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.     

Phagocytability of Pseudomonas pseudomallei

Experiments were conducted on the cell culture of macrophages of animals significantly differing by the extent of resistance to melioidosis; the presence of correlation between the extent of the animal natural immunity and the intensity of dying of the microbes in the test system was demonstrated. The causative agent of melioidosis proved to be more resistant to phagocytosis with guinea pig macrophages than E. coli. Ps. aeruginosa and A. mallei. It was impossible to establish any relationship between the efficacy of phagocytosis by animal macrophages and the virulence or morphology of the colonies in the Ps. pseudomallei species.     

Evaluation of preservative effectiveness in pharmaceutical products : the use of a wild strain of Pseudomonas cepacia

A sodium benzoate-sorbic acid preservative system of a pharmaceutical product was proved effective against a wild strain of Pseudomonas cepacia , following the official method of the Italian and British Pharmacopoeias. However, this preservative system was ineffective against a challenge of Ps. cepacia wild strain cells grown in the unpreserved pharmaceutical product and on culture media different from those described by the Pharmacopoeias. The adaptive resistance of the wild strain of Ps. cepacia was not demonstrated with a laboratory strain (ATCC 25609). In contrast, p- hydroxybenzoate-based preservative systems proved to be efficient in protecting the pharmaceutical product against a challenge of wild and laboratory strains of Ps. cepacia grown in the different conditions described above. The results obtained suggest the usefulness, in the official methods for testing pharmaceutical preservatives, of using wild microbial strains isolated from the pharmaceutical environment. Metabolic adaptive responses, capable of affecting the antimicrobial sensitivity of wild micro-organisms used to challenge the preserved product, can be detected by using cells grown in the unpreserved pharmaceutical product.     

Endotoxin of Pseudomonas pseudomallei detected by the body weight-decreasing reaction in mice and comparison of it with those of P. cepacia and P. aeruginosa.

The heat-treated whole cells, culture supernatants, and extracted endotoxin preparations of Pseudomonas pseudomallei were examined for endotoxin by the mouse body weight-decreasing (BWD) test. The experiments were conducted also with those of P. cepacia and P aeruginosa. Endotoxin was detected in all the samples of P. pseudomallei. Endotoxin of P. cepacia was detected in whole cells, but not in culture supernatant. The BWD activity of P. aeruginosa was 30 times as high as that of P. pseudomallei. This result was confirmed by the experiments with endotoxin preparations. In the limulus amebocyte lysate gelation (LAL) test, however, the endotoxin preparations of the two species showed the same level of activity.     

单克隆抗体对鼻疽和类鼻疽病原抗原及其血清学鉴别诊断的实验研究

应用抗鼻疽杆菌和抗类鼻疽杆菌的单克隆抗体(McAb),以间接ELISA,IFA以及免疫组织化学(下简称免疫组化)等技术方法,对来自不同地区的鼻疽杆菌(Ps.mallei)和类鼻疽杆菌(Ps.pseudmallei)的表面抗原进行了分析。在此基础上,又对鼻疽(Mallcus)和类鼻疽(Melioidosis)之间的血清学鉴别诊断等问题进行了研究。试验结果表明：(1)鼻疽杆菌和类鼻疽杆菌各自表达了不同的表面抗原反应类型,其闻并有一定的交叉关系;(2)鼻疽杆菌和类鼻疽杆菌各株均与McAb 2D4发生反应,说明表位2D4很可能为二菌所共有;(3)McAb4D4和lA9的类似抗体在鼻疽和类鼻疽血清都表现了较高的出现频率,说明其可能为二种血清的共有抗体成份;(4)McAb 3A1是仅同类鼻疽杆菌各株发生反应的特异性抗体。应用该McAb,以相应的实验技术,有可能解决长期以来存在的鼻疽和类鼻疽菌体间和血清间的免疫学鉴别诊断问题。     

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.     

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.     

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.     

Bacterial whole cell protein profiles of the rRNA group II pseudomonads

Studies on bacterial whole cell protein profiles showed that members of the rRNA group II pseudomonads were distinct from other non-fluorescent and fluorescent pseudomonads, including Pseudomonas aeruginosa, the type species of the genus Pseudomonas. Strains of Ps. andropogonis, Ps. caryophylli, Ps. gladioli pv. gladioli, Ps. pickettii, Ps. pseudomallei and Ps. rubrisubalbicans showed uniform and distinct protein patterns, while strains of Ps. solanacearum and Ps. cepacia displayed differences within species. Numerical analysis of their protein profiles with GelManager and Taxan programs generated dendrograms comprising 16 clusters at 89% similarity. Each cluster included strains belonging to the same species with the exception of Ps. solanacearum, which fragmented into three clusters. Pseudomonas solanacearum showed different protein patterns correlating with different biovars and the two divisions of Cook et al. (1989), as well as the results of 16S rRNA gene sequencing. The whole cell protein profiles of a total of 83 strains belonging to 14 bacterial species were numerically analysed.     

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.     

