L-Forms of Pseudomonas aeruginosa

L-forms of a strain of Pseudomonas aeruginosa were produced by serial subculture of the bacterial form on agar medium containing sucrose as an osmotic stabilizer and carbenicillin. L-forms eventually became stable, i.e., would not revert in the absence of antibiotic, and were adapted to grow well in broth with the osmotic stabilizer. Gross morphology and light microscopic colony morphology were typical of an L-form. L-form cells were approximately spherical and bounded in part by a plasma membrane; they lacked the triple-layer cell wall structure and coarse, electron-dense nucleoidal granules of the parent bacterial form. The L-form, but not the bacterial form, contained cores, organelles previously reported only in group D streptococci. Antibiotic disc-sensitivity studies showed the stable L-form to be as sensitive as, or more sensitive than, the bacterial form to most antibiotics. Exceptions were polymyxin B, colimycin sulfate, and gentamicin, which were more active against the bacterial form. The remainder of the aminoglycosides and cell wall-active antibiotics showed no inhibition of either form. The L-form was more susceptible to cidal activity of normal human serum than the parent form. The L-form exhibited fewer biochemical activities than the parent bacteria or bacterial forms derived by reversion at a time when the L-form was still unstable. L-form colonies appeared colorless, and chemical analysis demonstrated that, if the L-form produces pigment at all, which was not demonstrated, it could not have been more than 3.6% of that produced by the bacterial form.     

Pyocine typing of Pseudomonas aeruginosa

The growth-inhibitory effect of a small group of different pyocines has been used to differentiate Ps. aeruginosa isolates. A method is described which can be carried out with ordinary media and is easy for routine application. One hundred and one isolates of Ps. aeruginosa were differentiated by four pyocines into 10 pyocine patterns.     

Silver against Pseudomonas aeruginosa biofilms

Silver has been recognized for its antimicrobial properties for centuries. Most studies on the antibacterial efficacy of silver, with particular emphasis on wound healing, have been performed on planktonic bacteria. Our recent studies, however, strongly suggest that colonization of wounds involves bacteria in both the planktonic and biofilm modes of growth. The action of silver on mature in vitro biofilms of Pseudomonas aeruginosa, a primary pathogen of chronic infected wounds, was investigated. The results show that silver is very effective against mature biofilms of P. aeruginosa, but that the silver concentration is important. A concentration of 5-10 mug/mL silver sulfadiazine eradicated the biofilm whereas a lower concentration (1 mug/mL) had no effect. The bactericidal concentration of silver required to eradicate the bacterial biofilm was 10-100 times higher than that used to eradicate planktonic bacteria. These observations strongly indicate that the concentration of silver in currently available wound dressings is much too low for treatment of chronic biofilm wounds. It is suggested that clinicians and manufacturers of the said wound dressings consider whether they are treating wounds primarily colonized either by biofilm-forming or planktonic bacteria.     

Aeruginocine typing of Pseudomonas aeruginosa

Aeruginocine typing using eight indicator strains of Wahba on Bacto tryptone-glucose yeast extract Agar (Difco) with an incubation temperature of 32°C was found to be a reproducible and easy method which could be readily adopted by a diagnostic clinical laboratory. The results of aeruginocine typing of 1500 strains of Ps. aeruginosa suggest that hospitals differ in the range and patterns of aeruginocine types of strains isolated in them. In addition to representatives of nine of the 10 pyocine types of Wahba, a large number of strains isolated in India were found to fall into other groups not described in Wahba's system. Hence, 20 new inhibition patterns (aeruginocine types) are proposed.     

Immunological nonidentity of Pseudomonas paucimobilis with Pseudomonas aeruginosa and Pseudomonas cepacia.

This investigation determined the serum agglutination activity and serum bactericidal response after rabbit immunization with Pseudomonas paucimobilis. Agglutination activity of antisera showed a twofold increase in titer from before immunization to 4 weeks post-immunization and peaked at 8 weeks post-immunization with a titer of 1:512. 2-Mercaptoethanol reduction of immunoglobulin M decreased agglutination titers. No major antigens were found to be common from crude antigen preparations of P. paucimobilis, Pseudomonas aeruginosa and Pseudomonas cepacia when tested with antisera to P. paucimobilis. Serum bactericidal activity was found in post-immunization antisera at 8 and 12 weeks against P. paucimobilis, with no activity present before immunization or at 4 weeks post-immunization. Antisera against P. paucimobilis showed no bactericidal activity against P. aeruginosa or P. cepacia.     

Comparative studies on Pseudomonas aeruginosa endotoxin

Attempts to obtain toxic preparations of endotoxin from Pseudomonas aeruginosa (C₂) were initiated by employing five different extraction procedures which were subsequently assayed for mouse lethality using a variety of routes. The standard methods of extraction were the aqueous phenol, trichloracetic acid, ethylenediaminetetraacetate-lysozyme, ethyl ether, and hot water procedures. The aqueous phenol preparation was found to be the most toxic and exhibited an LD₅₀ value of 450 μg dry weight when administered intravenously and 840 μg intraperitoneally. No lethality was observed when endotoxin was administered intranasally. The second most lethal preparation was obtained by the trichloroacetic acid extraction and yielded LD₅₀ values of 589 μg intravenously and 947 μg intraperitoneally. The other three preparations were considerably less lethal. The aqueous phenol and trichloroacetic acid preparation tended to have a higher content of those carbohydrates associated with the core region of the lipopolyssacharide molecule than did the lesser toxic preparations. Some correlation between lethality and lipid content as determined by the alkaline hydroxylamine procedure was also observed.     

