Antimicrobial activity and effectiveness of a combination of sodium hypochlorite and hydrogen peroxide in killing and removing Pseudomonas aeruginosa biofilms from surfaces

AIMS: To evaluate both the antimicrobial activity and the effectiveness of a combination of sodium hypochlorite and hydrogen peroxide (Ox-B) for killing Pseudomonas aeruginosa ATCC 19142 cells and removing P. aeruginosa biofilms on aluminum or stainless steel surfaces. METHODS AND RESULTS: Pseudomonas aeruginosa biofilms were developed in tryptic soy broth containing vertically suspended aluminium or stainless steel plates. Biofilms were exposed to a mixed sodium hypochlorite and hydrogen peroxide solution as a sanitizer for 1, 5 and 20 min. The sanitizer was then neutralized, the cells dislodged from the test surfaces, and viable cells enumerated. Cell morphologies were determined using scanning (SEM) and transmission electron microscopy (TEM). Cell viability was determined by confocal scanning laser microscopy (CSLM). Biofilm removal was monitored by Fourier transform infrared (FTIR) spectrophotometry. Cell numbers were reduced by 5-log to 6-log after 1 min exposure and by 7-log after 5 min exposure to Ox-B. No viable cells were detected after a 20 min exposure. Treatment with equivalent concentrations of sodium hypochlorite reduced viable numbers by 3-log to 4-log after 1 min exposure and by 4-log to 6-log after 5 min, respectively. A 20 min exposure achieved a 7-log reduction. Hydrogen peroxide at test concentration treatments showed no effect. FTIR analysis of treated pseudomonad biofilms on aluminium or stainless steel plates showed either a significant reduction or complete removal of biofilm material after a 5 min exposure to the mixed sodium hypochlorite and hydrogen peroxide solution. SEM and TEM images revealed damage to cell wall and cell membranes. CONCLUSIONS: A combination of sodium hypochlorite and hydrogen peroxide effectively killed P. aeruginosa cells and removed biofilms from both stainless steel and aluminium surfaces. SIGNIFICANCE AND IMPACT OF THE STUDY: The combination of sodium hypochlorite and hydrogen peroxide can be used as an alternative disinfectant and/or biofilm remover of contaminated food processing equipment.     

A protease stable in organic solvents from solvent tolerant strain of Pseudomonas aeruginosa

A solvent tolerant strain of Pseudomonas aeruginosa (PseA) was isolated from soil samples by cyclohexane enrichment in medium. The strain was able to sustain and grow in a wide range of organic solvents. The adaptation of P. aeruginosa cell towards solvents was seen at membrane level in transmission electron micrographs. It also secreted a novel protease, which exhibited remarkable solvent stability and retained most of the activity at least up to 10 days in the presence of hydrophobic organic solvents (log P > or = 2.0) at 25% (v/v) concentrations. The protease was able to withstand as high as 75% concentration of solvents at least up to 48 h. P. aeruginosa strain and its protease, both seem promising for solvent bioremediation, wastewater treatment and carrying out biotransformation in non-aqueous medium.     

Susceptibility and intrinsic tolerance of Pseudomonas aeruginosa to selected plant volatile compounds

AIMS: To examine the causes for variations in sensitivity and intrinsic tolerance of Pseudomonas aeruginosa to plant volatile compounds. METHODS AND RESULTS: Minimum inhibitory concentrations were determined for a selection of volatile phytochemicals against P. aeruginosa using a microdilution assay. Effects on growth were also assessed in 100-ml broth cultures. The two strains of P. aeruginosa included in the study exhibited intrinsic tolerance to all compounds, with the exception of carvacrol and trans-cinnamaldehyde. The protonophore carbonyl cyanide m-chlorophenylhydrazone increased P. aeruginosa sensitivity to all compounds except trans-cinnamaldehyde, implicating an ATP-dependent efflux mechanism in the observed tolerance. Outer membrane integrity following treatment with test compounds was assessed by measuring sensitization to detergents. Only carvacrol caused damage to the outer membrane of P. aeruginosa. CONCLUSIONS: The intrinsic tolerance of P. aeruginosa strains to plant volatile compounds is associated with an active efflux mechanism and the barrier function of the outer membrane. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings offer an explanation for the intrinsic tolerance to plant volatile compounds exhibited by P. aeruginosa. The study also confirms that the outer membrane-permeabilizing action of carvacrol, previously reported for the gram-negative bacteria Escherichia coli and Salmonella, extends to monoterpene-tolerant strains of P. aeruginosa.     

