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Volatile compounds produced in cheese by pseudomonas strains of dairy origin belonging to six different species.

Production of volatile compounds by seven Pseudomonas strains belonging to six different species, Ps. brenneri, Ps. graminis, Ps. libanensis, Ps. lundensis, Ps. putida, and Ps. rhodesiae, was investigated, with the aim of elucidating their possible contribution to the volatile profile of cheese. Laboratory-scale cheeses were made from pasteurized milk of low bacterial counts separately inoculated with approximately 10(5) colony-forming units/mL of each Pseudomonas strain and ripened for 12 days at 10 degrees C. A total of 122 volatile compounds were identified in cheeses by GC-MS of the dynamic headspace. The abundance of 62 compounds, belonging to eight chemical groups (aldehydes, ketones, acids, esters, alcohols, hydrocarbons, benzene compounds, and sulfur compounds) increased during ripening for at least one of the strains. Most groups of volatile compounds were more abundant in the outer part of cheeses than in the inner part, in agreement with the aerobic metabolism of the genus Pseudomonas and coinciding with the higher counts in the outer part. Production of volatile compounds in cheese by Pseudomonas was shown to be species-dependent.

Alcohols↗

Hydroxylamine metabolism in Pseudomonas PB16: involvement of a novel hydroxylamine oxidoreductase.

Pseudomonas strain PB16, a Gram-negative heterotrophic nitrifying bacterium closely related to Pseudomonas azalaica on the basis of 16 S rDNA analysis, was able to use hydroxylamine as an additional energy source during growth in acetate limited chemostat cultures giving an increased biomass yield. In aerobically growing cells of Pseudomonas PB16 only 50% of supplemented hydroxylamine could be recovered as nitrite. In addition to nitrite, N2O could be detected in the chemostat off-gas, indicating combined heterotrophic nitrification and aerobic denitrification. The maximum specific hydroxylamine oxidizing activity observed was 450 nmol per min per mg dry weight, with a Ks of approximately 40 microM. Upon addition of hydroxylamine to the medium, Pseudomonas PB16 induced a soluble 132 KDa dimeric hydroxylamine oxidoreductase. The enzyme had a pH optimum of 9, and did not contain spectroscopic features typical for cytochromes, which is in contrast to hydroxylamine oxidoreductases found in autotrophic bacteria.

Acetates↗

Unusual traits of the pyoverdin-mediated iron acquisition system in Pseudomonas putida strain BTP1.

Fluorescent Pseudomonas species are characterized by the production of pyoverdin-type siderophores for Fe3+ acquisition in iron-limited environments. Since it produces a structurally specific pyoverdin, Pseudomonas putida strain BTP1 could represent a valuable tool in an attempt to correlate the structural features of these compounds with some specificity in their two main properties i.e. affinity for iron and recognition rate by other Pseudomonas strains. An uncommonly high affinity for iron of the pyoverdin synthetized by P. putida BTP1 was observed by comparing both the apparent stability constant and the decomplexation kinetic of its ferric complex with those of ferripyoverdins from other strains. On another hand, results from growth stimulation experiments and labeled ferripyoverdin uptake assays highlighted the very low recognition rate of BTP1 isopyoverdins by membrane receptors of foreign strains. By contrast, P. putida BTP1 was able to utilize a broad spectrum of structurally unrelated exogenous pyoverdins by means of multiple receptors that are likely constitutively expressed in its outer membrane. The unusual traits of its pyoverdin-mediated iron acquisition system should contribute to enlarge the ecological competence of Pseudomonas putida BTP1 in terms of colonization and persistence in the rhizosphere.

Amino Acid Sequence↗

The metabolism of cresols by species of Pseudomonas.

