Fourteen-year survival of Pseudomonas cepacia in a salts solution preserved with benzalkonium chloride.

A strain of Pseudomonas cepacia that survived for 14 years (1963 to 1977) as a contaminant in an inorganic salt solution which contained commercial 0.05% benzalkonium chloride (CBC) as an antimicrobial preservative, was compared to a recent clinical isolate of P. cepacia. Ammonium acetate was present in the concentrated stock CBC solution, and served as a carbon and nitrogen source for growth when carried over into the salts solution with the CBC. The isolate's resistance to pure benzalkonium chloride was increased step-wise to a concentration of 16%. Plate counts showed 4 x 10(3) colony-forming units per ml in the salts solution. Comparison of growth rates, mouse virulence, antibiotics resistance spectra, and substrate requirements disclosed no differences between the contaminant and a recently isolated clinical strain of P. cepacia. The results indicate that it is critical that pharmaceutical solutions containing benzalkonium chloride as an antimicrobial preservative be formulated without extraneous carbon and nitrogen sources or be preserved with additional antimicrobial agents.     

Evaluation of the preservative properties of Thymus vulgaris essential oil in topically applied formulations under a challenge test

The preservative properties of thyme essential oil (3%) with a known composition were evaluated in two types of final formulations, suitable for use as pharmaceutical or cosmetic vehicles, by means of a standard challenge test proposed by the latest European Pharmacopoeia. The required preservation efficacy criteria were satisfied against the bacterial strains, against the yeast in one of the formulations, but not against the mould strain involved in this study. Interactions between the essential oil compounds and other factors present in the final formulation might have influenced the activity of this essential oil, leading to an incomplete satisfaction of the criteria.     

Fourteen-year survival of Pseudomonas cepacia in a salts solution preserved with benzalkonium chloride.

A strain of Pseudomonas cepacia that survived for 14 years (1963 to 1977) as a contaminant in an inorganic salt solution which contained commercial 0.05% benzalkonium chloride (CBC) as an antimicrobial preservative, was compared to a recent clinical isolate of P. cepacia. Ammonium acetate was present in the concentrated stock CBC solution, and served as a carbon and nitrogen source for growth when carried over into the salts solution with the CBC. The isolate's resistance to pure benzalkonium chloride was increased step-wise to a concentration of 16%. Plate counts showed 4 x 10(3) colony-forming units per ml in the salts solution. Comparison of growth rates, mouse virulence, antibiotics resistance spectra, and substrate requirements disclosed no differences between the contaminant and a recently isolated clinical strain of P. cepacia. The results indicate that it is critical that pharmaceutical solutions containing benzalkonium chloride as an antimicrobial preservative be formulated without extraneous carbon and nitrogen sources or be preserved with additional antimicrobial agents.     

Resistance of a strain of Pseudomonas cepacia to esters of p-hydroxybenzoic acid.

Cells of a strain of Pseudomonas cepacia were isolated from an oil-in-water emulsion containing methyl and propyl p-hydroxybenzoates (methylparaben and propylparaben) as preservative additives. This strain demonstrated the ability to destroy these additives, to utilize the propyl ester as sole carbon source, and to hydrolyze the methyl ester. When the isolate was grown on Eugon agar, exposure to the methyl ester killed 99.9% of the inoculum, but the surviving cells grew logarithmically. On the other hand, cells grown on media containing propylparaben were less susceptible when subsequently exposed to emulsions containing methylparaben. These observations demonstrate one mechanism by which microorganisms develop resistance to antimicrobial preservatives.     

Resistance of a strain of Pseudomonas cepacia to esters of p-hydroxybenzoic acid.

Cells of a strain of Pseudomonas cepacia were isolated from an oil-in-water emulsion containing methyl and propyl p-hydroxybenzoates (methylparaben and propylparaben) as preservative additives. This strain demonstrated the ability to destroy these additives, to utilize the propyl ester as sole carbon source, and to hydrolyze the methyl ester. When the isolate was grown on Eugon agar, exposure to the methyl ester killed 99.9% of the inoculum, but the surviving cells grew logarithmically. On the other hand, cells grown on media containing propylparaben were less susceptible when subsequently exposed to emulsions containing methylparaben. These observations demonstrate one mechanism by which microorganisms develop resistance to antimicrobial preservatives.     

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.     

Production and specificity of poly- and monoclonal antibodies raised against Shewanella putrefaciens

B. FONNESBECH, H. FRØKIAER, L. GRAM AND C. MOSBY JESPERSEN. 1993. Polyclonal antibodies were raised in rabbits and mice against Shewanella putrefaciens. Murine monoclonal antibodies were produced against the type strain (ATCC 8071) as well as wild type strains isolated from fish products. The specificities of four polyclonal and 12 monoclonal antibodies were tested by dot-blotting, an indirect and a competitive ELISA against 16 Gram-negative strains; including six strains of S. putrefaciens and one strain of Pseudomonas rubescens (NC 10695). All polyclonal antibodies reacted strongly with S. putrefaciens and with Ps. rubescens and cross-reacted with the nine other bacteria ( Pseudomonas spp., Aeromonas spp. and Vibrio anguillarum ). The monoclonal antibodies could be divided into three groups with different patterns of specificity. The largest group (8 monoclonal antibodies) reacted strongly with S. putrefaciens and with Ps. rubescens and showed only weak reactions with the other strains. The results confirm that Ps. rubescens should be classified as S. putrefaciens.     

