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PubMed2004

Knockout of the alanine racemase gene in Lactobacillus plantarum results in septation defects and cell wall perforation.

A stable mutant of Lactobacillus plantarum deficient in alanine racemase (Alr) was constructed by two successive homologous recombination steps. When the mutant was supplemented with D-alanine, growth and viability were unaffected. Surprisingly, deprivation of d-alanine during exponential growth did not result in a rapid and extensive lysis as observed in Alr-deficient strains of Escherichia coli or Bacillus subtilis. Rather, the starved mutant cells underwent a growth arrest and were gradually affected in viability with a decrease in colony forming units over 99% in less than 24 h. Additionally, fluorescent techniques demonstrated a loss of cell envelope integrity in the starved cells. Prolonged d-alanine starvation resulted in cells with an aberrant morphology. Scanning and transmission electron microscopy analyses revealed an increase in cell length, deficiencies in septum formation, thinning of the cell envelope and perforation of the cell wall in the septum region. We discuss the involvement of peptidoglycan hydrolases in these phenotypic defects in the context of the crucial role played by D-alanine in peptidoglycan biosynthesis and teichoic acids substitution.

Alanine↗

PubMed2004

Cryptococcus neoformans methionine synthase: expression analysis and requirement for virulence.

This paper describes (i) the expression profile of the methionine synthase gene (MET6) in the human pathogenic fungus Cryptococcus neoformans and (ii) the phenotypes of a C. neoformans met6 mutant. In contrast to the MET3 gene, which showed no significant change in expression in any environmental condition tested, the MET6 gene showed a substantial induction in response to methionine and a dramatic transcriptional induction in response to homocysteine. Like a met3 mutant, the met6 mutant was a methionine auxotroph. However, relative to a met3 mutant, the met6 mutant grew very slowly and was less heat-shock resistant. In contrast to a met3 mutant, the met6 mutant lost viability when starved of methionine, and it was deficient in capsule formation. Like a met3 mutant, the met6 mutant was avirulent. In contrast to a met3 mutant, the met6 mutant was hypersensitive to fluconazole and to the calcineurin inhibitors FK506 and cyclosporin A. A synergistic fungicidal effect was also found between each of these drugs and met6. The phenotypic differences between the met3 and met6 mutants may be due to the accumulation in met6 mutants of homocysteine, a toxic metabolic intermediate that inhibits sterol biosynthesis.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed1986

Recovery of Mycobacterium avium after treatment with chemical decontaminants.

Cell suspensions of a pathogenic strain of Mycobacterium avium, serovar 1, were tested for susceptibility to chemical disinfectants that are frequently used for decontamination of environmental samples. Kinetic data were obtained by determining the viability at 15-min intervals during 60 min of exposure at room temperature with aeration. After 60 min exposure to a combination of 0.094% Zephiran, and 10% trisodium phosphate, only 0.3% of the initial number of colony-forming units survived. Survival after 60 min treatment with 2% sodium hydroxide, 1% sodium hydroxide, or 0.04% hexadecylpyridinium chloride was 20, 46, and 57%, respectively. To assess the practical applications, the same agents were employed to extract M. avium that had been seeded into soil. Recovery of more than 25% of the initial number of colony-forming units was achieved with any of the treatments. Several combinations of chloramphenicol and antifungal drugs were tested for inhibition of soil microbiota that survived the decontamination procedures.

Disinfectants↗

PubMed1996

Refinement of, and new applications for, Helicobacter pylori colonization in pig gastric biopsy specimens cultured in vitro.

