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Burkholderia cepacia complex genomovars: utilization of carbon sources, susceptibility to antimicrobial agents and growth on selective media.

AIMS: To investigate the relationship between genomovar status and carbon source utilization, antibiotic susceptibility and growth ability on selective media of 142 clinical and environmental Burkholderia cepacia complex (Bcc) isolates belonging to all nine genomovars. METHODS AND RESULTS: Carbon source utilization and growth on selective media were tested by agar plate multipoint inoculation. Antimicrobial minimum inhibitory concentration (MIC) values were determined by agar dilution. Of all carbon sources, l-arabinose was most frequently utilized, supporting growth of 90% of all isolates. Burkholderia cepacia genomovar VI failed to utilize azelaic acid, penicillin G, phtalate, salicin and tryptamine. Overall, B. vietnamiensis and B. anthina were most susceptible and B. cepacia genomovar VI most resistant to antimicrobial agents. Burkholderia cepacia selective agar (BCSA) and the Mast B. cepacia medium supported growth of Bcc isolates most efficiently. CONCLUSIONS: This study demonstrates phenotypic heterogeneity within the Bcc. Some trends can be observed at the genomovar level, but only B. cepacia genomovar VI could be differentiated unambiguously on the basis of its inability to grow on PCAT. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides an update on some differential phenotypic characteristics of all nine Bcc genomovars.

Burkholderia cepacia↗

Growth of Pseudomonas C on C1 compounds: enzyme activites in extracts of Pseudomonas C cells grown on methanol, formaldehyde, and formate as sole carbon sources.

Pseudomonas C can grow on methanol, formaldehyde, or formate as sole carbon source. It is proposed that the assimilation of carbon by Pseudomonas C grown on different C1 growth substrates proceeds via one of two metabolic pathways, the serine pathway or the allulose pathway (the ribose phosphate cycle of formaldehyde fixation). This contention is based on the distribution of two key enzymes, each of which appears to be specifically involved in one of the assimilation pathways, glycerate dehydrogenase (serine pathway) and hexose phosphate synthetase (allulose pathway). The assimilation of methanol in Pseudomonas C cells appears to occur via the allulose pathway, whereas the utilization of formaldehyde or formate in cells grown on formaldehyde or formate as sole carbon sources appears by the serine pathway. When methanol is present together with formaldehyde or formate in the growth medium, the formaldehyde or formate is utilized by the allulose pathway.

Alcohol Oxidoreductases↗

Variation of antimetabolite sensitivity with different carbon sources in Bacillus subtilis.

The susceptibility of Bacillus subtilis to amino acid analogues was found to be markedly influenced by the carbon source used in the test media. Thialysine inhibited the bacterium with a greater number of carbon sources than the other two analogues tested. 5-Hydroxylysine was inhibitory with glycerol, lactose, D-xylose, L-arabinose and soluble starch while ethionine showed toxicity with lactose, D-xylose and L-arabinose. None of these analogues were toxic at the levels tested when D-galactose was used as carbon source. The bacterium was not susceptible to thialysine with glycerol, to 5-hydroxylysine with L-arabinose and to ethionine with lactose.

Arabinose↗

[Effect of the cultivation temperature on the dynamics of the ATP and ADP levels and on the growth of P. nigricans Thom. Strains in the presence of various carbon sources].

The effect of the medium high temperature (28--28.6 degrees C) on the yield of the dry mycelium and the levels of ATP and ADP in the cells of the highly and low productive strains of P. nigricans Thom, 117 and B at different developmental periods (2, 5, 9 and 13 days) on the mineral medium using glucose, succinate or acetate as the carbon sources was studied. It was shown that the effect of the high temperature depended on the strain and the carbon source. The growth stimulating effect of this factor observed in strain 117 grown on the media with the above carbon sources and strain B grown on the media with glucose and acetate as the carbon sources was evident at different periods and later replaced by suppression. The growth of strain 177 in the presence of succinate was not inhibited, while that of strain B was suppressed during the whole developmental cycle. Such an effect of high temperature on the culture growth was the result of different supply of energy in the form of ATP to the strains which was confirmed by the study of the dynamics of the levels of ATP and ADP and their ratio in the strains during the developmental process.