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.     

Serum IgG response to Burkholderia cepacia outer membrane antigens in cystic fibrosis: Assessment of cross-reactivity with Pseudomonas aeruginosa

Abstract Burkholderia cepacia (Pseudomonas cepacia) is now recognised as an important pathogen in cystic fibrosis patients, and several reports have suggested that sputum-culture-proven colonisation occurs despite the presence of specific antibody. In an attempt to establish the use of antibody studies as diagnostic and prognostic indicators of B. cepacia infection, we have examined the IgG response to B. cepacia outer membrane proteins and lipopolysaccharide in patients also colonised with P. aeruginosa . The B. cepacia strains were grown in a modified iron-depleted chemically defined medium and outer membrane components examined by SDS-PAGE and immunoblotting. IgG antibodies were detected against B. cepacia outer membrane antigens, which were not diminished by extensive preadsorption with P. aeruginosa . The response to B. cepacia O-antigen could be readily removed by adsorption of serum either with B. cepacia whole cells or purified LPS, whereas we were unable to adsorb anti-outer membrane protein antibodies using B. cepacia whole cells. The inability to adsorb anti-outer membrane protein antibodies using B. cepacia whole cells maybe due to non-exposed surface epitopes. Several B. cepacia sputum-culture negative patients colonised with P. aeruginosa had antibodies directed against B. cepacia outer membrane protein. This study suggests that there is a specific anti- B. cepacia LPS IgG response, which is not due to antibodies cross-reactive with P. aeruginosa . Our studies indicate that much of the B. cepacia anti-outer membrane protein response is specific and not attributable to reactivity against co-migrating LPS.     

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.     

Efficiency of Calamintha officinalis essential oil as preservative in two topical product types

AIMS: To verify the efficiency of Calamintha officinalis essential oil as natural preservative in two current formulations. METHODS AND RESULTS: The 1.0 and 2.0% (v/v) C. officinalis essential oil was assayed for its preservative activity in two product types (cream and shampoo). The microbial challenge test was performed following the standards proposed by the European Pharmacopoeia Commission (E.P.) concerning topical preparations using standard micro-organisms and in addition wild strains, either in single or mixed cultures were used. The results clearly demonstrated that the C. officinalis essential oil at 2.0% concentration reduced the microbial inoculum satisfying the criterion A of the E.P. in the cream formulation and the criterion B in the shampoo formulation. Standard and wild strains showed a behaviour similar, both in cream and in shampoo formulation, with no significant difference (gerarchic variance, P > 0.05). CONCLUSION: C. officinalis essential oil confirmed its preservative properties but at higher concentration than that shown in previous studies on cetomacrogol cream. SIGNIFICANCE AND IMPACT OF THE STUDY: The nature of the formulation in which an essential oil is incorporated as preservative could have considerable effect on its efficacy.     

The role of catalases in protection of proteins against oxidation in Saccharomyces cerevisiae utilizing ethanol as a carbon source

The content of protein carbonyls and thiobarbituric acid reactive substances (TBARS) in the wild and catalase-deficient strains of the yeast Saccharomyces cerevisiae grown in glucose and ethanol media are compared. The deficient strain cells reproduced 10.6-fold slower in ethanol-containing medium. Activity of glucose-6-phosphate dehydrogenase in YWT1 cells was 1.7-fold lower when yeast are grown in ethanol, and content of protein carbonyls was 4.7-fold higher, than when they are grown in the medium with glucose. At the same time, reproduction of the wild type cells in ethanol was 2.7-fold slower and carbonyl groups of protein content was 2-fold lower, than under cultivation in glucose. TBARS content in both strains was similar when they were grown in ethanol and in glucose. It has been supposed that catalases play a certain role in the protection of S. cerevisiae proteins against oxidative modification when they are grown on the media with glucose and ethanol.     

Phylogenetic studies of the rRNA group II pseudomonads based on 16S rRNA gene sequences

Nearly complete sequences of 16S rRNA genes were determined for eight bacterial strains representing five species of the rRNA homology group II pseudomonads that are members of the beta subclass of the class Proteobacteria. Comparative analysis with published sequence data indicated that Pseudomonas andropogonis, Ps. caryophylli, Ps. gladioli pv. gladioli and Ps. cepacia aggregate in one coherent cluster at 94·2% sequence similarity; Ps. solanacearum and Ps. pickettii shared 95·3% and 92·8% similarity with Alcaligenes eutrophus in another cluster. Both clusters joined at 87·8% similarity, which is similar to that for genera in this subclass of Proteobacteria. Based on this study and on comparison with other works we suggest that these species are separated from authentic pseudomonads and constitute a new genus or possibly two related genera accommodating Ps. andropogonis, Ps. caryophylli, Ps. gladioli, Ps. cepacia, and Ps. solanacearum, Ps. pickettii and A. eutrophus, respectively. Four strains of Ps. solanacearum representing Biovars 1, 2, 3 and 4 were subdivided into two clusters at 99·1% sequence similarity, in agreement with other published phenotypic and genotypic studies. The two clusters may be potentially regarded as subspecies.     

