Behavior of bacteria and antibiotics under space conditions.

We have previously reported an increase of the "resistance" to antibiotics of bacteria during space missions. In the present experiment, we studied the growth of Escherichia coli cultured in vitro in space in the presence of dihydrostreptomycin: tritiated and nontritiated. This experiment was carried out during the STS 42 mission aboard the U.S. Space Shuttle Discovery (IML-1 program). Cells were cultured in plastic bags and growth was stopped at six different time points by lowering the temperature to 5 degrees C. Several methods were used: viable cell counting by Colony Forming Units; total cell number by optical densitometry; electron microscopy; radioactivity measurements. The investigations show no difference between flight and ground experiments for the cultures without antibiotic. The growth rate with antibiotic was accelerated in flight, the growth yield was not changed, and there were no differences in the ultrastructures. The results suggest some changes in antibiotic binding in space. We did not observe any differences between the cultures developed in flight in the 1-g centrifuge and the cultures placed in the static rack in microgravity.

Growth and division of Escherichia coli under microgravity conditions.

The growth rate in glucose minimal medium and time of entry into the stationary phase in pepton cultures were determined during the STS 42 mission of the space shuttle Discovery. Cells were cultured in plastic bags and growth was stopped at six different time points by lowering the temperature to 5 degrees C, and at a single time point, by formaldehyde fixation. Based on cell number determination, the doubling time calculated for the flight samples of glucose cells was shorter (46 min) than for the ground samples (59 min). However, a larger cell size expected for more rapidly growing cells was not observed by volume measurements with the electronic particle counter, nor by electron microscopic measurement of cell dimensions. Only for cells fixed in flight was a larger cell length and percentage of constricted cells found. An optical density increase in the peptone cultures showed an earlier entry into the stationary phase in flight samples, but this could not be confirmed by viability counts. The single sample with cells fixed in flight showed properties indicative of growth stimulation. However, taking all observations together, we conclude that microgravity has no effect on the growth rate of exponentially growing Escherichia coli cells.

[Determination of factors promoting, in vitro, the expression of adhesion of Pseudomonas aeruginosa to buccal cells]. / Détermination des facteurs favorisant, in vitro, l'expression de l'adhésion de Pseudomonas aeruginosa aux cellules buccales.

The composition of the bacterial flora in the upper respiratory tract is closely correlated with the type of pathogens recovered from the respiratory tract in patients. In intensive care patients, colonization of the oral cavity with Gram-negative organisms increases the risk of Gram-negative respiratory tract infection; the ability of bacterial cells to attach to buccal cells seems to play a central role in this correlation. Similar findings have been reported in chronic respiratory tract infections, including bronchiectasis and cystic fibrosis, with Pseudomonas aeruginosa colonization. This study was undertaken to determine the conditions best suited to in vitro detection of adhesion of P. aeruginosa to buccal cells. Use of brain-heart-infusion medium, incubation at 35 degrees C for 2 hours, and a bacterial concentration of 2 x 10(9) cells/ml were the factors correlated with improved detection of adhesion to buccal cells. Furthermore, attachment of bacteria to buccal cells was not found to vary across donors or over time in a given donor. Adhesion was independent of cell viability.

Adherence and hemagglutination of Corynebacterium group D2.

Sixty-one strains of Corynebacterium group D2 were examined for their ability to adhere to human uroepithelial cells and to agglutinate human and guinea-pig erythrocytes. Strains were isolated from samples of two origins: urine of bacteriuric patients and healthy skin of patients without urinary infection. In addition, the isolates were examined by scanning and transmission electron microscopy. Heavy adherence to the uroepithelial cells but weak hemagglutination were noted. No statistical association was demonstrated between the adherence and the origin of the strains (65.2% of urinary isolates and 80% of healthy skin isolates were adherent). On transmission electron microscopy, a close association was observed between adherent bacteria and cells on thin sections and only few strains were piliated with negative staining. These results do not support a role of adherence as a predictor of pathogenicity of Corynebacterium group D2 which seems to act as an opportunistic pathogen in urinary tract infections.

Identification of a novel EBV-induced membrane glycoprotein of 43 kDa with H667 MAb.

