The requirements of Pseudomonas aeruginosa dissociants for carbon, nitrogen, and phosphorus

Quantitative data on the nutritional requirements of microorganisms are necessary to predict the behavior of bacterial populations and to control their cultivation. The requirements of the R, S, and M dissociants of Pseudomonas aeruginosa for carbon, nitrogen, and phosphorus were derived from the results of 88 cultivation experiments. For each of the dissociants, we derived a coefficient that relates the optical density and the number of cells in the dissociant culture, determined the time when the cultures entered the stationary growth phase, studied cultural changes induced by transfer to the stationary phase, and determined what nutrients limit the growth of particular dissociants. The nutritional requirements of the dissociants are discussed in relation to our earlier data.     

Removal of bacteria from water by adhesion to cross-linked poly(vinylpyridinium halide).

Cross-linked poly(vinylpyridinium halide) was found to have a novel and remarkable ability to remove bacteria from water. For example, when 10 g (wet weight) of cross-linked poly(N-benzyl-4-vinylpyridinium bromide) was contacted with 20 ml of suspensions of Escherichia coli (9.7 X 10(4) to 9.7 X 10(7)/ml), Salmonella typhimurium (8.0 X 10(6) to 1.1 X 10(7)/ml), Streptococcus faecalis (5.0 X 10(7)/ml), Staphylococcus aureus (8.1 X 10(7)/ml), and Pseudomonas aeruginosa (3.2 X 10(5)/ml) under stirring in sterilized physiological saline at 37 degrees C, 99% of the viable cells of these bacteria were removed in 2 to 6 h. When suspensions of these bacteria (10(5) to 10(8) cells per ml) were passed through a column (20 mm by 100 cm) of cross-linked poly(N-benzyl-4-vinylpyridinium bromide) at 37 degrees C with a flow rate of 0.8 to 1.4 bed volumes per h, 97 to 100% of the viable cells were eliminated from the suspensions during the treatment. Mechanistic studies demonstrated that cross-linked poly(vinylpyridinium halide) irreversibly captured these bacteria alive during the treatment. That is, total organic carbon was removed during the treatment, and the bacteria which adhered to the resin proliferated on the bacterial medium. The adhesion capacity was estimated to be 10(10) cells per g (dry weight). Total organic carbon was also removed even when the bacteria were killed by heat treatment before the column studies.     

Paraffin baiting system for demonstration of growth and biofilm production in Pseudomonas aeruginosa

Pseudomonas aeruginosa is one of the commonest pathogens among the pseudomonads. This organism can grow in minimal nutritional requirements. Because of the ability of pseudomonads to grow on paraffin is not commonly found among other human pathogens and the primary human pathogen being P. aeruginosa, we studied the adaptation of this organism to paraffin baiting system for growth and biofilm formation. Strains were tested for the capacity to use paraffin as the sole source of carbon using Czapek's minimal salt medium. Of the 53 clinical isolates of P. aeruginosa, 20 strains exhibited growth by 24 hrs and 42 strains by 48 hrs. The remaining strains did not show any growth in the paraffin baiting system. The oxidase test with the paraffin baiting system was also performed. This simple and inexpensive method can be used to isolate and demonstrate the biochemical and biofilm forming capacity of the organism.     

Serotyping of Pseudomonas aeruginosa strains by slide coagglutination

Serological typing of Pseudomonas aeruginosa strains (228 strains) by slide coagglutination, using our own reagents (5 polyvalent and 22 monovalent ones, corresponding to the 22 serotypes in Meitert-Meitert scheme), led to identical results obtained by conventional slide agglutination. Utilization of live Ps. aeruginosa cells suspensions, killed by boiling or autoclaving, showed a 100% concordance of results, when using the second and the third suspension types and a 97.37% one between them and the live cells suspension. We noticed that reactions intensity was higher when using bacterial suspensions, boiled for 2.5 hours, in comparison with autoclaved cells suspensions, 30 minutes at 120 C. Compared to conventional slide agglutination, the slide coagglutination presents more advantages, being simple, rapid, specific and economical.     