Inositol polyphosphate-mediated iron transport in Pseudomonas aeruginosa

It has previously been shown that myo-inositol hexakisphosphate (myo-InsP6) mediates iron transport into Pseudomonas aeruginosa and overcomes iron-dependent growth inhibition. In this study, the iron transport properties of myo-inositol trisphosphate and tetrakisphosphate regio-isomers were studied. Pseudomonas aeruginosa accumulated iron (III) at similar rates whether complexed with myo-Ins(1,2,3)P3 or myo-InsP6. Iron accumulation from other compounds, notably D/L myo-Ins(1,2,4,5)P4 and another inositol trisphosphate regio-isomer, D-myo-Ins(1,4,5)P3, was dramatically increased. Iron transport profiles from myo-InsP6 into mutants lacking the outer membrane porins oprF, oprD and oprP were similar to the wild-type, indicating that these porins are not involved in the transport process. The rates of reduction of iron (III) to iron (II) complexed to any of the compounds by a Ps. aeruginosa cell lysate were similar, suggesting that a reductive mechanism is not the rate-determining step.     

Effects of three nickel salts on germinating seeds of Grevillea exul var. rubiginosa, an endemic serpentine Proteaceae

BACKGROUND AND AIMS: Serpentine soils are usually quite infertile, arid and toxic, mainly because they contain high levels of heavy metals such as Ni. The aim of the present work was to assess the effects of Ni on the germinating seeds of Grevillea exul var. rubiginosa, an endemic serpentine Proteaceae of New Caledonia. In addition, the distribution of macronutrients and the Ni levels in germinating seeds were examined. METHODS: Seeds were sown in glass Petri dishes and exposed to increasing concentrations of Ni (5 to 500 mg Ni L(-1)) using Ni chloride, Ni sulphate and Ni acetate. The germination percentage and root length were measured after 40 d. Longitudinal frozen sections of germinating seeds growing in the presence of Ni (500 mg L(-1) for all three salts) were used for X-ray microanalysis and X-ray elemental mapping using scanning electron microscopy (SEM). KEY RESULTS: Ni chloride resulted in the greatest reductions in germination and root growth, particularly at 500 mg L(-1), followed by Ni sulphate and Ni acetate. SEM images revealed Ca crystalline structures in the seed coat for all the samples. S/Ca and Mg/P/K/Mn were found to be distributed differently in Ni-treated samples, whereas they all followed the same pattern in the controls. For all three salts, the Ni added to the medium had accumulated in the seed coat, whereas the endosperm seemed to be devoid of Ni. CONCLUSIONS: It is assumed that the seed coat is able to reduce the amount of Ni entering the seed, and that a high level of Ni induced the mobilization of macronutrients.     

等离子体射流灭活液体中铜绿假单胞菌的研究

【目的】测定等离子射流对铜绿假单胞菌(Pseudomonas aeruginosa)的灭活效果,探究低温等离子体射流的杀菌机理。【方法】采用平板计数法测定等离子体射流的杀菌效果,荧光显微镜、透射电镜观察等离子体作用后菌体结构的变化,蛋白浓度测定和SDS-PAGE电泳检测菌液上清液中可溶性蛋白的泄漏量。【结果】等离子体射流处理铜绿假单胞菌菌液5 min,杀灭率可达到99.9%以上。透射电镜观察可见细菌菌体结构发生改变,细胞壁、细胞膜损伤破裂,细胞内容物泄露。进一步对处理铜绿假单胞菌上清液中的蛋白质含量变化进行检测,结果显示随着处理时间的增加,上清液中蛋白质含量持续增加,在2 min时达到最大值。【结论】等离子体射流可以通过破坏细胞结构造成细胞质泄露,使其丧失正常的细胞功能,从而达到快速有效地杀灭铜绿假单胞菌的效果。     

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.     