1. A comparison of rates of oxidation of various compounds by whole cells indicated that protocatechuate was a reaction intermediate when a non-fluorescent species of Pseudomonas oxidized p-cresol. In contrast, a fluorescent Pseudomonas oxidized 3-methylcatechol and 4-methylcatechol when grown with p-cresol, but did not oxidize protocatechuate. 2. Heat-treated extracts of the fluorescent Pseudomonas oxidized catechol, 3-methylcatechol and 4-methylcatechol to ring-fission products, the spectroscopic properties of which were recorded. Rates of enzymic degradation of these products were also measured. 3. Acetic acid and formic acid were obtained by the action of a Sephadex-treated extract on 3-methylcatechol and 4-methylcatechol respectively. In each case 0.8mol. of the carboxylic acid was formed from 1.0mol. of substrate. 4. Dialysed extracts converted 3-methylcatechol into acetaldehyde and pyruvate, with 4-hydroxy-2-oxovalerate as a reaction intermediate. 4-Methylcatechol was converted first into 4-hydroxy-2-oxohexanoate and then into propionaldehyde and pyruvate. 5. The ring-fission product of catechol was formed from phenol by a fluorescent Pseudomonas, that of 3-methylcatechol was formed from o-cresol and m-cresol, and the ring-fission product of 4-methylcatechol was given from p-cresol. Propionate was readily oxidized by these cells after growth with p-cresol, but this compound was not attacked when phenol, o-cresol or m-cresol served as source of carbon. 6. Cell extracts appeared to attack only one enantiomer of synthetic 4-hydroxy-2-oxohexanoate.

Acetaldehyde↗

Aspects of glycine and serine biosynthesis during growth of Pseudomonas AM1 on C compounds.

1. Methanol or formate can replace serine or glycine as supplements for growth on succinate of the auxotrophic mutants 20S and 82G of Pseudomonas AM1, showing that the organism can synthesize glycine and serine in net fashion from C(1) units. 2. Double mutants of Pseudomonas 20S and 82G have been prepared (20ST-1 and 82GT-1) that are unable to grow on succinate+1mm-glyoxylate, succinate+2mm-methanol or methanol alone. 3. Mutants 20ST-1 and 82GT-1 lacked serine-glyoxylate aminotransferase activity, and revertants to the phenotype of 20S and 82G regained serine-glyoxylate aminotransferase activity. A total revertant of 82GT-1 to wild-type phenotype regained activities of serine hydroxymethyltransferase and serine-glyoxylate aminotransferase. 4. The activity of serine-glyoxylate aminotransferase in methanol-grown Pseudomonas AM1 is eightfold higher than in the succinate-grown organism. 5. The combined results show that in Pseudomonas AM1 serine-glyoxylate aminotransferase is necessary for growth on C(1) compounds and is involved in the conversion of methanol into glycine via glyoxylate. 6. It is suggested that the phosphorylated pathway of serine biosynthesis from phosphoglycerate replenishes the supply of alpha-amino groups necessary for the flow of glyoxylate through the main assimilatory pathway during growth on C(1) compounds.

Carbon↗

Microbial metabolism of C 1 and C 2 compounds. The involvement of glycollate in the metabolism of ethanol and of acetate by Pseudomonas AM1.

Pseudomonas AM1 grows on ethanol with a mean generation time of about 10h. A single alcohol dehydrogenase is responsible for oxidation of both methanol and ethanol. It is proposed that the glyoxylate bypass does not operate in Pseudomonas AM1 during growth on ethanol. Although malate synthase is present in extracts of ethanol-grown Pseudomonas AM1, the activity of isocitrate lyase is negligible. Short-term incubation experiments with [(14)C]ethanol and [(14)C]acetate indicate that a novel pathway operates during growth of Pseudomonas AM1 on ethanol. Glycollate, glyoxylate and malate are probably intermediates in this pathway.

Acetates↗

The reaction of Pseudomonas aeruginosa cytochrome c oxidase with carbon monoxide.

The binding of CO to ascorbate-reduced Pseudomonas cytochrome oxidase was investigated by static-titration, stopped-flow and flash-photolytic techniques. Static-titration data indicated that the binding process was non-stoicheiometric, with a Hill number of 1.44. Stopped-flow kinetics obtained on the binding of CO to reduced Pseudomonas cytochrome oxidase were biphasic in form; the faster rate exhibited a linear dependence on CO concentration with a second-order rate constant of 2 X 10(4) M-1-s-1, whereas the slower reaction rapidly reached a pseudo-first-order rate limit at approx. 1s-1. The relative proportions of the two phases observed in stopped-flow experiments also showed a dependency on CO concentration, the slower phase increasing as the CO concentration decreased. The kinetics of CO recombination after flash-photolytic dissociation of the reduced Pseudomonas cytochrome oxidase-CO complex were also biphasic in character, both phases showing a linear pseudo-first-order rate dependence on CO concentration. The second-order rate constants were determined as 3.6 X 10(4)M-1-s-1 and 1.6 X 10(4)M-1-s-1 respectively. Again the relative proportions of the two phases varied with CO concentration, the slower phase predominating at low CO concentrations. CO dissociation from the enzyme-CO complex measured in the presence of O2 and NO indicated the presence of two rates, of the order of 0.03s-1 and 0.15s-1. When sodium dithionite was used as a reducing agent for the Pseudomonas cytochrome oxidase, the CO-combination kinetics observed by both stopped flow and flash photolysis were extremely complex and not able to be simply analysed.