Dynamics of the growth and population composition of the mixed cultures of R, S, and M dissociants of Pseudomonas aeruginosa

The population composition of polycultures of Pseudomonas aeruginosa dissociants (R + M and R + S + M) developing on media with various contents and ratios of nitrogen and phosphorus has been studied. Irrespective of its proportion (10 to 90%) in the inoculum, the R variant accounted for 65 to 84% of the whole population of linear-phase and stationary-phase binary cultures of R and M dissociants, which differ in terms of energy metabolism and nutritional requirements. After prolonged cultivation, the population in the binary culture contained only R cells (100%), which are characterized by minimum requirements with respect to the main biogenic elements. These data agree with the predictive data of model studies and can be attributed to regulation of the population composition of bacterial cultures by trophic factors. It was established that the proportion of M cells, which are distinguished by maximum nutrient requirements and enhanced stability, increased during two developmental stages of the Ps. aeruginosa polycultures (R + M and R + S + M): the lag phase and the decay stage. This result cannot be due to the influence of trophic factors and presumably results from changes in the levels of autoregulatory factors (anabiosis autoinducers) involved in stress resistance and plausibly in the adaptive interconversion of dissociants upon transfer to a new medium (during the lag phase) and under starvation conditions (at the onset of the decay phase).     

Use of ethidium bromide monoazide for quantification of viable and dead mixed bacterial flora from fish fillets by polymerase chain reaction

Ethidium bromide monoazide (EMA) was utilized to selectively allow conventional PCR amplification of target DNA from viable but not dead cells from a broth culture of bacterial mixed flora derived from cod fillets. The universal primers designated DG74 and RW01 that amplify a 370-bp sequence of a highly conserved region of all eubacterial 16S rDNA were used for the PCR. The use of 10 microg/ml or less of EMA did not inhibit the PCR amplification of DNA derived from viable bacteria. The minimum amount of EMA to completely inhibit the PCR amplification of DNA derived from dead bacterial cells was 0.8 microg/ml. Amplification of target DNA from only viable cells in a suspension with dead cells was selectively accomplished by first treating the cells with 1 microg/ml of EMA. A standard curve was generated relating the intensity of fluorescence of DNA bands to the log of CFU of mixed bacterial cultures for rapidly assessing the number of genomic targets per PCR derived from the number of CFU. A linear range of DNA amplification was exhibited from 1 x 10(2) to 1 x 10(5) genomic targets per PCR. The viable/dead cell discrimination with the EMA-PCR method was evaluated by comparison with plate counts following freezing and thawing. Thawing frozen cell suspensions initially containing 1 x 10(5) CFU/ml at 4, 20, and 37 degrees C yielded a 0.8 log reduction in the number of viable cells determined by both plate counts and EMA-PCR. In contrast, thawing for 5 min at 70 degrees C resulted in a 5 log reduction in CFU derived from plate counts (no CFU detected) whereas the EMA-PCR procedure resulted in only a 2.8 log reduction in genomic targets, possibly reflecting greater damage to enzymes or ribosomes at 70 degrees C to a minority of the mixed population compared to membrane damage.     

Suppression of Drosophila cellular immunity by directed expression of the ExoS toxin GAP domain of Pseudomonas aeruginosa

We show here that transgenic Drosophila can be used to decipher the effect of a bacterial toxin on innate immunity and demonstrate the contribution of blood cells in fly resistance to bacterial infection. ExoS is a Pseudomonas aeruginosa exotoxin directly translocated into the host cell cytoplasm through the type III secretion system found in many Gram-negative bacteria. It contains a N-terminal GTPase activating (GAP) domain that prevents cytoskeleton reorganization by Rho family of GTPases in cell culture. Directed expression of the ExoS GAP domain (ExoSGAP) during fly eye morphogenesis inhibited Rac1-, Cdc42- and Rho-dependent signalling, demonstrating for the first time its activity on RhoGTPases in a whole organism. We further showed that fly resistance to P. aeruginosa infections was altered when ExoSGAP was expressed either ubiquitously or in haemocytes, but not when expressed into the fat body, the major source of NF-(kappa)B-dependent anti-microbial peptide synthesis. Fly sensitivity to infection was also observed with Gram-positive Staphylococcus aureus strain and was associated to a reduced phagocytosis capacity of ExoSGAP-expressing haemocytes. Our results highlight the major contribution of cellular immunity during the first hours after Drosophila infection by P. aeruginosa, an opportunist pathogen affecting patients with pathologies associated to a reduced leukocyte number.     