Identification of RegA protein from Pseudomonas aeruginosa using anti-RegA antibody

The product of Pseudomonas aeruginosa regA gene acts as a positive regulator of exotoxin A expression. The protein, RegA, was overproduced in E. coli transformed with an expression vector containing the regA gene. The overproduced RegA accumulated in E. coli in the form of inclusion bodies. The latter were isolated and served as a source of antigen for raising polyclonal antibodies. The antibodies reacted specifically with a P. aeruginosa protein whose molecular weight corresponded to that predicted for RegA from its known DNA sequence, and whose response to modulating factors matched that expected for RegA. The immunodetectable RegA was localized in the membrane fraction of P. aeruginosa strain PA103.     

Oligomerization of type III secretion proteins PopB and PopD precedes pore formation in Pseudomonas

Pseudomonas aeruginosa is the agent of opportunistic infections in immunocompromised individuals and chronic respiratory illnesses in cystic fibrosis patients. Pseudomonas aeruginosa utilizes a type III secretion system for injection of toxins into the host cell cytoplasm through a channel on the target membrane (the 'translocon'). Here, we have functionally and structurally characterized PopB and PopD, membrane proteins implicated in the formation of the P.aeruginosa translocon. PopB and PopD form soluble complexes with their common chaperone, PcrH, either as stable heterodimers or as metastable heterooligomers. Only oligomeric forms are able to bind to and disrupt cholesterol-rich membranes, which occurs within a pH range of 5-7 in the case of PopB/PcrH, and only at acidic pH for PcrH-free PopD. Electron microscopy reveals that upon membrane association PopB and PopD form 80 A wide rings which encircle 40 A wide cavities. Thus, formation of metastable oligomers precedes membrane association and ring generation in the formation of the Pseudomonas translocon, a mechanism which may be similar for other pathogens that employ type III secretion systems.     

Isolation and characterization of a transposon mutant of Pseudomonas aeruginosa affecting uptake of dibenzothiophene in n-tetradecane

AIMS: Isolation and characterization of a transposon mutant of Pseudomonas aeruginosa affecting the uptake of dibenzothiophene (DBT) in n-tetradecane (n-TD). METHODS AND RESULTS: The dsz desulphurization gene cluster from Rhodococcus erythropolis KA2-5-1 was transferred to the chromosome of P. aeruginosa NCIMB9571 using a transposon vector. A recombinant (named PARM1) was obtained which was able to desulphurize DBT in water, but not in n-TD. CONCLUSIONS: PARM1 is a mutant deficient in a DBT transport system operational in n-TD. This transport system is independent of rhamnolipids and of the n-alkane transport system. SIGNIFICANCE AND IMPACT OF THE STUDY: Pseudomonas aeruginosa NCIMB9571 seems to have a specific system of transporting hydrophobic compounds such as DBT in oil.     

Cellular response mechanisms in Pseudomonas aeruginosa PseA during growth in organic solvents