Adenosine Diphosphate↗

Production of medium-chain-length polyhydroxyalkanoates by Pseudomonas aeruginosa with fatty acids and alternative carbon sources.

In this study, medium-chain-length polyhydroxyalkanoates (mcl-PHAs) were produced by Pseudomonas aeruginosa using different carbon sources. Decanoic acid induced the highest (9.71% [+/- 0.7]) mcl-PHAs accumulation in bacterial cells at 47 h. The cells preferred to accumulate and degrade the polyhydroxyoctanoate than polyhydroxydecanoate (PHD) during early stage and final stage of the growth, respectively. The production cost of mcl-PHAs can be reduced by using edible oils as the carbon source. The bacteria accumulated 6% (+/- 0.7) of mcl-PHAs in the presence of olive oil. Besides, reused oil was another potential carbon source for the reduction of the production cost of mcl-PHAs. Overall, PHD was the major constituent in the accumulated mcl-PHAs.

Bioreactors↗

Influence of carbon source on nitrate removal by nitrate-tolerant Klebsiella oxytoca CECT 4460 in batch and chemostat cultures.

The nitrate-tolerant organism Klebsiella oxytoca CECT 4460 tolerates nitrate at concentrations up to 1 M and is used to treat wastewater with high nitrate loads in industrial wastewater treatment plants. We studied the influence of the C source (glycerol or sucrose or both) on the growth rate and the efficiency of nitrate removal under laboratory conditions. With sucrose as the sole C source the maximum specific growth rate was 0.3 h-1, whereas with glycerol it was 0.45 h-1. In batch cultures K. oxytoca cells grown on sucrose or glycerol were able to immediately use sucrose as a sole C source, suggesting that sucrose uptake and metabolism were constitutive. In contrast, glycerol uptake occurred preferentially in glycerol-grown cells. Independent of the preculture conditions, when sucrose and glycerol were added simultaneously to batch cultures, the sucrose was used first, and once the supply of sucrose was exhausted, the glycerol was consumed. Utilization of nitrate as an N source occurred without nitrite or ammonium accumulation when glycerol was used, but nitrite accumulated when sucrose was used. In chemostat cultures K. oxytoca CECT 4460 efficiently removed nitrate without accumulation of nitrate or ammonium when sucrose, glycerol, or mixtures of these two C sources were used. The growth yields and the efficiencies of C and N utilization were determined at different growth rates in chemostat cultures. Regardless of the C source, yield carbon (YC) ranged between 1.3 and 1.0 g (dry weight) per g of sucrose C or glycerol C consumed. Regardless of the specific growth rate and the C source, yield nitrogen (YN) ranged from 17.2 to 12.5 g (dry weight) per g of nitrate N consumed. In contrast to batch cultures, in continuous cultures glycerol and sucrose were utilized simultaneously, although the specific rate of sucrose consumption was higher than the specific rate of glycerol consumption. In continuous cultures double-nutrient-limited growth appeared with respect to the C/N ratio of the feed medium and the dilution rate, so that for a C/N ratio between 10 and 30 and a growth rate of 0.1 h-1 the process led to simultaneous and efficient removal of the C and N sources used. At a growth rate of 0.2 h-1 the zone of double limitation was between 8 and 11. This suggests that the regimen of double limitation is influenced by the C/N ratio and the growth rate. The results of these experiments were validated by pulse assays.

Culture Media↗

[Kinetic aspects of the relation of the growth of the producer and the biosynthesis of heliomycin depending on the carbon source in the medium].

Kinetic parameters of Streptomyces olivocinereus 11-98 growth and biosynthesis of heliomycin were studied. It was shown that carbon sources such as glycerol, mannitol and ramnose were the most favourable for the antibiotic biosynthesis. These carbon sources belonged to the group of substances providing high growth rates of the culture. Ranging of the culture growth rates and antibiotic production levels revealed a set of carbon sources providing a converse relationship between the growth rate and antibiotic biosynthesis i.e. L-arabinose, potassium gluconate, raffinose and sucrose. It was suggested that these compounds were catabolic type regulators of heliomycin biosynthesis.

