Filamentous growth ofPseudomonas aeruginosa

Summary The growth of two strains ofPseudomonas aeruginosa in stirred batch cultures was monitored by optical density, DNA concentration, and acridine orange direct cell count measurements. Growth of adherent bacteria in pure culture was also observed on suspended glass discs by light and scanning electron microscopy. Strain MUCOID produced significant numbers of filamentous cells in broth culture and in the adherent population, while strain PAO 381 did not produce elongated cells. Filamentous growth of MUCOID could be prevented by the addition of 5 × 10^–2 M Mg²⁺. However, the addition of 0.66 mM EDTA caused an increased proportion of the population (>50%) of MUCOID cells to become filamentous in broth culture. The results are discussed and related to theories regarding bacterial plasticity, and filamentation of normally bacillary cells.     

Properties ofPseudomonas aeruginosa exhibiting self-lysis

Spontaneous lysis leading to the production of turbid, iridescent auto-plaques (AP⁺) was noted in 46 out of 50 strains ofPseudomonas aeruginosa. Strain Pa-1 which is mucoid and is a non-auto-plaque former (M⁺AP^–) on rare occasions lyses; the surviving cells are non-mucoid and always exhibited plaques on itself (M^–AP⁺) as well as on the M⁺AP^– culture. In addition, the non-mucoid culture gave rise to a mucoid, auto-plaque producing variant (M⁺AP⁺). Biochemical characterization of the cultures indicated no other qualitative differences, although AP⁺ cultures were more proteolytic, but less hemolytic than the M⁺AP^– strain. All three cultures synthesized the bacteriocin pyocin, but were immune to both their own and each other's agents. In addition, they exhibited the same lysogenic host range when streaked against 18 other cultures ofP. aeruginosa. Treatment of the auto-plaque forming strains with non-inhibitory levels of penicillin, streptomycin, chloromycetin, or polymyxin, stimulated cultures to produce increased numbers of auto-plaques in proportion to antibiotic concentrations, while no lysis was noted with the non-autolytic strain (M⁺AP^–). However, treatment of the three cultures with varying doses of ultra-violet did not stimulate the production of auto-plaques beyond the normal level of non-irradiated cultures, and in some cases suppressed their appearance. Filtrates obtained from the non-mucoid, auto-plaque producing culture (M^–AP⁺) formed iridescent, turbid plaques on M⁺AP^–, M⁺AP⁺, and M^–AP⁺. Similar results were obtained with the M⁺AP⁺ filtrates.     

The influence ofPseudomonas aeruginosa on liposomes

The interaction of liposomes loaded with Ponceau red (used as a marker) withPseudomonas aeruginosa cells was observed and it resulted in marker leakage. The marker leakage from liposomes was low in physiological fluids. The interaction was independent of secreted phospholipase C level and the serotype of the tested strain. Six of 37 examined isolates did not cause any release of the marker from the liposomes. Marker release of over 50% of total encapsulated material was observed only for ten of the strains tested.     

Some remarks on autolysis ofPseudomonas aeruginosa

Summary Several observations on the autolysis in cultures ofPs. aeruginosa are described. Autolysis appears to be caused by thermolabile and non-dialysable substances, which are identified as excreted proteolytic enzymes. These enzymes only digest dead cells.     

The mechanism of pathogenicity ofPseudomonas aeruginosa

Extra-cellular proteinases ofPseudomonas aeruginosa toxic for larvae of the greater wax moth were separated from the culture filtrate by means of ammonium sulphate precipitation, chromatography on DEAE cellulose, gel filtration on Sephadex G-75 and preparative disc-electrophoresis respectively. The five proteinases isolated were characterized by pH optima, molecular weights, elastase and collagenase activities.     

The mechanism of pathogenicity ofPseudomonas aeruginosa

Proteinases produced byPseudomonas aeruginosa are toxic for galleria larvae and their LD₅₀ differ from one enzyme to the other. The most toxic enzyme has a specific affinity to some protein fractions of insect haemolymph as shown by disc electrophoresis.     