Aims:  Solvent-tolerant bacteria have emerged as a new class of micro-organisms able to grow at high concentrations of toxic solvents. Such bacteria and their solvent-stable enzymes are perceived to be useful for biotransformations in nonaqueous media. In the present study, the solvent-responsive features of a lipase–producing, solvent-tolerant strain Pseudomonas aeruginosa PseA have been investigated to understand the cellular mechanisms followed under solvent-rich conditions.
Methods and Results:  The solvents, cyclohexane and tetradecane with differing log P -values (3·2 and 7·6 respectively), have been used as model systems. Effect of solvents on (i) the cell morphology and structure (ii) surface hydrophobicity and (iii) permeability of cell membrane have been examined using transmission electron microscopy, atomic force microscopy and other biochemical techniques. The results show that (i) less hydrophobic (low log P -value) solvent cyclohexane alters the cell membrane integrity and (ii) cells adapt to organic solvents by changing morphology, size, permeability and surface hydrophobicity. However, no such changes were observed in the cells grown in tetradecane.
Conclusions:  It may be concluded that P. aeruginosa PseA responds differently to solvents of different hydrophobicities. Bacterial cell membrane is more permeable to less hydrophobic solvents that eventually accumulate in the cytoplasm, while highly hydrophobic solvents have lesser tendency to access the membrane.
Significance and Impact of the Study:  To the best of our knowledge, these are first time observations that show that way of bacterial solvent adaptability depends on nature of solvent. Difference in cellular responses towards solvents of varying log P -values (hydrophobicity) might prove useful to search for a suitable solvent for carrying out whole-cell biocatalysis.     

Antimicrobial action of silver nitrate

Silver nitrate 3 mug/ml prevented the separation into two daughter cells of sensitive dividing cells of Pseudomonas aeruginosa growing in nutrient broth plus the chemical. Cell size of sensitive cells was increased and the cytoplasmic contents, cytoplasmic membrane and external cell envelope structures were all abnormal. P. aeruginosa cells grown in the presence of silver nitrate 9 mug/ml showed all these changes to a marked degree except inhibition of cell division was not observed. Silver nitrate (1.5 mug/ml) in distilled water inactivated bacteriophage T2 particles as determined by their infectivity to Escherichia coli B cultures. Lysozyme (50 mug/ml) reduced, and sodium chloride (0.9%) blocked this activity.     

Effect of pH on the biosorption of nickel and other heavy metals by Pseudomonas fluorescens 4F39

Accumulation of heavy metals by Pseudomonas fluorescens 4F39 was rapid and pH-dependent. The affinity series for bacterial accumulation of metal cations decreased in the order Ni>>Hg>U>>As>Cu>Cd>Co>Cr>Pb. Metal cations were grouped into those whose accumulation increased as the pH increased, with a maximum accumulation at the pH before precipitation (Ni, Cu, Pb, Cd, Co), and those whose maximum accumulation was not associated with precipitation (Cr, As, U, Hg). High Ni²⁺ accumulation was studied. Electron microscopy indicated that at pH 9, Ni²⁺ accumulated on the cell surface as needle and hexagon-like precipitates, whose crystalline structure was confirmed by electron diffraction analysis and corresponded to two different orientations of the nickel hydroxide crystals. Crystals on cells showed marked anisotropy by X-ray powder diffraction, which differentiated them from crystals observed in nickel solution at pH 10 and 11 and from commercial nickel hydroxide. Nickel biosorption by Pseudomonas fluorescens 4F39 was a microprecipitation consequence of an ion exchange. Journal of Industrial Microbiology & Biotechnology (2000) 24, 146–151. Received 22 June 1999/ Accepted in revised form 04 December 1999     

Evidence for the location of OprM in the Pseudomonas aeruginosa outer membrane

Abstract OprM with a M _r of 49 K is associated with the multidrug resistance of Pseudomonas aeruginosa . Detergent fractionation of bacterial cells has demonstrated that OprM is located in the outer membrane from which it sediments with the other major outer membrane proteins. In this study we have determined the location of OprM as the P. aeruginosa outer membrane. Western immunoblots of cell fractions, obtained by sucrose density gradient centrifugation of whole cell lysates, were probed with an OprM-specific murine polyclonal antiserum.     