Using purified B95-8 Epstein-Barr virus (EBV), a MAb designated H667 was produced. We demonstrated by indirect membrane immunofluorescence (IF) on six EBV producer cell lines and by immunoelectron microscopy that H667 reacted with a membrane antigen. H667 recognized a 43-kDa EBV protein (p43) as determined by immunoblotting using purified EBV from the six producer cell lines. Phosphonoacetic acid treatment of B95-8 cells was associated with the disappearance of p43, indicating that it was a late antigen. This antigen was shown to be a glycoprotein by incorporation of [14C]glucosamine and was shown to contain an N-asparagine-linked glycosyl group by its sensitivity to tunicamycin. It was named gp43. The H667 MAb inhibited B95-8 EBV cord blood lymphocyte transformation only when a low inoculum was used but failed to inhibit EA induction in Raji cells by P3HR1 EBV. Human sera reactivity against the gp43 antigen was studied. By the immunoblotting method, using H667 immunoaffinity chromatography-purified gp43, we showed that 70.9% of the human sera tested had antibodies directed against gp43. By IF blocking tests, we found that only 12.5% of the sera tested were reactive, indicating that the epitope corresponding to the H667 MAb was not the most immunogenic gp43 epitope.

Effects of five antibiotics on adhesion and haemagglutinating properties of Pseudomonas aeruginosa isolated from cystic fibrosis patients.

The aim of this study was to evaluate the in vitro effect of five antibiotics at sub-inhibitory concentrations on the adhesive and haemagglutinating properties of Pseudomonas aeruginosa isolated from cystic fibrosis sputa. Eleven isolates (mucoid and non-mucoid) from cystic fibrosis, and four isolates (mucoid and non-mucoid) from other chronic respiratory infections were tested. The adhesion test was performed on human lymphoblastoid cell-lines; the haemagglutination test used human O+ and guinea-pig erythrocytes. The antibiotics were tested at six sub-inhibitory concentrations, from MIC/2 to MIC/64. Among the five antibiotics, cefsulodin and pefloxacin were the most active in decreasing the adhesive properties: this effect was statistically significant at MIC/2 and MIC/4 for cefsulodin and at all sub-inhibitory concentrations for pefloxacin. No differences appeared between mucoid and non-mucoid strains, and no correlation was noted with their clinical origins. The three other antibiotics (ceftazidime, latamoxef and imipenem) had no significant effect on the adhesion of all the strains tested, but their effect was rather strain-dependent. This fact and the heterogeneity found in adherence and haemagglutinating activity of each strain suggest that the adhesins and the haemagglutinins of P. aeruginosa are very complex structures.

[In vitro study of the effects of oxolinic acid at sub-inhibitory concentrations on the activity of hemagglutinins and adhesion to uroepithelial cells by Escherichia coli isolated from urine]. / Etude in vitro des effets de l'acide oxolinique à concentrations sub-inhibitrices sur l'activité des hémagglutinines et de l'adhésion aux cellules uro-épithéliales des Escherichia coli isolés des urines.

The aim of the present study was to investigate the effects of sub-MIC doses of oxolinic acid (quinolone), widely used in the treatment of urinary tract infections, on both haemagglutinating activity and adhesion capacity of 13 Escherichia coli strains isolated from urine during acute cystitis or pyelonephritis. All these strains adhered to uroepithelial cells and showed mannose-sensitive and/or mannose-resistant haemagglutinating activity. Sub-MIC doses of oxolinic acid induced filaments in most of the bacterial cultures; however, inhibition of haemagglutination and adhesion was variable in vitro. When inhibition did take place in any one strain, both haemagglutination and adhesion were affected. These results confirm those of other authors and indicate that the effect of sub-MIC doses of a given antibiotic is strain-specific; they also indirectly show the heterogeneity of E. coli strains isolated from urine. It thus seems unlikely that, in clinical conditions, a single antibiotic is capable of reducing adhesion, given the diversity of the adhesins found in pathogenic E. coli strains.

Antibiotic activity in space.

Environmental factors in space exert an influence on the behaviour of bacteria, particularly on their sensitivity to antibiotics. Thus, G. Taylor and S. Zaloguev observed that bacterial samples collected on the crew during flight in the Apollo-Soyouz Test Project Mission presented higher antibiotic resistance than controls. This paper presents the results of two experiments performed in 1982 and 1985 (Cytos 2 during the French-Soviet Mission and "Antibio" in the Biorack programme of the European Space Agency). The results show an increase of antibiotic resistance in bacteria growth in flight and a modification in the structure of the cell wall. All these modifications are transitory. Two hypotheses are put forward to explain the phenomenon.

Study of minimal inhibitory concentration of antibiotics on bacteria cultivated in vitro in space (Cytos 2 experiment).