铜绿假单胞菌高产鼠李糖脂菌株的筛选

从多种来源筛选高产鼠李糖脂的菌株,并研究菌种发酵特性和鼠李糖脂产物的理化性质。采用CTAB平板初步筛选鼠李糖脂合成菌株,通过分析菌株的16S r RNA基因序列确定细菌种属,采用薄层色谱、红外光谱分析产物性质。结果显示,利用CTAB平板初筛获得163株阳性菌株,初步发酵确定10株高产细菌鼠李糖脂的产量为12.2-17.7 g/L,10株细菌均鉴定为铜绿假单胞菌。挑选产量最高的菌株B12,分别以甘油、菜籽油、花生饼粉或葵花籽饼粉为碳源进行发酵,发现菜籽油为合成鼠李糖脂的最佳碳源。进一步对比在35℃、37℃和40℃的发酵水平,发现37℃条件下鼠李糖脂产量最高,为26.8 g/L。最后,对鼠李糖脂发酵产物进行了初步纯化,并进行了薄层色谱和红外光谱分析。菌株B12能够合成较高水平的鼠李糖脂,可能成为工业生产的候选菌株。     

Resistance ofPseudomonas aeruginosa to β-lactam antibiotics

A series of experiments were performed withP. aeruginosa to demonstrate which of the biochemical mechanisms are responsible for the resistance to the β-lactam antibiotics. The constitutive β-lactamase was isolated and characterized for the strain used as type OXA whose pI was 7.1, with a molar mass of 49 kg/mol. The strain was also submitted to a series of treatments with Tris and Tris -EDTA to disrupt the outer membrane. The treated cells demonstrated a ten-fold reduction in the MIC with cloxacillin, six-fold with penicillin, and five-fold with oxacillin. At least two different biochemical mechanisms were responsible for the resistance to the β-lactams studied which could be important in the prevalence ofP. aeruginosa in nosocomial infections.     

Biosurfactant production by two isolates ofPseudomonas aeruginosa

Two strains of biosurfactant-producing bacteria, identified asPseudomonas aeruginosa, were isolated from injection water and crude oil-associated water in Venezuelan oil fields. Both biosurfactants resembled rhamnolipids and produced stable emulsions of heavy and extra-heavy crude oils, reducing the surface tension of water from 72 to 28 dynes/cm. Tenso-active properties of the biosurfactants were not affected by pH, temperature, salinity or Ca²⁺ or Mg²⁺ at concentrations in excess of those found in many oil reservoirs in Venezuela.     

Inactivation of allantoinase ofPseudomonas aeruginosa in vivo

The total and specific activity of allantoinase decreased in the stationary growth phase whenPseudomonas aeruginosa was cultured in a medium containing allantoin or allantoate as the sole source of carbon, nitrogen and energy. The enzyme was not instableper se. Inactivation was proven to be due to the synthesis of a protein, which exhibited no proteolytic activity toward allantoinase. The synthesis of a specific binding protein is postulated.     

Resistance ofPseudomonas aeruginosa to sodium dimethyldithiocarbamate by adaptation

Resistance and the development thereof inPseudomonas aeruginosa to the bactericide sodium dimethyldithiocarbamate (SMT) was investigated.P. aeruginosa was cultured in nutrient-poor broth in the presence of subinhibitory concentrations of SMT. It adapted over 21 days of exposure from 250 g·ml^–1 to 490 g·ml^–1. The initial high MIC was ascribed to exclusion of SMT by the lipopolysaccharide layer, since removal thereof by EDTA rendered cells highly susceptible. The alginate-producing mutant PAO 579 was much more susceptible to SMT than was its parent PAO 381, indicating that extracellular polysaccharide does not act as an exclusion barrier to SMT. Following 24 h of exposure to SMT,P. aeruginosa had an altered profile of outer membrane proteins as determined by SDS-PAGE. Resistant cells had a further altered profile. Resistance ofP. aeruginosa is ascribed to a change in the outer membrane protein profile, leading to improved exclusion of SMT.     