Isolation and characterization of a bio-agent antagonistic to diatom, Stephanodiscus hantzschii

AIMS: Identification of bacterium HYK0203-SK02 and its lysis of Stephanodiscus hantzschii. METHODS AND RESULTS: In an effort to identify a bio-agent capable of controlling S. hantzschii blooms, we used the algal lawn method to identify 76 bacteria in relevant water samples. Of these, the seven isolate showed algicidal activity against S. hantzschii; isolate HYK0203-SK02 exhibited the strongest algicidal activity, and was used for further analysis. 16S rDNA sequencing of this isolate allowed us to identify HYK0203-SK02 as a strain of Pseudomonas putida (99.2%). Growth of S. hantzschii was strongly suppressed by bacteria in all growth phases, with the strongest algicidal activity noted against diatoms in the exponential stage (5-18 days). Host range assays revealed that isolate HYK0203-SK02 also strongly inhibited the growth of Microcystis aeruginosa, but stimulated growth of the diatom Cyclotella sp., which has a similar structure to that of S. hantzschii. Biochemical assays revealed that the algicidal substance seemed to be localized in the cytoplasmic membrane of this newly identified algicidal bacterium. CONCLUSION: The algicidal bacteria P. putida HYK0203-SK02 caused cell lysis and death of not only diatom S. hantzschii but also cyanobacteria M. aeruginosa, dramatically. Algicidal substance might be located at the compartment of cytoplasmic membrane. SIGNIFICANCE AND IMPACT OF THE STUDY: Taken together, our results indicate that P. putida HYK0203-SK02 may be a potential bio-agent for future use in controlling freshwater diatomic blooms.     

Formation of polyesters consisting of medium-chain-length 3-hydroxyalkanoic acids from gluconate by Pseudomonas aeruginosa and other fluorescent pseudomonads.

Pseudomonas aeruginosa PAO and 15 other strains of this species synthesized a polyester with 3-hydroxydecanoate as the main constituent (55 to 76 mol%) if the cells were cultivated in the presence of gluconate and if the nitrogen source was exhausted; 3-hydroxyhexanoate, 3-hydroxyoctanoate, and 3-hydroxydodecanoate were minor constituents of the polymer. The polymer was deposited in granules within the cell and amounted to 70% of the cell dry matter in some strains. Among 55 different strains of 41 Pseudomonas species tested, P. aureofaciens (21.6% of cellular dry matter), P. citronellolis (78.0%), P. chlororaphis (8.5%), P. marginalis (11.4%), P. mendocina (50.7%), P. putida (33.5%), and Pseudomonas sp. strain DSM 1650 (54.6%) accumulated this type of polymer at significant levels (greater than 5%) during cultivation on gluconate. In two strains of P. facilis and P. fluorescens, as well as in one strain of P. syringae, this polymer was detected as a minor constituent (much less than 5%). All other strains accumulated either poly(3-hydroxybutyrate) or a polymer consisting mainly of 3-hydroxyoctanoate with octanoate but no polyester with gluconate as the carbon source. Only a few species (e.g., P. stutzeri) were unable to accumulate poly(hydroxyalkanoic acids) (PHA) at all. These results indicated that the formation of PHA depends on a pathway which is distinct from all other known PHA-biosynthetic pathways. The polyesters accumulated by gluconate- or octanoate-grown cells of recombinant strains of P. aeruginosa and P. putida, which harbored the Alcaligenes eutrophus poly(3-hydroxybutyrate)biosynthetic genes, contained 3-hydroxybutyrate as an additional constituent.     

Formation of polyesters consisting of medium-chain-length 3-hydroxyalkanoic acids from gluconate by Pseudomonas aeruginosa and other fluorescent pseudomonads