Binding Sites↗

A non-modular endo-beta-1,4-mannanase from Pseudomonas fluorescens subspecies cellulosa.

Pseudomonas fluorescens subsp. cellulosa when cultured in the presence of carob galactomannan degraded the polysaccharide. To isolate gene(s) from P. fluorescens subsp. cellulosa encoding endo-beta-1,4-mannanase (mannanase) activity, a genomic library of Pseudomonas DNA, constructed in lambda ZAPII, was screened for mannanase-expressing clones using the dye-labelled substrate, azo-carob galactomannan. The nucleotide sequence of the pseudomonad insert from a mannanase-positive clone revealed a single open reading frame of 1257 bp encoding a protein of M(r) 46,938. The deduced N-terminal sequence of the putative polypeptide conformed to a typical prokaryotic signal peptide. Truncated derivatives of the mannanase, lacking 54 and 16 residues from the N- and C-terminus respectively of the mature form of the enzyme, did not exhibit catalytic activity. Inspection of the primary structure of the mannanase did not reveal any obvious linker sequences or protein motifs characteristic of the non-catalytic domains located in other Pseudomonas plant cell wall hydrolases. These data indicate that the mannanase is non-modulator, comprising a single catalytic domain. Comparison of the mannanase sequence with those in the SWISSPROT database revealed greatest sequence homology with the mannanase from Bacillus sp. Thus the Pseudomonas enzyme belongs to glycosyl hydrolase Family 26, a family containing mannanases and endoglucanases. Analysis of the substrate specificity of the mannanase showed that the enzyme hydrolysed mannan and galactomannan, but displayed little activity towards other polysaccharides located in the plant cell wall. The enzyme had a pH optimum of approx. 7.0, was resistant to proteolysis and had an M(r) of 46,000 when expressed by Escherichia coli.

Amino Acid Sequence↗

Intrinsic and acquired resistance to quaternary ammonium compounds in food-related Pseudomonas spp.

AIMS: To determine the sensitivity of a strain used for disinfectants testing (Pseudomonas aeruginosa ATCC 15442) and food-associated isolates to benzalkonium chloride and didecyl dimethylammonium chloride (DDAC). To determine whether the increase in bacterial resistance after adaptation to DDAC can be associated with phenotypic changes. To test the activity of alternative disinfectants to eliminate resistant Pseudomonas spp. METHODS AND RESULTS: Pseudomonas aeruginosa ATCC 15442 was among the most resistant strains tested using a bactericidal suspension test. Growth of a sensitive Ps. fluorescens in gradually higher concentrations of DDAC resulted in stable higher resistance and to some cross-resistance to several antibacterial agents, with the exception of disinfectants containing chloramine T, glutaraldehyde or peracetic acid. It was shown by microscopy that adaptation was followed by loss of flagella, and slime formation. Removal of the slime by sodium dodecyl sulphate resulted in partial loss of the acquired resistance. CONCLUSIONS: Pseudomonas spp. may adapt to survive against higher concentrations of quaternary ammonium compounds (QACs), but resistant strains can be eliminated with chemically unrelated disinfectants. SIGNIFICANCE AND IMPACT OF THE STUDY: The work supports the rotation of disinfectants in food processing environments for avoiding the development of bacterial resistance to QACs. The alternating disinfectants should be chosen carefully, because of possible cross-resistance.

Anti-Bacterial Agents↗

Impact of biocontrol agents Pseudomonas fluorescens CHA0 and its genetically modified derivatives on the diversity of culturable fungi in the rhizosphere of mungbean.