Acids↗

[Expression of xylose-metabolic key genes of Trichoderma reesei on various carbon sources measured by a series of Northern hybridizations].

The expression of xylose reductase (XR), xylitol dehydrogenase (XDH) and transaldolase (TAL) genes from Trichoderma reesei, measured by Northern hybridization, were studied by adding different carbon sources (20 kinds, including single and mixed carbon sources) separately into the basal medium on which T. reesei QM9414 was grown. The experiment results indicated that the two disaccharides: sophrose and xylibiose act as a strong inducer for the expression of xr and xdh. The lactose and arabinose were identified as inducer also. The presence of glucose repressed the transcription of xr and xdh. When glucose depleted. xr and xdh were expressed at certain level, implying that expression of xr and xdh are controlled by the carbon catabolite repression mechanism and there existed a constitutive base level's expression of xr and xdh when the catabolite repression mechanism was derepressed. On the other hand, transaldolase gene strongly expressed on all the carbon sources used.

Aldehyde Reductase↗

Oxygen consumption by Metarhizium anisopliae during germination and growth on different carbon sources.

Respirometry was used to monitor the germination and growth of the entomopathogenic deuteromycete Metarhizium anisopliae on media containing carbon sources of different kinds (monosaccharides, polysaccharides, amino acids, and proteins). As also observed in several other species of fungi, M. anisopliae germination was found to be marked by a significant increase in O(2) consumption, which started a few hours before germ tube emergence. The exponential consumption of the carbon source and O(2) coincided with the exponential growth phase of the cultures. QO(2) reached a maximum value during the exponential growth phase and was drastically reduced after the depletion of the exogenous carbon source. Taking glucose as reference, we observed that casein, hydrolyzed casein, and N-acetylglucosamine accelerated germination, reduced the lag phase, and increased the growth rate. This fact demonstrates that the fungus can readily use amino acids and N-acetylglucosamine, which are the monomers of the major constituents of the insect cuticle (proteins and chitin), a property that represents an important physiological adaptation to entomopathogenicity.

Biomass↗

Identification of Rhodococcus, Gordona and Dietzia species using carbon source utilization tests ("Biotype-100" strips).

The "Biotype-100" identification system (BioMérieux, La Balme-Ies-Grottes, France) based on carbon source utilization was evaluated for its ability to discriminate among 10 species of Rhodococcus, 7 species of Gordona and one species of Dietzia. The type strains of three species of Tsukamurella and 8 species of Nocardia were also included in the study. Results were compared with chemotaxonomic and conventional data. Carbon source utilization was shown to be reliable, rapid and easy to use when compared with standard identification methods. The 29 species tested were unambiguously separated by carbon source utilization tests. Rhodococcus equi was found to be heterogenous.

Bacterial Typing Techniques↗

Chlamydia trachomatis lacks an adaptive response to changes in carbon source availability.

Most bacteria coordinately regulate gene expression as an adaptive response to a variety of environmental changes. One key environmental cue is the carbon source necessary for central metabolism. We used microarray analysis to monitor the global transcriptional response of the obligate intracellular pathogen Chlamydia trachomatis to the presence of glycolytic and gluconeogenic carbon sources. In contrast to free-living bacteria, changing the carbon source from glucose to glutamate or alpha-ketoglutarate had little effect on the global gene transcription of C. trachomatis.

Adaptation, Physiological↗

The production of gamma-linolenic acid by selected members of the Dikaryomycota grown on different carbon sources.

