Nucleotide sequence of the aliphatic amidase regulator gene (amiR) of Pseudomonas aeruginosa

The nucleotide sequence of a 1001 bp ClaI/XhoI DNA fragment encoding the amidase regulator gene (amiR) from Pseudomonas aeruginosa has been determined. The sequence derives from strain PAC433, a constitutive high expressing amidase mutant, and contains two overlapping open reading frames. Analysis of the sequence has identified one of the reading frames as amiR. The gene encodes a 196 amino acid polypeptide which shows a strong bias towards codons with G or C in the third position. The amiR gene shows no sequence homology with other bacterial regulator proteins.     

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 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.     

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.     

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.     

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.     

Association of MexAB-OprM with intrinsic resistance ofPseudomonas aeruginosa to aminoglycosides

MexAB-OprM is known to pump out mostly lipophilic and amphiphilic drugs. But in low-ionic-strength medium, nutrient broth (NB), this pump has been shown to contribute to hydrophilic antibiotic (aminoglycosides) resistance, via active efflux. The association of the MexAB-OprM efflux system to aminoglycosides resistance inPseudomonas aeruginosa were assessed using a drug susceptibility test carried out in NB, in presence and absence of protonophore, carbonyl cyanide m-chlorophenyl hydrazone (CCCP) by 23 multidrug resistant strains were selected from 104 clinical isolates ofP. aeruginosa. Active efflux was assessed using EtBr accumulation assays. PCR was used to identify themexAB-oprM and MexAB-OprM-dependent efflux of aminoglycosides and the results were confirmed by continuous fluorescence assay. A multidrug resistant mutant ofmexAB-oprM, derivative of PAO1, was selected by ciprofloxacin and subjected to the same analysis as described above for the clinical isolates. In this study, CCCP reduced the level of MICs in at least 1 dilution. Ethidium bromide accumulation assays confirmed the presence of efflux mechanism in all clinical isolates and PCR demonstrated that 17% of our isolates had themexAB-oprM operon. Results of aminoglycosides accumulation showed, in addition to amphiphilic antibiotics in NB medium, MexAB-OprM extrudes aminoglycosides (hydrophilic) drugs.     

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。     

In vitro PYOCIN activity ofPseudomonas aeruginosa strains pretreated with antibiotics

Fifty-six selected strains of Pseudomonas aeruginosa belonging to 8 different pyocin types (H, I, 15, 6, PTI-1, PTI-2, PTI-3, PTI-4) were treated with subinhibitory concentrations (MIC/2) of either gentamicin or carbenicillin. Both treatments induced changes in pyocin patterns for all types but at different levels. The percentage of strains that retained their pyocin pattern were more or less equal in both treatments. In treated and untreated producers, the growth inhibition ability for 5 different strains of Enterobacteriaceae (Escherichia coli K12, E. coli EB, Proteus vulgaris, Salmonella typhi, Shigella flexneri) was also investigated. In all pyocin patterns the number of producers that inhibit the growth of these strains was lower after treatment with gentamicin or with carbenicillin, a smaller decrease was detected in the latter treatment. It appeared that the subinhibitory concentrations of these antibiotics are capable of protecting the Enterobacteriaceae strains from the action of the pyocins.     

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.     

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.     

Substrate interaction with recombinant amidase from Pseudomonas aeruginosa during biocatalysis

The interaction of a variety of substrates with Pseudomonas aeruginosa native amidase (E.C. 3.5.1.4), overproduced in an Escherichia coli strain, was investigated using difference FTIR spectroscopy. The amides used as substrates showed an increase in hydrogen bonding upon association in multimers, which was not seen with esters. Evidence for an overall reduction or weakening of hydrogen bonding while amide and ester substrates are interacting with the enzyme is presented. The results describe a spectroscopic approach for analysis of substrate–amidase interaction and in situ monitoring of the hydrolysis and transferase reaction when amides or esters are used as substrates.     

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.     

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.     

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.