Fatty acid profile and acid phosphatase activity of fresh isolates of Pseudomonas pseudomallei.

Eighty-one fresh isolates of Pseudomonas pseudomallei from melioidosis patients were subjected to the analysis for the fatty acid composition by gas-liquid chromatography (GLC) and pH-dependent pattern of nonspecific phosphatase activity. All the test strains were identical in the GLC profile showing the three peaks of characteristic hydroxy acids (3-OH 14:0, 2-OH 16:0, 3-OH 16:0) and the two prominent peaks of cyclopropane acids (17:0 delta, 19:0 delta). They had also basically the same pH-dependent curves of the enzymatic activity with paranitrophenyl phosphate as substrate, showing two to three peaks or shoulders only in the acidic side of the curve. These two biochemical characteristics could differentiate P. pseudomallei distinctly from P. aeruginosa, but not from P. cepacia.     

Growth and survival of Pseudomonas pseudomallei in acidic environments.

A study was made on the growth and survival of Pseudomonas pseudomallei in culture environments differing in nutrients, initial pH, and aeration, in comparison with Pseudomonas cepacia and Pseudomonas aeruginosa. The observations led us to a view that P. pseudomallei has the highest adaptability to acidic environments among the three species. Unlike the other species, it grew in heart infusion broth of initial pH 4.5 under aeration and survived keeping a high level (10(9) per ml) of viable counts for as long as 30 days. This sort of adaptation was found to be more evident in the media of poor nutrition and under limited aeration.     

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.     

High activity of acid phosphatase of Pseudomonas pseudomallei as a possible attribute relating to its pathogenicity

Phosphatase activities were compared quantitatively among selected species of pseudomonads. P. pseudomallei showed the highest activity of a bell-shaped pH pattern with a peak at around pH 5.0. P. cepacia had a similar pattern of milder intensity. In contrast, P. aeruginosa revealed an alkaline phosphatase activity with a pH optimum higher than 8.0, but the level of activity was much lower than those of the above two species. The enzymatic reactions of other species were slight or negligible at their optimum pH in the same test system. These data were discussed in reference to their growth behavior in different pH environments and also in connection with such recent information that the high activity of microbial acid phosphatase may be a favorable attribute to their intracellular parasitism.     

Endotoxin stimulates bronchial epithelial cells to release chemotactic factors for neutrophils. A potential mechanism for neutrophil recruitment, cytotoxicity, and inhibition of proliferation in bronchial inflammation.

To test the effect of endotoxin on bronchial epithelial cells (BEC), BEC were isolated from bovine lungs and cultured in the presence of bacterial endotoxin. The BEC culture supernatant fluids were harvested, and neutrophil chemotactic activity (NCA) was determined with a blindwell chamber technique; cytotoxicity determined by lactate dehydrogenase release and BEC proliferation determined by Coulter counting. Endotoxin caused a dose- and time-dependent release of NCA from BEC cultures compared with media alone (82.3 +/- 8.1 vs 12.0 +/- 3.1 cells/high power field, p less than 0.001). To further characterize this activity, reverse phase HPLC analysis of release eicosanoid metabolites after [3H]arachidonic acid incorporation was performed. Endotoxin stimulated the release of the neutrophil chemoattractants, leukotriene B4 and 12-hydroxyeicosatetraenoic acids. Endotoxin also resulted in a dose and time dependent release of lactate dehydrogenase (42.9 +/- 4.2 vs 20.2 +/- 2.2 U/liter, p less than 0.001) although higher doses were required to cause cytotoxicity than to stimulate chemotaxis. Finally, endotoxin resulted in a dose dependent inhibition of BEC proliferation (176 x 10(3) +/- 16 x 10(3) vs 1,080 x 10(3) +/- 38 x 10(3) cells/ml measured at day 14, p less than 0.001). These data suggest that bacterial release of endotoxin may contribute to the pathophysiologic changes observed in bronchial inflammation by stimulating BEC to release NCA, denuding airway epithelium by causing cytotoxicity of BEC, and inhibiting epithelial repair by inhibiting BEC proliferation.     