In this study, seven fungal strains, representing different phylogenetic groups within the Dikaryomycota, were tested for the presence of gamma-linolenic acid [18:3(omega 6)], when grown in synthetic liquid media devoid of fatty acids, on a series of 40 different carbon sources. The fungal strains represented the species Dipodascopsis uninucleata, Eurotium rubrum, Galactomyces geotrichum, Neurospora crassa, Saccharomyces cerevisiae, Spongipellis unicolor and Talaromyces flavus. Cultures were periodically harvested during growth and the fatty acids in the total lipids analysed as methyl esters, using gas chromatography and mass spectrometry. It was found that 18:3(omega 6) is present in E. rubrum CBS 350.65, S. unicolor CBS 117.16 and in T. flavus CBS 310.38NT, when these strains were grown on certain carbon sources. No correlation between the growth phase of the organism and the presence of 18:3(omega 6) could be detected. In order to confirm the production of 18:3(omega 6), the lipid metabolism of two unrelated dikaryomycotan fungi (S. unicolor CBS 117.16 and E. rubrum CBS 350.65) grown on two different carbon sources each, was examined. Cultures of E. rubrum CBS 350.65 were grown on glucose and sorbose and cultures of S. unicolor CBS 117.16 on glucose and sucrose in synthetic liquid media with a C:N ratio of 50:1 (w/w). The total lipids of these cultures were fractionated and the fatty acids in the fractions analysed as methyl esters, using gas chromatography and mass spectrometry. The lipid metabolism of both E. rubrum CBS 350.65 and S. unicolor CBS 117.16 differed on the two carbon sources used. The ab initio production of 18:3(omega 6) by E. rubrum CBS 350.65 in synthetic liquid media was confirmed. In contrast, the ab initio production of 18:3(omega 6) by S. unicolor CBS 117.16 in synthetic liquid media could not be confirmed.

Acids↗

Oxygen, pH value, and carbon source induced changes of the mode of oscillation in synchronous continuous culture of Saccharomyces cerevisiae.

Oscillations of measured process parameters occur in continuous cultures of Saccharomyces cerevisiae owing to a partial synchronization of budding. Intentional changes of the oxygen concentration, pH value, and carbon source cause effects on the period length similar to those known from variations of the dilution rate. The generation times of parent and daughter cells frequently differ in synchronous culture. To analyze the oscillation the term mode IJ of oscillation is used, which is defined as the ratio IJ of the generation times of parent and daughter cells. When the dissolved oxygen concentration was reduced to zero, the mode of oscillation changed within two periods from mode 12 to mode 11, caused by a decrease of the generation time of daughter cells and an increase of that of the parent cells. When the pH value was slowly reduced from 5.0 to 3.9, a change from mode 112 to mode 13 was observed. Mode 13, representing one parent and three daughter cell populations (the start of budding of each of the three being delayed by one period), denotes an elongated generation time of the daughter cells compared to mode 112, marked by one parent and two different daughter cell classes. When the carbon source galactose was replaced by glucose a mode change from mode 12 to mode 11 was observed. This alteration of the mode was found to be dependent on the status of the cell cycle at the time when the carbon source is changed. The population distribution in batch cultures with glucose or galactose as a substrate was analysed by dyeing the DNA and counting the bud scars. Galactose provoked higher growth rates for the older cells. According to the model for stationary synchronous growth parameters like DO, pH value or the type of carbon source can be varied within a certain range without effecting the period length. If the variation imposes a certain stress, the culture switches to a new mode. These kinds of parameters therefore provide selective measures to influence the period lengths and the modes of oscillation.

Bioreactors↗

Characterization of Aspergillus oryzae fermentation extract effects on the rumen fungus Neocallimastix frontalis, EB 188. Part 2. Carbon source utilization and effects on zoospore production.

The effect of a commercial Aspergillus oryzae fermentation extract on the utilization of carbon source and zoospore production by the rumen fungus Neocallimastix frontalis EB 188 was determined. In addition, the composition of a soluble extract prepared from the commercial product was analyzed. This extract was added to N. frontalis EB 188 cultures grown on a variety of substrates and periodically assayed for protein, enzymes, zoospore production, and carbon source utilization. The powdered product contained 93% dry matter, more than 3,000 A. oryzaespores per gram, and did not contain strong buffers or high concentrations of salt. Measurable concentrations of DNA, protein, carbohydrate and several enzymes including cellulase and amylase were also found. Soluble extract increased fungal physiology and treated cultures produced significantly higher levels of supernatant protein and enzymes including amylase, cellulase and beta-glucosidase. The fungal response depended on culture carbon source. However, culture zoospore production was increased regardless of substrate provided. Culture utilization of glucose was more rapid in treated cultures, yet high levels of the extract greatly inhibited glucose utilization.