AIMS: To assess whether Pseudomonas fluorescens strain CHA0 and its genetically modified derivatives, CHA0/pME3424 (antibiotic over-producer) and CHA89 (antibiotic-deficient) could have an impact on the fungal community structure and composition in the rhizosphere of mungbean. METHODS AND RESULTS: Under glasshouse conditions, mungbean was grown repeatedly in the same soil, which was inoculated with CHA0, CHA0/pME3424, CHA89 or was left untreated. Treatments were applied to soil at the start of each 36-day mungbean growth cycle, and their effects on the diversity of the rhizosphere populations of culturable fungi were assessed at the end of the first, second and third cycles. The effects of CHA0 and CHA0/pME3424 did differ from the controls while CHA89 did not. Whereas all major fungal species were frequently isolated from both bacterized and nonbacterized rhizospheres, certain fungal species were exclusively promoted or specifically suppressed from Pseudomonas-treated soils. In general, fungal diversity and equitability tended to decrease with time while species richness slightly increased. Whilst a total of 29 fungal species were isolated from the mungbean rhizosphere, only eight species colonized the root tissues. CONCLUSIONS: Soil inoculation with Ps. fluorescens CHA0 or CHA0/pME3424 altered fungal community structure in mungbean rhizosphere but strain CHA89 failed to produce such effect. SIGNIFICANCE AND IMPACT OF THE STUDY: Pseudomonas fluorescens-mediated alteration in the composition and structure of fungal communities might have acute or lasting effects on ecosystem functioning. Furthermore, the study provides useful data pertinent to characterization of the fate of genetically modified inoculants (e.g. antibiotic-overproducing Pseudomonas strains) released into the environment.

Antibiosis↗

Purification, characterization, and identification of a sphingomyelin synthase from Pseudomonas aeruginosa. PlcH is a multifunctional enzyme.

Sphingomyelin synthase is the enzyme that synthesizes sphingomyelin (SM) in mammalian cells by transferring a phosphorylcholine moiety from phosphatidylcholine to ceramide. Despite its importance, the gene and/or the protein responsible for this activity has not yet been identified. Here we report the purification, identification, and biochemical characterization of an enzymatic activity that synthesizes SM in Pseudomonas aeruginosa. SM synthase-like activity was found secreted in the culture medium of P. aeruginosa, strains PA01 and PAK, whereas it could not be detected in cultures of Escherichia coli. From the medium of PAK cultures, SM synthase was purified through sequential chromatographic columns. After separation on polyacrylamide-SDS gels and visualization by silver staining, the purified enzyme showed two bands, one of approximately 75 kDa and one of 30-35 kDa. Interestingly, the highly purified SM synthase preparation also showed neutral sphingomyelinase activity. We therefore investigated whether the protein we purified as SM synthase could actually be the previously identified PlcH, a 78-kDa phospholipase C known to hydrolyze phosphatidylcholine and SM in P. aeruginosa. First, the purified SM synthase preparation contained a 78-kDa protein that reacted with monoclonal antibodies raised against purified PlcH. Second, purified PlcH showed SM synthase activity. Third, using different knockout mutant strains for the PlcH operon, PlcH was found to be necessary for SM synthase activity in P. aeruginosa. Interestingly, SM synthase activity was specific to the Pseudomonas PlcH as other bacterial phospholipases did not display SM synthase activity. Biochemical studies on the Pseudomonas SM synthase confirmed that it is a transferase, similar to the mammalian enzyme, that specifically recognizes the choline head-group and the primary hydroxyl on ceramide. This SM synthase did not have reverse transferase activity. In conclusion, the Pseudomonas PlcH also exerts SM synthase activity; therefore, for the first time, we have identified a structural gene for a SM synthase.

Chromatography↗

Chitinolytic and cellulolytic Pseudomonas sp. antagonistic to fungal pathogens enhances nodulation by Mesorhizobium sp. Cicer in chickpea.