Immunological and biological relationship among flagellin of Pseudomonas aeruginosa, Burkholderia cepacia and Stenotrophomonas maltophilia

The flagellar protein (flagellin) was isolated and purified from strains of Pseudomonas aeruginosa, Burkholderia cepacia and Stenotrophomonas maltophilia. A significant difference was observed in the molecular weight of different flagellin preparations obtained from these bacterial isolates. Antiserum prepared against S. maltophilia flagellin did not react with flagellin of P. aeruginosa or/and B. cepacia on Immunoblot or in indirect ELISA. In addition the anti-flagellin did not agglutinate P. aeruginosa and B. cepacia. No inhibition of motility of P. aeruginosa and B. cepacia was observed in presence of antiserum; though the latter inhibited the motility of S. maltophilia. The results of the present study prove that no specific relationship existed among all the studied flagellar proteins obtained from closely related bacteria.     

Immigration and emigration of Burkholderia cepacia and Pseudomonas aeruginosa between and within mixed biofilm communities

AIMS: To investigate the dynamics of binary culture biofilm formation through use of both the Sorbarod model of biofilm growth and the constant depth film fermenter (CDFF). METHODS AND RESULTS: Pseudo steady-state biofilm cultures of laboratory and clinical strains of Pseudomonas aeruginosa, selected on the basis of their ability to produce a Burkholderia cepacia growth-inhibitory substance, were established on Sorbarod filters and challenged with corresponding planktonic grown cultures of B. cepacia. Reverse challenges were also conducted. Both B. cepacia and P. aeruginosa were able to form steady-state monoculture biofilms after 48 h growth. When steady-state biofilms of B. cepacia NTCT 10661 were challenged with planktonically grown P. aeruginosa PAO1 known to produce a B. cepacia growth-inhibitory substance, the immigrant population was rapidly and almost completely bound to the biofilm, displacing B. cepacia. By contrast, established biofilms of P. aeruginosa PAO1 resisted immigration of B. cepacia 10661. Similar experiments conducted with a nongrowth inhibitory substance producing clinical pairing of P. aeruginosa 313113 and B. cepacia 313113 led to the formation of stable, mixed biofilm populations in both instances. Moreover, co-inoculation with these clinical isolates resulted in a stable, mixed steady-state biofilm. Similar observations were made for biofilms generated in CDFFs. In such instances following pan-swapping between two monoculture CDFFs, B. cepacia 313113 was able to integrate into an established P. aeruginosa 313113 biofilm to form a stable binary biofilm. CONCLUSIONS: Establishment of a mixed species community follows a specific sequence of inoculation that may either be due to some degree of match between co-colonizers or that P. aeruginosa predisposes uncolonized sections of the surface to permit B. cepacia colonization. SIGNIFICANCE AND IMPACT OF THE STUDY: Colonization of a surface with one bacterial species confers colonization resistance towards other species. Disinfection of a surface might well increase the probability of pathogen harbourage.     

Comparative analysis of total cell protein electrophoregram of pathogenic Burkholderia

Whole-cell proteins of 22 strain of Burkhoderia pseudomallei, including 13 B. mallei, 5 B. cepacia strains and 14 strains of opportunistically pathogenic Pseudomonas defined by 1D SDC-PAAG electrophoresis. Electrophoregrams contained 35 to 45 protein fractions sized 19 to 130 kDa, which were highly reproductive. On the basis of computer-aided comparative analysis of protein patterns the interspecies and intraspecies grouping of studied microorganisms was made. The cluster analysis of the similarity matrix of protein spectra made it possible to allocate two groups of strains at the level of similarity of 78%. Group I was formed by Burkholderia species that previously belonged to the II RNA-DNA homology group of Pseudomonas: B. pseudomallei, B. mallei, B. cepacia. All Pseudomonas species were added to the 2nd Group: P. aeruginosa, P. stutzeri, P. testosterone, P. fluorescens, P. putida, P. mendocina. Four phenons were isolated among the strains of B. pseudomallei and 2 phenons--among the strains of B. mallei at the threshold similarity level (89%). The authors conclude that the comparative analysis of electrophoregrams of whole-cell proteins can be useful in the identification and typing of pathogenic Burkholderia.     