BACKGROUND: As we have previously reported, pig gastric biopsy specimens cultured in vitro are a highly useful model for studies of Helicobacter pylori growth and adhesion. The aim of this study was to further refine the model in terms of mucosal specificity, culture time, bacterial adhesion, and drug delivery. METHODS: H. pylori-inoculated antral and corporeal pig gastric specimens were cultured for up to 96 h. Biopsy viability, bacterial growth, and adhesion were determined every 24 h. Bismuth subcitrate and omeprazole were added to the top of the specimens via a bio-adhesive gel. RESULTS: Corporeal and antral specimens could be cultured for 72 h and 96 h, respectively, without affecting the viability. In parallel experiments from the same pig the percentage adhesion and total number of adhering H. pylori was higher in corporeal than in antral specimens at 72 h (28% and 4 x 10(5) versus 15% and 4 x 10(4), respectively). Removal of loosely attached H. pylori by rinsing at 24 h doubled the percentage H. pylori adhered during the subsequent 48 h. Bismuth subcitrate had a dose-dependent inhibitory effect on H. pylori; when added to the mucosal side, omeprazole had no effect. CONCLUSION: The pig in vitro biopsy model can be used for detailed H. pylori adhesion studies and for the screening of drugs added to the mucosal or serosal side.

Animals↗

PubMed1996

Optional elements in the chloroplast DNAs of chlamydomonas eugametos and C. moewusii: unidirectional gene conversion and co-conversion of adjacent markers in high-viability crossses.

Unlike most polymorphic markers in the Chlamydomonas eugametos and Chlamydomonas moewusii chloroplast DNAs (cpDNAs), the C. moewusii 6- and 21-kb extra sequences and the C. eugametos-specific CeLSU small middle dot 5 intron are transmitted to all of the few viable progeny in reciprocal crosses between the two green algae. To determine whether this unidirectional transmission pattern is due to gene conversion or to selection for F1 hybrid survival, we followed the inheritance of the parental alleles at the loci featuring these three deletions/additions and at several other polymorphic cpDNA loci in zygospore clones derived from high-viability crosses. The great majority of the zygospore clones examined inherited exclusively the long alleles from the mt- parent at the loci containing the three optional cpDNA elements, but as expected, they preferentially inherited the markers from the mt+ parent at most other loci. Our results therefore indicate that all three optional cpDNA sequences propagate themselves very efficiently by gene conversion in crosses between strains differing by the presence of these elements. The co-conversion tracts associated with these sequences are longer (>3 kb) than those previously reported for mobile elements spreading by gene conversion. Our results also revealed that less efficient gene conversion events occurred at two other cpDNA loci.

Alleles↗

PubMed2005

The vital status of human buccal epithelial cells and the bacteria associated with them.

OBJECTIVE: We have shown that buccal epithelial cells (BEC) from humans can contain a polymicrobial intracellular flora. Members of that flora can induce proinflammatory responses. However, our subjects all had healthy oral mucosa. This might reflect tolerance of bacterial invasion by live BEC. Alternatively, inflammation might not occur if invaded cells were mostly dead, and thus unable to mount a response. This study addressed that issue, by determining the vital status of BEC and the bacteria associated with them. DESIGN: Initial experiments indicated that BEC were anomalously permeable to the DNA stain propidium iodide. We used that property to develop a protocol that combined the DNA stains SYTO 9 and propidium iodide (indicators of bacterial viability) with the esterase substrate calcein blue AM (an indicator of BEC viability), and Annexin V Alexa Fluor 647 conjugate (an apoptosis marker). That protocol was applied to BEC collected from 36 human subjects. RESULTS: On average, 70% of BEC displayed calcein blue staining, with no binding of Annexin V, 25% showed signs of apoptosis, and 5% did not stain with calcein blue. The mean percent of BEC with live cell-associated bacteria was 29%. Collectively, 25% of total BEC displayed calcein blue staining and live (SYTO 9 stained) bacteria. Only 1% of total BEC were negative for calcein blue and associated with live bacteria. CONCLUSIONS: Our findings suggest that live BEC are tolerant of bacterial invasion. This may be due to complex interactions between members of the polymicrobial flora and their host BEC.

Adult↗

PubMed1987

Implications of disaggregation procedures on biological representation of human solid tumours.