Some physiological aspects of the autecology of the suspension-feeding protozoanTetrahymena pyriformis

Feeding, growth, and reproductive responses of the suspension-feeding protozoanTetrahymena pyriformis to shifts up or down of the density of its bacterial food were observed. The rates of feeding, growth, and reproduction were determined by measuring the rates of uptake of viable bacterial cells, of change of mean volume of the protozoan cells, and of change of number of protozoan cells, respectively. The effects of the nutritional status of the protozoans at the time of shifting were observed also. Results are interpreted in terms of the limited polymorphism exhibited in the life cycle of this organism. Responses in all cases seem to reflect a strategy for exploiting a patchy, transient environment, a conclusion already reached by several earlier investigators.     

Biosynthetic Enhancement of the Detection of Bacteria by the Polymerase Chain Reaction

Molecular viability testing (MVT) was previously reported to specifically detect viable bacterial cells in complex samples. In MVT, brief nutritional stimulation induces viable cells, but not non-viable cells, to produce abundant amounts of species-specific ribosomal RNA precursors (pre-rRNA). Quantitative polymerase chain reaction (qPCR) is used to quantify specific pre-rRNAs in a stimulated aliquot relative to a non-stimulated control. In addition to excluding background signal from non-viable cells and from free DNA, we report here that MVT increases the analytical sensitivity of qPCR when detecting viable cells. Side-by-side limit-of-detection comparisons showed that MVT is 5-fold to >10-fold more sensitive than standard (static) DNA-targeted qPCR when detecting diverse bacterial pathogens (Aeromonas hydrophila, Acinetobacter baumannii, Listeria monocytogenes, Mycobacterium avium, and Staphylococcus aureus) in serum, milk, and tap water. Sensitivity enhancement may come from the elevated copy number of pre-rRNA relative to genomic DNA, and also from the ratiometric measurement which reduces ambiguity associated with weak or borderline signals. We also report that MVT eliminates false positive signals from bacteria that have been inactivated by moderately elevated temperatures (pasteurization), a condition that can confound widely-used cellular integrity tests that utilize membrane-impermeant compounds such as propidium iodide (PI) or propidium monoazide (PMA) to differentiate viable from inactivated bacteria. MVT enables the sensitive and specific detection of very small numbers of viable bacteria in complex matrices.     

Serratia indica as a Bacterial Tracer for Water Movements

Summary: Methods are described for obtaining suitable liquid cultures of Serratia indica for transporting mature cultures in the field and for viable cell determination using membrane filters. The effect of culture medium composition on growth curves and on viability of very dilute suspensions of the organism in vitro in distilled and sea water was investigated. The results of a field investigation at a sewage outfall are described.     

Hydrogenase activity in aged, nonviable Desulfovibrio vulgaris cultures and its significance in anaerobic biocorrosion

Batch cultures of Desulfovibrio vulgaris stored at 32 degrees C for 10 months have been found to retain 50% of the hydrogenase activity of a 1-day culture. The hydrogenase found in old cultures needs reducing conditions for its activation. Viable cell counts are negative after 6 months, showing that the hydrogenase activity does not depend on the presence of viable cells. These observations are of importance in the understanding of anaerobic biocorrosion of metals caused by depolarization phenomena.     

Hydrogenase activity in aged, nonviable Desulfovibrio vulgaris cultures and its significance in anaerobic biocorrosion.

Batch cultures of Desulfovibrio vulgaris stored at 32 degrees C for 10 months have been found to retain 50% of the hydrogenase activity of a 1-day culture. The hydrogenase found in old cultures needs reducing conditions for its activation. Viable cell counts are negative after 6 months, showing that the hydrogenase activity does not depend on the presence of viable cells. These observations are of importance in the understanding of anaerobic biocorrosion of metals caused by depolarization phenomena.     