Extraction and characterization of lipopolysaccharide from Pseudomonas pseudomallei

The best yield of lipopolysaccharide (LPS) of Pseudomonas pseudomallei GIFU 12046 was obtained by extraction of defatted cells by phenol/chloroform/petroleum ether. The LPS showed a smooth character on SDS-polyacrylamide gel electrophoresis and contained D-glucose, L-glycero-D-manno-heptose, and D-glucosamine as the main sugar components, and 3-hydroxypalmitic acid as an amide-linked fatty acid. The growth conditions did not affect the electrophoresis profile and chemical composition of LPS. 2-Keto-3-deoxyoctonic acid was not detectable, and mild acid hydrolysis could not liberate free lipid A, suggesting that the linkage between inner core and lipid A was stable against acid hydrolysis, and the structure of this region is similar to that of P. cepacia, which has close taxonomic relationship with P. pseudomallei.     

Development of a multiplex PCR assay for rapid identification of Burkholderia pseudomallei, Burkholderia thailandensis, Burkholderia mallei and Burkholderia cepacia complex

We have developed a multiplex PCR assay for rapid identification and differentiation of cultures for Burkholderia pseudomallei, Burkholderia thailandensis, Burkholderia mallei and Burkholderia cepacia complex. The assay is valuable for use in clinical and veterinary laboratories, and in a deployable laboratory during outbreaks.     

Identification of Pseudomonas spp. Isolated from Milk Produced in South Eastern Queensland

S ummary . Bacteria were isolated from raw and pasteurized milks produced throughout S. E. Queensland. Milk samples were plated initially on penicillin agar or on milk agar and incubated at 30° and 7°, respectively. On the basis of primary characterization, 167 of the 330 isolates obtained were identified as Pseudomonas spp. The pseudomonads were further characterized in accordance with the taxonomic studies of the genus by Stanier, Palleroni & Doudoroff (1966). Species designations were ascribed to the Pseudomonas isolates on the basis of distinctive species characteristics in conjunction with similarity coefficients between each isolate and the ideal species phenotype, as follows: Ps. fluorescens (121), Ps. aeruginosa (16), Ps. putida (12), Ps. maltophilia (9), Ps. pseudoalcaligenes (5), Ps. cepacia (3) and Ps. alcaligenes (1). A dendrogram obtained by cluster analysis of the Pseudomonas isolates is included.     

Pseudomonas Cepacia Septicemia Associated with Intravenous Therapy

Four cases of Pseudomonas cepacia septicemia were found in one hospital in 1971. Two were related to severe phlebitis of the arms due to intravenous catheters, a third to an infected central venous pressure catheter. The infections resolved after the catheters were removed in these three cases.Prophylactic antibiotics may play a partial role in predisposing to this kind of infection. Ps. cepacia may be a more common pathogen than previously recognized. Its antibiotic sensitivity pattern distinguishes it from other members of the Pseudomonas family. Nosocomial infection with this bacteria has been traced to quaternary ammonium solutions but the source of infections in the present cases was not found.     

Identification of Pseudomonas tolaasi: the White Line in Agar and Mushroom Tissue Block Rapid Pitting Tests

A sharply defined white line of precipitate forms in Pseudomonas Agar F (Difco) between the opaque white colonies of Pseudomonas tolaasi and translucent colonies of certain unidentified pseudomonads. This visible interaction has been utilized in a specific and reliable method for the identification of Ps. tolaasi. The white line test was positive when 113 isolates of Ps. tolaasi from five different countries were examined, whereas 154 isolates of pseudomonads other than Ps. tolaasi , including Ps. corrugata, Ps. delphinii, Ps. fluorescens, Ps. lachrymans, Ps. marginalis, Ps. pastinaceae, Ps. phaseolicola, Ps. aeruginosa, Ps. putida, Ps. syringae, Ps. mors-prunorum, Ps. cichorii, Ps. antirrhini, Ps. viridiflava, Ps. caryophylli, Ps. cepacia, Ps. mendocina, Ps. stutzeri, Ps. acidivorus and Ps. lemoignei did not give the white line reaction with a reacting translucent colony pseudomonad. Browning of mushrooms in host tests does not help in the identification of Ps. tolaasi , but a conspicuous pitting produced in less than 10 min at the cut surface of mushroom tissue is as specific as the white line test in detecting Ps. tolaasi in suspension in distilled water.     

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.     

Detection of tabtoxin-producing strains of Pseudomonas syringae by PCR

AIMS: The present study describes a system based on PCR to distinguish tabtoxin-producing strains of Pseudomonas syringae from other Ps. syringae plant pathogens that produce chlorosis-inducing phytotoxins. METHODS AND RESULTS: Thirty-two strains of Ps. syringae and related species were examined. Two sets of PCR primers were developed to amplify genes (tblA and tabA) required for tabtoxin production. Only a PCR product of 829 bp or 1020 bp was produced in PCR reactions with the tblA or tabA primer sets, respectively, and cells from tabtoxin-producing pathovars of Pseudomonas syringae. All known non-tabtoxin producing bacterial species failed to produce an amplification product with either primer set. CONCLUSIONS: PCR of genes required for tabtoxin production is a simple, rapid and reliable method for identifying tabtoxin-producing strains of Ps. syringae. SIGNIFICANCE AND IMPACT OF THE STUDY: The protocol can effectively distinguish tabtoxin-producing strains of Ps. syringae from other Ps. syringae pathovars and Ps. syringae pv. tabaci strains from other tabtoxin-producing Ps. syringae pathovars.     

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.     

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.     

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.