Pseudomonas aeruginosa PAO and 15 other strains of this species synthesized a polyester with 3-hydroxydecanoate as the main constituent (55 to 76 mol%) if the cells were cultivated in the presence of gluconate and if the nitrogen source was exhausted; 3-hydroxyhexanoate, 3-hydroxyoctanoate, and 3-hydroxydodecanoate were minor constituents of the polymer. The polymer was deposited in granules within the cell and amounted to 70% of the cell dry matter in some strains. Among 55 different strains of 41 Pseudomonas species tested, P. aureofaciens (21.6% of cellular dry matter), P. citronellolis (78.0%), P. chlororaphis (8.5%), P. marginalis (11.4%), P. mendocina (50.7%), P. putida (33.5%), and Pseudomonas sp. strain DSM 1650 (54.6%) accumulated this type of polymer at significant levels (greater than 5%) during cultivation on gluconate. In two strains of P. facilis and P. fluorescens, as well as in one strain of P. syringae, this polymer was detected as a minor constituent (much less than 5%). All other strains accumulated either poly(3-hydroxybutyrate) or a polymer consisting mainly of 3-hydroxyoctanoate with octanoate but no polyester with gluconate as the carbon source. Only a few species (e.g., P. stutzeri) were unable to accumulate poly(hydroxyalkanoic acids) (PHA) at all. These results indicated that the formation of PHA depends on a pathway which is distinct from all other known PHA-biosynthetic pathways. The polyesters accumulated by gluconate- or octanoate-grown cells of recombinant strains of P. aeruginosa and P. putida, which harbored the Alcaligenes eutrophus poly(3-hydroxybutyrate)biosynthetic genes, contained 3-hydroxybutyrate as an additional constituent.     

Subcellular localization of chromium and nickel in root cells of Allium cepa by EELS and ESI

The ultrastructural investigation of the root cells of Allium cepa L. exposed to two different concentrations of chromium + nickel (Cr+Ni) (10 micromol/L and 100 micromol/L) revealed that toxic symptoms were induced by increasing heavy metal concentration and treatment time. Several significant ultrastructural changes were caused by 100 micromol/L Cr+Ni - deposition of electron dense material in cell walls; larger vacuolar precipitates surrounded by membranes inside vacuoles; increment of disintegrated organelles and high vacuolization in cytoplasm. The localization of the precipitates in which the metal ions were detected by electron energy loss spectroscopy (EELS) and electron spectroscopic imaging (ESI) was investigated. Chromium and nickel were localized in the electron dense precipitates of the root cells exposed to only 100 micromol/L Cr+Ni. None were found in the root cells exposed to 10 micromol/L Cr+Ni. Higher amounts of Cr+Ni were mainly accumulated in the cell walls and vacuoles of the fourth or fifth cortical layer.     

Amikacin disrupts the cell envelope of Pseudomonas aeruginosa ATCC 9027

Amikacin, an aminoglycoside known to inhibit protein synthesis, was found to perturb the outer membrane of a sensitive Pseudomonas aeruginosa strain (ATCC 9027). This perturbation was monitored using electron microscopy and biochemical analyses. Following exposure to 20 micrograms amikacin/mL for 15 min, the outer membrane of exponentially growing cells lost 15% of its protein, 18% of its lipopolysaccharide, and 18% of its phosphate. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed that the whole spectrum of outer membrane protein and lipopolysaccharide was affected. Similarly, atomic absorption spectrophotometry revealed that magnesium and calcium were also lost. When cells were treated with amikacin, electron microscopy of negative stains showed a substantial increase in outer membrane blebbing. Freeze fractures revealed changes in membrane fracture pattern and particle distribution, and thin sections revealed a sequential disruption of the cell envelope beginning at the outer membrane and ending at the plasma membrane. This study supports the proposal that aminoglycoside antibiotics cross the outer membrane of Pseudomonas aeruginosa by displacing metal cations necessary to stabilize the organic constituents of the membrane. Their removal results in loss of the outer membrane and the formation of transient small holes which permit the antibiotic access to the cytoplasmic membrane where it is transported into the cytoplasm.     

Outer membrane proteins of Pseudomonas

In this review, we describe the outer membrane proteins of Pseudomonas aeruginosa and related strains from the Pseudomonas fluorescens rRNA homology group of the Pseudomonadaceae, with emphasis on the physiological function and biochemical characteristics of these proteins. The use of opr (for outer membrane protein) is proposed as the genetic designation for the P. aeruginosa outer membrane proteins and letters are assigned, in conjunction with this designation, to known outer membrane proteins. Proteins whose primary functions involve pore formation, transport of specific substrates, cell structure determination and membrane stabilization are discussed. The conservation of selected proteins in the above Pseudomonas species is also examined.