Pseudomonas strains isolated from the rhizosphere of chickpea (Cicer arietinum L.) and green gram (Vigna radiata L.) were screened for the production of chitinases and cellulases. Five Pseudomonas strains were found to produce appreciable amounts of both enzymes in culture-free supernatants and showed growth inhibition of the two fungi Pythium aphanidermatum (Oomycete) and Rhizoctonia solani (Basidiomycete) in plates on potato dextrose agar medium. The fungal growth inhibition was not correlated with cell wall-degrading enzyme activity, which suggested that other antifungal compounds produced by these rhizobacteria were also involved in antagonism. Coinoculation of the Pseudomonas strains with the Mesorhizobium sp. Cicer strain Ca 181 resulted in a significant increase in nodule biomass when grown under sterilized chillum jar conditions. The results suggest that hydrolytic enzymes produced by Pseudomonas sp. contribute to suppression of plant diseases by inhibiting growth of phytopathogenic fungi and also promote nodulation of legumes by rhizobia.

Antibiosis↗

Effects of combined 405-nm and 880-nm light on Staphylococcus aureus and Pseudomonas aeruginosa in vitro.

OBJECTIVE: The aim of this study was to determine the effect of a combination of 405-nm blue light and 880-nm infrared light on Staphylococcus aureus and Pseudomonas aeruginosa in vitro. BACKGROUND DATA: Reports indicate that certain wavelengths and treatment parameters of light promote the growth of bacteria, but our earlier study indicates that light at specific wavelengths and intensities are bactericidal for specific organisms (1). METHODS: Two common aerobes, Staphylococcus aureus and Pseudomonas aeruginosa were tested because of their frequent isolation from skin infections and wounds. Each organism was treated simultaneously with a combination of 405-nm and 880-nm light emitted by a cluster of Super Luminous Diodes (SLDs). Doses of 1, 3, 5, 10, and 20 Jcm2 were used. Colony counts were performed and compared to untreated controls using Student t tests and one-way ANOVA with Tukey and Scheffe post hoc analyses. RESULTS: The results revealed significant dose-dependent bactericidal effects of the combined blue and infrared light on Staphylococcus aureus (F 4,94 = 5.38, p = 0.001) and Pseudomonas aeruginosa (F 4,95 = 21.35, p < 0.001). With P. aeruginosa, the treatment reduced the number of bacteria colonies at all doses, achieving statistical significance at 1, 3, and 20 J cm2 doses and reducing bacterial colony by as much as 93.8%; the most effective dose being 20 J cm2. Irradiation of S. aureus resulted in statistically significant decreases in bacterial colonies at all dose levels; the most decrease, 72%, was also achieved with 20 Jcm2. CONCLUSION: Appropriate doses of combined 405-nm and 880-nm phototherapy can kill Staphylococcus aureus and Pseudomonas aeruginosa in vitro, suggesting that a similar effect may be produced in clinical cases of bacterial infection.

Colony Count, Microbial↗

Comparison of antibiotic regimens for treatment of experimental pneumonia due to Pseudomonas.

The high mortality associated with pneumonia due to Pseudomonas aeruginosa prompted a comparative trial of several currently available antibiotic regimens for this infection in a guinea pig model. Normal guinea pigs receiving an intratracheal challenge of 10(8) colony-forming units of Pseudomonas routinely died within 3-48 hr when treated with saline injections. Treatment with carbenicillin or ticarcillin did not affect this uniformly fatal outcome. Groups of animals treated with gentamicin or tobramycin had survival rates of 39% and 67%, respectively. The addition of either carbenicillin or ticarcillin to an aminoglycoside failed to enhance further the survival rates or durations of survival after infection. These survival data were supported by studies showing superior clearance of viable Pseudomonas from lung tissues in aminoglycoside-treated animals chosen at random for sacrifice 3 hr after infection. Thus, in animals experimentally challenged with P. aeruginosa to cause pneumonia and in which only a single isolate of Pseudomonas was evaluated, protection from pulmonary infection was best provided by an aminoglycoside rather than by a beta-lactam antibiotic.

Aminoglycosides↗

Oral ciprofloxacin and a monoclonal antibody to lipopolysaccharide protect leukopenic rats from lethal infection with Pseudomonas aeruginosa.