Nitrogen fixation activity of a filamentous,nonheterocystous cyanobacterium in the presence and absence of exogenous,organic substrates

Gallionella ferruginea is able to utilize Fe(II) and the reduced sulfur compounds sulfide and thiosulfate as electron donor and energy source. Tetrathionate and elemental sulfur, on the other hand, are not metabolized. In sulfide-O₂ microgradient cultures G. ferruginea grows at the interface between the oxidizing and the reducing zones. Optimal growth depends on low oxygen and sulfide concentrations. Establishing within the gradient protects the bacterium from too high sulfide concentrations. G. ferruginea excretes extracellular polymeric substances (EPS). While in FeS-gradient cultures 2×10⁶ cells/ml were obtained the bacterial mass could be increased to 1–3×10⁸ cells/ml in shaken batch cultures using thiosulfate as substrate. A further increase of bacterial mass by adding an organic carbon source was not possible confirming that G. ferruginea is an obligate autotrophic organism. When growing on sulfide or thiosulfate the otherwise characteristic twisted stalk consisting of ferric hydroxide is lacking. It is thus shown to be a metabolic end product of Fe(II) oxidation rather than metabolically active cellular material.     

Establishment of stable cell lines producing anti-Pseudomonas aeruginosa monoclonal antibodies and their protective effects for the infection in mice.

Human-human hybridomas producing monoclonal antibodies (MoAbs) specific for five major serotypes of Pseudomonas aeruginosa were developed by fusing P. aeruginosa primed and Epstein-Barr virus-transformed cells with human myeloma P109 cells using polyethyleneglycol. The MoAbs which were produced by the hybridomas were protective against lethal intraperitoneal (i.p.) challenge of P. aeruginosa (10 LD50) in mice. The 50% effective dose (ED50) values of MoAbs ranged from 0.5 to 10.2 micrograms/mouse and were 26 to 240 times more protective than a commercial human IgG preparation. MoAb administration to mice promoted bacterial clearance in peritoneal cavity, and prevented bacterial invasion into blood in the way of increasing both the number of bacteria trapped by a macrophage and the ratio of macrophages that trapped bacteria. MoAbs also showed protective effects against lethal infection of P. aeruginosa in the mice which were decreased in polymorphonuclear cells (PMN) by cyclophosphamide (CY). All MoAbs showed serotype-specific binding to the clinical isolates of P. aeruginosa as well as to the immunized strains. The hybridoma cell lines maintained their capacity to produce MoAb continuously for more than 12 months and produced 10 to 60 micrograms MoAbs per 10(6) cells in 24 hr. It is practicable to use these cell lines for large-scale production of anti-P. aeruginosa MoAbs and such MoAbs must be useful for the therapeutics of patients with P. aeruginosa infection.     

Use of autoradiography to assess viability of Helicobacter pylori in water.

Autoradiographic methods have been developed to detect metabolic activity of viable but nonculturable cells of Helicobacter pylori in water. Four strains of H. pylori were studied by using microcosms containing suspensions of 72-h cultures in water. The suspensions of aged, nonculturable cells of H. pylori were incubated with [3H]thymidine for 24 to 72 h, after which the cell suspensions were exposed to Kodak NTB2 emulsion for 3 to 28 days. Each sample was processed with three separate controls to rule out false-positive reactions. The organism remains viable and culturable under these conditions for up to 48 h and, in some cases, 20 to 30 days, depending on physical conditions of the environment. We found that temperature was a significant (P < or equal to 0.01) environmental factor associated with the viability of H. pylori cells in water. Autoradiographs of tritium-labeled cells of H. pylori revealed aggregations of silver grains associated with uptake by H. pylori of radiolabelled substrate. Findings based on the autoradiographic approach give strong evidence supporting the hypothesis that there is a waterborne route of infection for H. pylori. The possibility that H. pylori may persist in water in a metabolically active stage but not actively growing and dividing is intriguing and relevant to public health concerns.     

The Requirements of Pseudomonas aeruginosa Dissociants for Carbon,Nitrogen, and Phosphorus

Quantitative data on the nutritional requirements of microorganisms are necessary to predict the behavior of bacterial populations and to control their cultivation. The requirements of the R, S, and M dissociants of P. aeruginosa for carbon, nitrogen, and phosphorus were derived from the results of 88 cultivation experiments. For each of the dissociants, we derived a coefficient that relates the optical density and the number of cells in the dissociant culture, determined the time when the cultures entered the stationary growth phase, studied cultural changes induced by transfer to the stationary phase, and determined what nutrients limit the growth of particular dissociants. The nutritional requirements of the dissociants are discussed in relation to our earlier data.     