Non-flagellate Pseudomonas aeruginosa in pathological material

Three isolates of non-flagellate Pseudomonas aeruginosa obtained from pathological material of human cases are described. Two were found in urinary tract infections and the third in a respiratory infection. They were shown by typing methods to be distinct strains.     

Vascular Permeability Factor of Pseudomonas aeruginosa

The production of vascular permeability factor (PF) by certain strains of Pseudomonas aeruginosa has been demonstrated in rabbits injected intradermally with culture filtrates followed by intravenous injection with Pontamine Sky Blue 6BX. The dose-response curve was found to be rectilinear when lesion diameters, within the range of 10 to 20 mm, were plotted against log dose. Thus, PF in test filtrates can be measured with reasonable accuracy by the concomitant testing of a reference PF. In contrast to the titers of PF obtained with Vibrio cholerae cultures, those with strains of P. aeruginosa were rather low. Thus far, PF has been demonstrated only in shallow still cultures of P. aeruginosa and not in shake cultures. A variety of commercial media were tested for the production of PF, but none was satisfactory. A synthetic medium that gave more reproducible and higher yields of PF was developed. Cultivation at 30 C generally gave higher yields of PF than at 37 C. PF was destroyed by heating at 60 C for 30 min or by digesting with trypsin or Pronase. Strains producing larger amounts of PF appeared to have greater virulence when inoculated onto the surface of burns in mice than those yielding little or no PF.     

Clinical analysis of Pseudomonas aeruginosa bacteremia

To determine the outcome of Pseudomonas aeruginosa bacteremia and to identify risk factors for these infections in our University hospital, 46 cases (65 episodes) of Pseudomonas aeruginosa bacteremia were retrospectively investigated. The most frequent underlying diseases or cases were from Emergency and Critical care center (18 cases, including 11 case of cerebrovascular accident and head injury) followed by hematologic malignancies (11 cases) but none of the HIV infection was included in this study. The overall crude mortality rate was 50% and mortality rate within the first 1 week was 17%. Clinical analysis of those cases revealed that possible risk factors were neutropenia, sever sepsis and prior use of antibiotics (antipseudomonal antibiotics were administered before positive blood culture episodes in 90% cases). But these factors were not statistically significant between dead and survived cases. CONCLUSION: To improve the prognosis of Pseudomonas aeruginosa bacteremia, we must change the management of the hospital infection, such as the more rational use of new antipseudomonal antibiotics and the more clean and reasonable management of central venous catheters.     

In vivo-induced genes in Pseudomonas aeruginosa

In vivo expression technology was used for testing Pseudomonas aeruginosa in the rat lung model of chronic infection and in a mouse model of systemic infection. Three of the eight ivi proteins found showed sequence identity to known virulence factors involved in iron acquisition via an open reading frame (called pvdI) implicated in pyoverdine biosynthesis, membrane biogenesis (FtsY), and adhesion (Hag2).     

Phospholipase A activity in Pseudomonas aeruginosa

Our study describes the production, purification and properties of an enzyme from Pseudomonas aeruginosa displaying the properties of phospholipase A. Maximal amounts of enzyme could be detected in the culture supernatant when the bacterium was grown for 3 to 5 days at 37°C in stirred flask cultures containing brain heart infusion. The enzyme was purified by polyethylenimine precipitation and ammonium sulfate precipitation followed by gel filtration. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the enzyme preparation exhibited two bands with molecular weights of 13.5 and 60 kD, respectively. Correspondingly, two peaks of the same molecular weight could be demonstrated by high performance size exclusion chromatography. The activity toward the sn-2 ester binding of phospholipids was characterized and found to be highest towards phosphatidylcholine. Enzymatic activity was not influenced by the addition of calcium or EDTA while magnesium and strontium caused a decrease of activity. The lyophilized enzyme was found to be stable when stored at −70°C and most active at pH 8.0.     

Electron microscopic observations of Pseudomonas aeruginosa

Preparative techniques were developed for the purpose of visualizing, by negative contrast, some of the cytological characteristics of unsectioned cells of Pseudomonas aeruginosa. Concentrated fimbriae free from cells had a length of approximately 1.5 μm and a diameter of 6 nm. Pyocin-like structures ranging in length from 32-64 nm with a width of 8.6 nm were “attached” to the terminal ends of a small percentage of these fimbriae. Microtubules and ringlets having a diameter of 10-20 nm were also visualized in the bacteria. Budding or evagination of cell wall material in multiple units up to 135 nm in length was frequently observed in older cultures grown on agar media.