Amylases↗

Production of docosahexaenoic acid by Crypthecodinium cohnii grown in a pH-auxostat culture with acetic acid as principal carbon source.

Crypthecodinium cohnii, a marine alga used for the commercial production of docosahexaenoic acid (DHA), was cultivated in medium containing sodium acetate as principal carbon source; the pH was maintained at a constant value by addition of acetic acid, which also provided an additional carbon source in a controlled manner. The accumulation of lipid by C. cohnii in this pH-auxostat culture was significantly greater than previously reported for batch cultures using glucose as principal carbon source. Of six strains tested in pH-auxostat cultures, C. cohnii ATCC 30772 was the best, with the cells reaching 20 to 30 g dry weight per liter after 98 to 144 h and containing in excess of 40% (w/w) total lipid, with DHA representing approximately half of the total fatty acids in the triacylglycerol fraction. A productivity of 36 mg DHA L(-1) h(-1) was achieved during cultivation for 98 h using a 5% (vol/vol) inoculum, and DHA production was in excess of 3 g per liter of culture. Most of the DHA was present in neutral lipids.

Acetic Acid↗

A bacterium capable of using phytol as its sole carbon source, isolated from algal sediment of Mud Lake, Florida.

A species of Flavobacterium that consistently attacks pure phytol and can use it as a sole source of carbon has been isolated from the blue-green algal sediment of Mud Lake, Florida. Biochemical tests demonstrate that this bacterium also readily uses various other organic compounds. This bacterium may account for the degradation products of chlorophyll and its side chain phytol, which have been found in the Mud Lake algal sediment. Phytol and its degradation products play a role in Refsum's disease, but phytol is also the most promising precursor of the isoprenoid hydrocarbons found in oil shale of the Green River Formation (Eocene) of Colorado, Utah, and Wyoming. The discovery of this species of Flavobacterium is a significant product of a protracted study of the bacteriology, phycology, zoology, and geochemistry of the algal sediment forming in Mud Lake, which is believed to be a modern analogue of the kind of algal sediment that, through geologic time, became oil shale.

Chlorophyll↗

Characterization of the primary starch utilization operon in the obligate anaerobe Bacteroides fragilis: Regulation by carbon source and oxygen.

The opportunistic pathogen Bacteroides fragilis is a commensal organism in the large intestine, where it utilizes both dietary and host-derived polysaccharides as a source of carbon and energy. In this study, a four-gene operon required for starch utilization was identified. The operon also was found to be oxygen responsive and thus was designated osu for oxygen-induced starch utilization. The first three genes in the operon were predicted to encode outer membrane proteins involved in starch binding, and a fourth gene, osuD, encoded an amylase involved in starch hydrolysis. Insertional mutation of the osuA gene (Omega osuA) resulted in the inability to utilize starch or glycogen and an insertional mutation into the osuD gene (Omega osuD) was severely impaired for growth on starch media. Transcriptional studies indicated that maltose, maltooligosaccharides, and starch were inducers of osu expression and that maltose was the strongest inducer. A transcriptional activator of osuABCD, OsuR, was identified and found to mediate maltose induction. The Omega osuA and Omega osuD mutants were able to grow on maltose but not starch, whereas a mutation in osuR abolished growth on both substrates, indicating that additional genes under the control of OsuR are needed for maltose utilization. The osuABCD operon also was induced by exposure to oxygen and was shown to be part of the oxidative stress response important for aerotolerance of B. fragilis. Transcriptional analyses showed that osuA was induced 20-fold by oxygen, but OsuR was not required for this activation. Analysis of osu mutants suggested that expression of the operon was important for survival during oxygen exposure but not to hydrogen peroxide stress.

Amylases↗