Use of ethidium bromide monoazide for quantification of viable and dead mixed bacterial flora from fish fillets by polymerase chain reaction

Ethidium bromide monoazide (EMA) was utilized to selectively allow conventional PCR amplification of target DNA from viable but not dead cells from a broth culture of bacterial mixed flora derived from cod fillets. The universal primers designated DG74 and RW01 that amplify a 370-bp sequence of a highly conserved region of all eubacterial 16S rDNA were used for the PCR. The use of 10 μg/ml or less of EMA did not inhibit the PCR amplification of DNA derived from viable bacteria. The minimum amount of EMA to completely inhibit the PCR amplification of DNA derived from dead bacterial cells was 0.8 μg/ml. Amplification of target DNA from only viable cells in a suspension with dead cells was selectively accomplished by first treating the cells with 1 μg/ml of EMA. A standard curve was generated relating the intensity of fluorescence of DNA bands to the log of CFU of mixed bacterial cultures for rapidly assessing the number of genomic targets per PCR derived from the number of CFU. A linear range of DNA amplification was exhibited from 1 × 10² to 1 × 10⁵ genomic targets per PCR. The viable/dead cell discrimination with the EMA-PCR method was evaluated by comparison with plate counts following freezing and thawing. Thawing frozen cell suspensions initially containing 1 × 10⁵ CFU/ml at 4, 20, and 37 °C yielded a 0.8 log reduction in the number of viable cells determined by both plate counts and EMA-PCR. In contrast, thawing for 5 min at 70 °C resulted in a 5 log reduction in CFU derived from plate counts (no CFU detected) whereas the EMA-PCR procedure resulted in only a 2.8 log reduction in genomic targets, possibly reflecting greater damage to enzymes or ribosomes at 70 °C to a minority of the mixed population compared to membrane damage.     

Secretion of an antibacterial factor during resuscitation of dormant cells inMicrococcus luteus cultures held in an extended stationary phase

A high proportion ofMicrococcus luteus cells in cultures starved for 3–6 months in spent medium following growth to stationary phase in batch culture lost the ability to grow and form colonies on agar plates, but could be resuscitated from dormancy by incubation in liquid medium containing supernatant taken from the late log phase of viable cultures of the same organism (Kaprelyants et al. 1994). In the present work, we found that during the first 50–70 h of such resuscitation the dormant cells actually divide for 10–17 generations in lactate minimal medium containing yeast extract whilst remaining nonculturable on agar plates. Further incubation results in a decrease in the total cell number in liquid medium. The addition of viable (culturable)Micrococcus luteus cells in concentrations of up to 10⁴ ml^–1 to test tubes containing either resuscitating cells or supernatant from these cultures revealed the excretion of a factor or factors which inhibited the proliferation of otherwise viable cells. The maximum production of this factor took place after some 96 h of incubation of starved cells in resuscitation medium. Supernatant from late logarithmic phase batch cultures ofM. luteus abolished the antibacterial effect of starved cultures incubated in resuscitation medium. It is concluded that the stimulating effect of viable cells, and of supernatant taken from batch cultures, on the resuscitation of dormant cells might be connected in part with overcoming the activity of an antibacterial factor causing self-poisoning of dormant cells during their resuscitation.     

In vitro culture of embryonic cells from the shrimp, Penaeus chinensis

Studies were carried out in our laboratory to develop cell cultures from embryonic tissue of Penaeus chinensis. Good yields of dissociated, uncontaminated, viable cell suspensions were obtained by physical disruption of harvested embryonic tissues. Primary cultures in the form of proliferating foci of cells were readily initiated using MPS medium (osmolality: 2.4%) with 20% heat-inactivated fetal bovine serum. Insulin-like growth factor-II (IGF-II) and basic fibroblast growth factor (bFGF) at different concentrations were also added into the culture medium. Passage of primary cultures resulted in rapid proliferation and good adherence of lymphocytes in the presence of IGF-II at 80 ng/ml and bFGF at 20 ng/ml. Cells maintained in subculture for up to 10 passages still had good cellular morphology and division rates.