Numerical systematics of bacteria of the genus Pseudomonas

In 290 strains of bacteria belonging to the genus Pseudomonas, 120 morphological and physiologo-biochemical characters were studied and the results obtained thereby were analyzed by the methods of numerical taxonomy using computers. The majority of strains were subdivided into 11 clusters: Ps. aeruginosa (1), Ps. putida (2), Ps. rathonis (5), Ps. syringae (8), Ps. pseudoalcaligenes (9), Ps. maltophilia (10), Ps. acidovorans (11), Ps. testosteroni (12), Ps. mendocina (13), Ps. cepacia (14), Ps. fluorescens (3). The latter cluster included also the strains identified earlier as Ps. aurantiaca, Ps. lemonnieri, Ps. fluoro-violaceus, and Ps. aureofaciens. Three clusters contained strains which could not be identified and probably should be regarded as distinct species. The characteristics have been selected useful for diagnostics of the above Pseudomonas bacteria and the subgroups of Ps. fluorescens.     

A modified titration technique for the evaluation of preservative effectiveness in liquid pharmaceutical preparations

The original titration technique proposed for testing preservatives in cosmetics was modified and used for the evaluation of preservative efficiency in liquid pharmaceutical preparations. The modified technique has demonstrated that while a product is bactericidal to a small microbial challenge, it can be bacteriostatic or even nutritional to a larger challenge level.     

Factors contributing to the survival of a strain of Pseudomonas cepacia in chlorhexidine solutions

Adsorption of Pseudomonas cepacia onto the surface of glass containers enables cells to survive in the presence of user strength chlorhexidine (0.05% w/v). The organisms were found to be firmly attached to the glass and colonised glass slides could be used to infect uncontaminated chlorhexidine solutions. The efficacy of chlorhexidine against Ps. cepacia decreased with decreasing pH. Storing infected solutions at low temperatures reduced the toxicity of chlorhexidine and promoted longer survival.     

Plasmids of Pseudomonas cepacia strains of diverse origins.

Thirty-seven strains of Pseudomonas cepacia from clinical, pharmaceutical-industrial, and environmental origins were analyzed for the presence of plasmid DNA by a modification of the rapid alkaline extraction method of Birnboim (H. C. Birnboim, Methods Enzymol. 100:243-255, 1983). Plasmids were present in 31 strains (84%) from all sources, with no one source showing less than 75% plasmid carriage among its strains. The plasmid profiles indicated that the presence of large plasmids (146 to 222 kb) was the norm. Those strains with greater antibiotic resistance were mainly in the clinical and pharmaceutical groups and carried large plasmids (222 kb) that appeared essentially identical by restriction digest analysis. The ability for conjugative transfer was shown with the broad-host-range plasmid R751 carrying the gene for resistance to trimethoprim, one of the few antimicrobial agents effective against P. cepacia. The plasmid was transferred from Pseudomonas aeruginosa to P. cepacia strains as well as from P. cepacia transconjugants to other P. cepacia strains.     

Plasmids of Pseudomonas cepacia strains of diverse origins.

Thirty-seven strains of Pseudomonas cepacia from clinical, pharmaceutical-industrial, and environmental origins were analyzed for the presence of plasmid DNA by a modification of the rapid alkaline extraction method of Birnboim (H. C. Birnboim, Methods Enzymol. 100:243-255, 1983). Plasmids were present in 31 strains (84%) from all sources, with no one source showing less than 75% plasmid carriage among its strains. The plasmid profiles indicated that the presence of large plasmids (146 to 222 kb) was the norm. Those strains with greater antibiotic resistance were mainly in the clinical and pharmaceutical groups and carried large plasmids (222 kb) that appeared essentially identical by restriction digest analysis. The ability for conjugative transfer was shown with the broad-host-range plasmid R751 carrying the gene for resistance to trimethoprim, one of the few antimicrobial agents effective against P. cepacia. The plasmid was transferred from Pseudomonas aeruginosa to P. cepacia strains as well as from P. cepacia transconjugants to other P. cepacia strains.     

Construction of species-specific primers for Pseudomonas andropogonis based on 16S rDNA sequences

Approximately 94% of the total 16S rDNA of Pseudomonas andropogonis strain ACH 01053A was sequenced and compared with that of strain ATCC 23061 obtained from the GenBank database. The two sequences were highly homologous with 1·3% difference. Alignment of sequences with those from closely related bacteria revealed two possible regions for design of a specific primer suitable for detection of Ps. andropogonis . Using primers designed to these variable regions in a PCR test, an amplification product of approximately 410 bp was specifically produced by 40 strains of Ps. andropogonis . No other bacterial species showed an amplification product under optimized PCR conditions. As few as 1000 cells per reaction were detected.