli. The cDNAs were fused to the MS2-polymerase gene in an expression vector, pEx31. The fusion proteins, expressed under the control of the λP_L-promoter, showed the chitinase activity independent of the existence of the hevein domain. The enzymatic hydrolysis of colloidal chitin by the fusion proteins showed that the proteins were endo-type enzymes.