Substrate response in acid phosphatase activity of Pseudomonas pseudomallei and Pseudomonas cepacia, with special reference to tyrosine phosphatase.

The substrate response in acid phosphatase activity of Pseudomonas pseudomallei and Pseudomonas cepacia was examined with different phosphate esters including hexose phosphates and phosphoaminoacids in a whole cell assay system. The enzymatic activity against each substrate was evaluated in terms of percent activity to that against para-nitrophenyl phosphate set as 100. A remarkable finding was that the phosphatase reaction was the highest with phosphotyrosine or phosphoserine as substrate showing 180% activity. This tyrosine phosphatase activity was resistant to heating at 60 C for 20 min and inhibited greatly by 0.1% ZnCl2. Pseudomonas cepacia showed the same pattern of substrate response and the same characteristics of tyrosine phosphatase activity.     

Burkholderia spp. alter Pseudomonas aeruginosa physiology through iron sequestration

Pseudomonas aeruginosa and members of the Burkholderia cepacia complex often coexist in both the soil and the lungs of cystic fibrosis patients. To gain an understanding of how these different species affect each other's physiology when coexisting, we performed a screen to identify P. aeruginosa genes that are induced in the presence of Burkholderia: A random gene fusion library was constructed in P. aeruginosa PA14 by using a transposon containing a promoterless lacZ gene. Fusion strains were screened for their ability to be induced in the presence of Burkholderia strains in a cross-streak assay. Three fusion strains were induced specifically by Burkholderia species; all three had transposon insertions in genes known to be iron regulated. One of these fusion strains, containing a transposon insertion in gene PA4467, was used to characterize the inducing activity from Burkholderia: Biochemical and genetic evidence demonstrate that ornibactin, a siderophore produced by nearly all B. cepacia strains, can induce P. aeruginosa PA4467. Significantly, PA4467 is induced early in coculture with an ornibactin-producing but not an ornibactin-deficient B. cepacia strain, indicating that ornibactin can be produced by B. cepacia and detected by P. aeruginosa when the two species coexist.     

Interspecies biofilms of Pseudomonas aeruginosa and Burkholderia cepacia.

The leading cause of morbidity and mortality in cystic fibrosis (CF) continues to be lung infections with Pseudomonas aeruginosa biofilms. Co-colonization of the lungs with P aeruginosa and Burkholderia cepacia can result in more severe pulmonary disease than P. aeruginosa alone. The interactions between P. aeruginosa biofilms and B. cepacia are not yet understood; one possible association being that mixed species biofilm formation may be part of the interspecies relationship. Using the Calgary Biofilm Device (CBD), members of all genomovars of the B. cepacia complex were shown to form biofilms, including those isolated from CF lungs. Mixed species biofilm formation between CF isolates of P. aeruginosa and B. cepacia was readily achieved using the CBD. Oxidation-fermentation lactose agar was adapted as a differential agar to monitor mixed biofilm composition. Scanning electron micrographs of the biofilms demonstrated that both species readily integrated in close association in the biofilm structure. Pseudomonas aeruginosa laboratory strain PAO1, however, inhibited mixed biofilm formation of both CF isolates and environmental strains of the B. cepacia complex. Characterization of the soluble inhibitor suggested pyocyanin as the active compound.     

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.     

Characterization of Pseudomonas Species Isolated from Clinical Specimens

More than 90 morphological and physiological characters of 227 strains of pseudomonads isolated from clinical specimens and 16 reference strains are described. The clinical isolates included P. aeruginosa (apyocyanogenic), P. fluorescens, P. putida, P. pseudomallei, P. cepacia, P. acidovorans, P. alcaligenes, P. pseudoalcaligenes, P. stutzeri, P. putrefaciens, P. maltophilia, and P. diminuta.     