Substrate specificity of the paraffin hydroxylase ofPseudomonas aeruginosa

The paraffin hydroxylating enzyme system isolated fromPseudomonas aeruginosa (strain 473) grown onn-heptane has been studied with respect to its substrate specificity. It has been found that cell-free extracts of this organism can hydroxylate a wide variety of hydrocarbons. In order to function as substrates, molecules should be able to assume a more or less planar conformation. The compounds used in this study can be divided into three classes: (a) alkanes, (b) alkylbenzenes and (c) (alkyl)cycloalkanes. In each of these groups hydroxylation occurs at a specific site in the molecule. Noteworthy is the hydroxylation of isopropyl groups at a methyl carbon and that of monoalkylcyclohexanes at the 4-transposition.From the geometry of the substrate and non-substrate molecules the conclusion was drawn that the active centre of the hydroxylase is a cleft in the enzyme surface of about 5 Å wide and 8 Å deep.We gratefully acknowledge the skillfull assistance of Miss W. T. Hempel.     

Effect ofPseudomonas aeruginosa rhamnolipid on human neutrophil migration

The effect ofPseudomonas aeruginosa heat-stable hemolysin (rhamnolipid) on human neutrophil migration has been investigated. Rhamnolipid was prepared from culture filtrate and characterized by thin-layer chromatography. The lytic activity of rhamnolipid was quantitated by titration against neutrophils. Leukocyte migration response was measured using⁵¹Cr-labeled neutrophils with a double-filter technique in modified Boyden chambers. The results suggest rhamnolipid stimulated chemotaxis as well as chemokinesis. Moreover, rhamnolipid impaired a chemotactic response toN-formyl-methionyl-leucyl-phenylalanine. These effects may be important in host-parasite interactions.     

Heavy metal resistance in clinical isolates ofPseudomonas aeruginosa

One hundred clinical isolates of Pseudomonas aeruginosa were checked for their sensitivity towards silver nitrate. Majority of the isolates were resistant at 20 mg/L and the resistance decreased with increasing concentration of silver nitrate, only 5% of the organisms showed resistance above 70 mg/L. These silver-resistant isolates were further checked for their resistance towards mercury and cadmium at 20 mg/L of concentration and the level of resistance was found to be 33 and 40%, respectively. A correlation between silver ion resistance and concurrent mercury and cadmium ion resistance was observed, suggesting a possible linkage between resistance towards various metal ions.     

Permeability factor,cytotoxicity and serotyping ofPseudomonas aeruginosa strains

We analyzed in detail the permeability and cytotoxic activity as well as the serotypes of 127Pseudomonas aeruginosa strains. Sixty-seven strains were isolated from immunocompromised patients (51 from patients with tumors and 16 from patients after transplantation) and 60 strains were isolated from patients’ ears. Culture filtrates of strains isolated from patients after transplantation were responsible for the highest part of permeability reactions corresponding to an intermediate toxin production (68.8%) (categories 2 and 3) and culture filtrates of strains isolated from patients with tumors caused the highest percentage of permeability reactions corresponding to a strong toxin production. Culture filtrates of strains isolated from ears of patients were responsible for the highest percentage of negative permeability reactions (15%). With positive permeability reaction size (categories 2–6) increased also the percentage of cytotoxicity as well as the intensity of morphological changes on Vero cells after 1 and 2 d. We did not observe any relationship between a particular permeability reaction category and the most frequent serotypes (O4, O6) or nontypable strains of the tested groups.     

Effect of mucoid property on antibiotic susceptibility ofPseudomonas aeruginosa

Clinical isolates ofPseudomonas aeruginosa from patients with cystic fibrosis (CF) were examined for susceptibility to the antibiotics carbenicillin, ticarcillin, tobramycin, gentamicin, and tetracycline. Minimal inhibitory concentrations of the antibiotics were determined for mucoid and nonmucoid isolates from the same patient by a single-colony replica plating method. This method allows the rapid determination of antibiotic susceptibility of a single cell’s progeny and the individual screening of each colony against all antibiotics. Twenty of 34 (58%) cystic fibrosis patients had a mucoid isolate which was more susceptible to antibiotics than their nonmucoid isolate of the same serotype. Nonmucoid revertant segregants of mucoid strains isolated from 50% of the patients demonstrated greater resistance to at least one antibiotic than the original mucoid strain. Multiple isolates from 25 patients were serotyped by Difco (Liu) or Homma antiserum; only 2 patients harbored multiple strains with no common serotyping antigens. Serotypes of the nonmucoid revertants were the same as the original mucoid isolate even if the susceptibilities of the two strains were not similar.     