The aim of the Cytos 2 experiment, carried out during the French-Soviet manned flight in July 1982, was to study the bacteria's sensitivity to antibiotics cultivated in vitro during the orbital flight, using the bacterial method of minimal inhibitory concentration (MIC). Two species of bacteria were tested with various antibiotics: Staphylococcus aureus with Oxacillin, Chloramphenicol and Erythromycin; Escherichia coli with Colistin and Kanamycin. The results show an increase in resistance to antibiotics particularly strong in E. coli and weaker in Staphylococcus aureus. Considering these results, we think that there might be a relationship between the increase in resistance to antibiotics and a stimulating effect on growth rate by the factors of environmental space.

Preliminary results of Cytos 2 experiment.

Cytos 2 experiment, carried out during the French-Soviet manned flight (July 1982), has studied the antibiotics sensitivity of bacteria cultivated in vitro during the orbital flight. The results show an increase of the antibiotics resistance and a larger thickness of the cellular envelope for the inflight cells. The increase of antibiotics resistance can be related to a stimulating effect of space on the cell growth rate or to changes of the cellular envelope structure.

Influence of dicarboxylic phosphatidylcholines on phosphatidylcholine liposomes as revealed by gel chromatography and electron microscopy.

The effect of dicarboxylic phosphatidylcholines (glutarylphosphatidylcholine) on the structural changes of phosphatidylcholine liposomes is examined by using multilamellar liposomes prepared with egg phosphatidylcholine or dipalmitoylphosphatidylcholine and by varying the surface charge by addition of dicetyl phosphate. Investigations are performed by gel chromatography and electron microscopy. Glutarylphosphatidylcholine is in micellar form (rod-like micelles or globular micelles). The structures obtained depend on the fatty acid saturation of liposomes and on the charge of liposome (addition or not of dicetyl phosphate). With egg phosphatidylcholine/glutarylphosphatidylcholine dispersions, an aspect more similar to myelinic figures than liposomes is observed, while in the presence of dicetyl phosphate, liposomes similar to control egg phosphatidylcholine liposomes are obtained. Gel chromatography on Sepharose 4B and turbidity measurements prove that dicetyl phosphate increases the stability of egg phosphatidylcholine/glutarylphosphatidylcholine mixtures. On the other hand, in dipalmitoylphosphatidylcholine/glutarylphosphatidylcholine dispersions, incorporation of dicetyl phosphate destabilizes bilayer structure and the formation of mixed micelles occurs. Viscosity measurement shows, in the presence of dicetyl phosphate, an increased fluidity for dipalmitoylphosphatidylcholine/glutarylphosphatidylcholine dispersions, in agreement with the micellar organization. These data confirm that the disorganization of liposomal membranes by dicarboxylic phosphatidylcholine depends on the fatty acid composition of phosphatidylcholine and on the presence of dicetyl phosphate.

[Bacteriophage occurrence in "Mycobacterium phlei" (author's transl)]. / Présence d'une bactériophage chez Mycobacterium phlei.

Surface growth of synchronized bacteria was obtained by means of a suspension of Mycobacterium phlei cells in pentane, the dispersion of which resulted from passage through glass (Ballotini) column. By using standardized conditions, a series of identical cultures were obtained, suitable for studying their evolution as a function of time. By counting colonies every twenty minutes, during ten hours, two doublings were observed, with a generation time of five hours. At the end of a plateau, just before the next doubling, the curve exhibited a marked decrease. Bacteriophages were found in culture medium at the time corresponding to this decrease. In thin sections of the pellicles collected at this time, condensations resembling DNA from phage heads could be noticed within the bacterial cells, as well as free phages in th close neighbourhood of burst cells. The relations between phage and bacteria, and the possible relation between the presence of the phage and the synthesis of phleates has not been determined.

Reclassification of Mycobacterium phlei PN-bb as Rhodococcus bronchialis PN-bb.

Mycobacterium phlei PN-bb, induced in the parental strain PN by gamma-radiation and ethyl methanesulfonate, should be reclassified as Rhodococcus bronchialis PN-bb. The reclassification is based on the results of lipid analysis, DNA-DNA hybridization and several biochemical tests, performed in comparison with the parental strain PN, earlier reclassified as R. bronchialis, and R, bronchialis (N654).

Comparative studies of the strains PA and PN of Mycobacterium phlei leading to their reclassification: examination of lipids and DNA, biochemical tests and phage typing.

Study of lipid and DNA, biochemical tests and phage typing performed on the strain PA previously labelled Mycobacterium phlei, lead to the conclusion that this strain belongs to the species M. smegmatis. Parallel studies performed on strain PN, isolated from a culture of strain PA, as well as DNA homology percentage of the two strains, do not support the assumption that strain PN could have resulted from a mutation of strain PA Strain PN produces mycolic acids similar to those found in Rhodococcus bronchialis; the few biological tests applied quite agree with such a classification.