The influence of bacteria of the Burkholderia cepacia and Pseudomonas aeruginosa complex on cell-mediated immune reactions in experimental animals

The evidence has been obtained that various species, as well as individual strains having pathogenicity factors, produced different effect on the functional activity of immunocompetent B and T lymphocytes of mice infected intraperitoneally. The injection of live P. aerruginosa PA 103 and B. cepacia 8240 cells resulted in imunosuppression of antibody-forming cells, synthesizing antibodies to heterologous antigens. On the contrary, in the animals infected with B. cepacia 8236 the functional activity of B lymphocytes increased. An increase in the proliferative activity of spleen cells was noted in the presence of T and B mitogens after the infection of mice with P. aeruginosa PA 103 in comparison with B. cepacia 8236 and B. cepacia 8240 which produced a faintly pronounced modulating effect. The pathogenesis mechanisms of infections induced by these microorganisms as well as the development of chronic, persisting forms of the infectious process are discussed.     

Iodine sensitivity of bacteria isolated from iodinated water systems

Fourteen bacterial isolates, predominantly Pseudomonas sp., from two water systems disinfected by iodinated anion-exchange resins were studied and compared with an isolate of Pseudomonas aeruginosa from a povidone-iodine solution and four other isolates. Pseudomonas cepacia and P. aeruginosa grown in brain heart infusion were 3 to 5 logs less sensitive to 1 mg/L I2 (pH 7.2, 1 min) when compared with cultures grown in phosphate buffer. Another P. cepacia isolate was the least sensitive culture when grown in brain heart infusion (1 log decrease) but was more sensitive after cultivation in phosphate buffer (5 logs). Isolates from an iodinated potable water system, including P. cepacia, Staphyloccus warneri, and a Bacillus sp., were all less sensitive to iodine than a "resistant" P. aeruginosa and three other isolates when grown in brain heart infusion. A clinical isolate of P. aeruginosa exhibited intermediate sensitivity. The sensitivity of bacteria to iodine is thus highly variable, depending on the organism as well as the growth conditions.     

Study of the structure and function of endotoxins with the aid of enzymes. 2. Modification of Salmonella paratyphi B endotoxin with lysozyme and lipase]

Endotoxin preparations from the S. paratyphi B cultures, isolated by various methods, were treated with lysozyme (splitting of beta-1,4-glycoside links of lipid A) and lipase of the pancreas (splitting of complex ester links of glycerophosphatides). Lysozyme and, to a lesser extent, lipase, were capable of partial depression of the toxic endotoxin function. The process of enzymatic detoxication coursed selectively, without influencing the serological and immunological activity of the preparations. Suppositions are put forward on the complicated nature of the toxic endotoxin function manifestation and possibility of provision of detoxication effect by specific actions differing by the point of application.     

Electron microscope study of the effect of benzalkonium, chlorhexidine and polymyxin on Pseudomonas cepacia

Electron micrographs of Pseudomonas cepacia cell grown in nutrient broth show an external membrane which is distinctly wavy, when compared with similar preparations of Pseudomonas aeruginosa, and which is not affected by growing in the presence of broth containing benzalkonium (10 microgram/ml), chlorhexidine (10 microgram/ml) or polymyxin (25 units/ml). Both benzalkonium (10 microgram/ml) and chlorhexidine (10 microgram/ml) damage the cytoplasmic membrane of P. cepacia cell grown in the presence of the chemicals. Contrasts are shown between the effect of polymyxin (chlorhexidine and benzalkonium) on the outer membrane of P. cepacia and P. aeruginosa.     