Dynamic interactions ofPseudomonas aeruginosa and bacteriophages in lake water

The persistence and interaction between newly isolated strains ofPseudomonas aeruginosa and resident bacteriophages indigenous to a freshwater environment was monitored over 45 days in lake water microcosms. The interaction between susceptible and resistant bacteria with pure phage (UT1) particles or a mixed phage population (M1) was investigated by following temporal changes in host density, phage-to-bacteria ratio (PBR), and the appearance of apparent prophage carriers within the host population. Decay rates of the phage (UT1) ranged from 0.054 hour^–1 in natural water to 0.027 hour^–1 in filtered lake water. About 45% of sensitive bacteria incubated with phase UT1 were pseudolysogenic within 12 hours of incubation in natural lake water. This process was delayed until 72 hours in the steile lake water control, suggesting that host-phage interaction is promoted in the presence of a viable natural microbial community. Phage UT1 appeared to stabilize the density of host bacteria in lake water at a level of 10⁴ colony-forming units (cfu) ml^–1. Bacterial coexistence with the mixed phage (M1) population resulted in an oscillating equilibrium with the PBR stabilizing at about 3. The presence of extraneous homoimmune phages appeared to be detrimental to the stability of the pseudolysogens, which were maintained at a lower population density than prophage-free cells in lake water containing the mixed phage (M1) population.     

Characteristics ofPseudomonas aeruginosa strains isolated from urinary tract infections

Thirty-three uropathogenic strains ofPseudomonas aeruginosa were investigated for hemolytic activity in both bacterial broth culture filtrates and isolate lyzates, resistance to bactericidal activity of fresh human serum, resistance to six antibiotics and plasmid DNA profile. Twenty-four of the 33 (73%) bacterial filtrates showed lysis of rabbit erythrocytes, as did the three after guinea-pig erythrocyte treatment. Twelve of 33 isolate lysates showed in parallel lysis of both types of erythrocytes used. Serum resistance was found in 17 (52%) isolates, intermediate resistance in 15 (45%) isolates and only one isolate showed serum sensitivity. Resistance to antibiotics was detected as follows (in %): tetracycline 94, kanamycin 79, chloramphenicol 76, septrin 73, ampicillin 64, streptomycin 45, gentamicin 18. None of the isolates investigated showed resistance to colistine. With the exception of one isolate, plasmid DNA was detected in allP. aeruginosa strains.     

Glycerol catabolism in wild-type and mutant strains ofPseudomonas aeruginosa

Glycerol uptake, glycerol kinase (EC 2.7.1.30) and glycerol-3-phosphate dehydrogenase (EC 1.1.99.5) activities are specifically induced during growth ofPseudomonas aeruginosa PAO on either glycerol or glycerol-3-phosphate. Mutants of strain PAO unable to grow on both glycerol and glycerol-3-phosphate were isolated. Mutant PFB 121 was deficient in an inducible, membrane-bound, pyridine nucleotide-independent, glycerol-3-phosphate dehydrogenase activity and PFB 82 was deficient in glycerol uptake and glycerol kinase and glycerol-3-phosphate dehydrogenase activities. Each mutant spontaneously reverted to wild phenotype, which indicates that each contained a single genetic lesion. These results demonstrate that membrane-bound, inducible glycerol-3-phosphate dehydrogenase is required for catabolism of both glycerol and glycerol-3-phosphate and provide suggestive evidence for a single regulatory locus that controls the synthesis of glycerol uptake, glycerol kinase, and glycerol-3-phosphate dehydrogenase inP. aeruginosa.