To study the treatment of infection with Pseudomonas aeruginosa, a leukopenic rat model was developed that closely mimics the pathogenesis of Pseudomonas infection in man. This model achieved approximately 90% mortality within 10 d of infection. Pseudomonas organisms were inconsistently shed from the feces despite gastrointestinal colonization (9 fecal v. 23 cecal cultures positive for challenge strain in 28 rats). Treatment of rats with oral ciprofloxacin at 40 mg/kg afforded complete protection. A suboptimal dose of ciprofloxacin (20 mg/kg), achieving peak levels of 0.31 micrograms/mL in serum and 26.3 micrograms/mL in stool, resulted in survival of 8 (40%) of 20 rats. Intraperitoneal administration of a monoclonal antibody directed at the lipopolysaccharide of the challenge strain of Pseudomonas resulted in survival of 5 rats (26%). The combination of the two increased the survival to 75% (15 of 20, P less than 0.05 compared to either treatment alone). Thus, the combination of suboptimal doses of ciprofloxacin and a monoclonal antibody appears to protect leukopenic rats from lethal infection better than either treatment alone.

Administration, Oral↗

Treatment of serious Pseudomonas infections with azlocillin.

Azlocillin is a semisynthetic acylureidopenicillin with increased activity against most strains of Pseudomonas aeruginosa. It was given as the sole antibacterial agent in the treatment of 21 patients with serious pulmonary, wound, bone or joint, or urinary tract infections, endocarditis, or malignant external otitis caused by Pseudomonas sp. In preliminary in vitro tests, azlocillin inhibited 90% of 36 clinical isolates, while carbenicillin and ticarcillin inhibited only 60% and 73%, respectively. Mean MIC of azlocillin against Ps. aeruginosa isolated from the 21 study patients was 9.8 mg/l; more than 50% of the strains were inhibited by a concentration of 6.25 mg/l. Intravenous administration of the antibiotic at a mean dosage of 17 g/day for 6 to 59 days resulted in an excellent or good clinical response in 90% (19) of the patients treated. Pseudomonas sp. was eliminated from the site of infection in 67% (14) of the patients. Azlocillin therapy was well tolerated; in only two patients, both of whom had penicillin-type rashes, was it necessary to discontinue therapy. Azlocillin was a safe and effective antimicrobial agent for the treatment of serious infections caused by strains of Pseudomonas sp., primarily Ps. aeruginosa.

Adult↗

Ceftazidime in patients with Pseudomonas infections.

Ceftazidime was administered to 41 patients with serious infections caused by Pseudomonas aeruginosa (24 cases) and other bacteria (17 cases). The clinical response rate of pseudomonas infections (88%) was similar to that of other bacteria (94%) with microbiological eradication of 83% of initial pseudomonas isolates compared to 82% of non-pseudomonas strains. The development of resistance to ceftazidime during therapy was observed in 3 cases (Enterobacter agglomerans, Enterobacter cloacae, and Ps. aeruginosa), and superinfection by a resistant Enterobacter agglomerans occurred in one case. Adverse reactions of clinical significance included two cases of leukopenia and one case each of azotaemia, diarrhoea (Clostridium difficile toxin positive), and rash.

Abscess↗

The treatment of Pseudomonas aeruginosa urinary tract infections with norfloxacin.

Norfloxacin, a new quinolone compound has marked activity against strains of Pseudomonas aeruginosa being superior to that of gentamicin, carbenicillin, ticarcillin, the ureidopenicillins and cephalosporins, and similar to that of tobramycin. Nineteen patients with complicated urinary tract infections caused by Pseudomonas spp. were treated with norfloxacin and 16 (84%) responded to therapy. No side or toxic effects were seen. Two of the three failures of treatment were due to underlying uripenicillins and cephalosporins, and similar to that of tobramycin. Nineteen patients with complicated urinary tract infections caused by Pseudomonas spp. were treated with norfloxacin and 16 (84%) responded to therapy. No side or toxic effects were seen. Two of the three failures of treatment were due to underlying uripenicillins and cephalosporins, and similar to that of tobramycin. Nineteen patients with complicated urinary tract infections caused by Pseudomonas spp. were treated with norfloxacin and 16 (84%) responded to therapy. No side or toxic effects were seen. Two of the three failures of treatment were due to underlying urinary tract disease. The other failure was due to a resistant strain of Ps. aeruginosa. Norfloxacin is a new valuable oral antimicrobial agent with a wide range of bacterial activity which includes many strains of bacteria resistant to other agents. Due to its high urinary concentrations, norfloxacin is particularly indicated in the treatment of urinary tract infections.

Aged↗