A study of action of subinhibiting concentrations of antibiotics on the expression of genes regulating production of pathogenicity factors in Burkholderia cepacia and Pseudomonas aeruginosa

Described in the paper are the results of a study of differential expression of pathogenicity of B. cepacia and P. aeruginosa in subinhibiting concentrations (SIC) of cyprofloxacyne. While identifying genes expressing differentially in the antibiotic SIC, genes cepR B. cepacia and P. aeruginosa expressing without cyprofloxacyne in the cultivation medium and not expressing in the antibiotic SIC were detected. Finally, involvement of cepR B. cepacia in regulating the pathogenicity expression factors according to "quorum sensing" is under discussion.     

Regulation of endotoxin-induced proinflammatory activation in human coronary artery cells: expression of functional membrane-bound CD14 by human coronary artery smooth muscle cells

Low-level endotoxemia has been identified as a powerful risk factor for atherosclerosis. However, little is known about the mechanisms that regulate endotoxin responsiveness in vascular cells. We conducted experiments to compare the relative responses of human coronary artery endothelial cells (HCAEC) and smooth muscle cells (HCASMC) to very low levels of endotoxin, and to elucidate the mechanisms that regulate endotoxin responsiveness in vascular cells. Endotoxin (10-fold higher in magnitude at >10-fold lower threshold concentrations (10-30 pg/ml) compared with HCAEC. This remarkable sensitivity of HCASMC to very low endotoxin concentrations, comparable to that found in circulating monocytes, was not due to differential expression of TLR4, which was detected in HCAEC, HCASMC, and intact coronary arteries. Surprisingly, membrane-bound CD14 was detected in seven different lines of HCASMC, conferring responsiveness to endotoxin and to lipoteichoic acid, a product of Gram-positive bacteria, in these cells. These results suggest that the low levels of endotoxin associated with increased risk for atherosclerosis are sufficient to produce inflammatory responses in coronary artery cells. Because CD14 recognizes a diverse array of inflammatory mediators and functions as a pattern recognition molecule in inflammatory cells, expression of membrane-bound CD14 in HCASMC implies a potentially broader role for these cells in transducing innate immune responses in the vasculature.     

Burkholderia pseudomallei, B. thailandensis, and B. ambifaria produce 4-hydroxy-2-alkylquinoline analogues with a methyl group at the 3 position that is required for quorum-sensing regulation

4-Hydroxy-2-alkylquinolines (HAQs), especially 3,4-dihydroxy-2-heptylquinoline (Pseudomonas quinolone signal) and its precursor, 4-hydroxy-2-heptylquinoline, are attracting much attention, mainly because of their role as signaling molecules in Pseudomonas aeruginosa. The pqsABCDE operon is centrally involved in their biosynthesis. The presence of a homologous operon in Burkholderia pseudomallei and B. thailandensis was recently reported. Thus, we have investigated the abilities of 11 Burkholderia species to produce HAQ-like molecules by liquid chromatography/mass spectrometry. We have identified 29 different HAQ derivatives produced by the only three Burkholderia species where a pqsABCDE homologue was found among available sequenced Burkholderia species genomes, including B. ambifaria, a member of the Burkholderia cepacia complex. In contrast with those of P. aeruginosa, Burkholderia HAQs typically bear a methyl group, hence their designation as 4-hydroxy-3-methyl-2-alkylquinolines (HMAQs). We identified three families of HMAQs with a saturated or unsaturated alkyl chain at the 2' position, in contrast with the 1' position of P. aeruginosa, including one with an N-oxide group. Furthermore, the operon in these species contains two more genes downstream of the pqsE homologue, resulting in the hmqABCDEFG operon. While the inactivation of hmqA inhibits the production of HMAQs, the methylation of the quinoline ring requires a putative methyltransferase encoded by hmqG. Interestingly, hmqA or hmqG mutations increase the production of acyl homoserine lactones and, consequently, phenotypes under the control of quorum sensing in B. ambifaria: antifungal activity, siderophore production, and proteolytic activity. These results indicate that only HAQs bearing a methyl group (HMAQs) are involved in quorum-sensing regulation.