This study was designed to define some biological aspects of cell suspensions, obtained by mechanical or enzymatic disaggregations, and to verify whether single cell suspensions are representative of original solid tumours. The study was performed on a series of 25 human solid tumours including breast carcinoma, ovarian carcinoma and malignant melanoma. A higher cell viability and a loss of aneuploid subpopulations, or a lower fraction of aneuploid cells, were observed in enzymatically-released samples than in samples obtained by the mechanical procedure. Moreover, the proliferative activity, which was generally similar for the cell suspensions obtained by the two disaggregation procedures, was always markedly lower in the cell suspensions than in solid samples from the same tumour. In conclusion, the results from this study indicate that many changes, such as selective release of cell populations from the tumour matrix, damage and destruction of aneuploid and proliferating cells can be induced to various extents by different disaggregation procedures.

Breast Neoplasms↗

PubMed1997

[Wide occurrence of enterohemorragic Eschrichia coli O157 in natural freshwater environment].

Recent outbreaks of hemorrhagic colitis by Escherichia coli O157 urge us to clarify the source, distribution, and fate of those pathogens. We hypothesized that E. coli O157 enter into viable but nonculturable state (VBNC) so that the conventional culture method fails to detect those strains from natural environment. In the microcosm using sterilized river water, E. coli O157 gradually lost their viability. Total count using epifluorescent microscopy and the direct viable count (DVC), however, stayed almost constant for more than a month, indicating that the cells fell into the VBNC state. In order to see the distribution of E. coli O157 in natural environment, the DVC method and fluorescent antibody technique were applied. All river water samples from five different places in and near Tokyo metropolitan area showed occurrence of cells reacted with the antibody applied. This indicates that E. coli O157 may be widely distributed in freshwater environment as normal inhabitants. The understanding of VBNC state of E. coli O157 should be essential to investigate on the behavior of this pathogen in nature and find out a suitable method to prevent from further outbreaks.

Cell Survival↗

PubMed2004

Antagonistic activity against Helicobacter pylori infection in vitro by a strain of Enterococcus faecium TM39.

Lactic acid bacteria (LAB) strains from infant feces were screened for anti-Helicobacter pylori use. In the beginning, we selected the strains based on their capability to adhere to the human intestinal epithelial cell (Int-407), colonial enterocyte-like Caco-2 cell, human cervical epithelioid carcinoma cell (HeLa), and human gastric carcinoma cell (TSGH 9201). Then, acid and bile salt tolerance of these LAB strains was evaluated. In addition, the ability of these LAB strains to inhibit the growth of H. pylori and to expel H. pylori cells from TSGH 9201 were studied. The spent culture supernatant (SCS) of a selected strain TM39, i.e., TM39-SCS, significantly inhibited the viability of H. pylori in vitro. It also inhibited the urease activity of H. pylori in vitro. For these antagonistic effects, in addition to pH and lactic acid, some factors in TM39-SCS might play the major role. Treatment of H. pylori with the SCS or cells of strain TM39 significant reduced its binding to TSGH 9201 cells. Although strain TM39 is identified as Enterococcus faecium, it is not vancomycin resistant and is proved to be safe through the invasion study and a 28-day feeding study with Wistar rats.

Animals↗

PubMed2002

Bacteriocin-like activity of Butyrivibrio fibrisolvens JL5 and its effect on other ruminal bacteria and ammonia production.

When ruminal bacteria from a cow fed hay were serially diluted into an anaerobic medium that had only peptides and amino acids as energy sources, little growth or ammonia production was detected at dilutions greater than 10(-6). The 10(-8) and 10(-9) dilutions contained bacteria that fermented carbohydrates, and some of these bacteria inhibited Clostridium sticklandii SR, an obligate amino acid-fermenting bacterium. Phylogenetic analysis indicated that the most active isolate (JL5) was closely related to Butyrivibrio fibrisolvens B835. Strain JL5 inhibited B. fibrisolvens 49 and a variety of other gram-positive organisms, but it had little effect on most gram-negative ruminal bacteria. Strain JL5 did not produce a bacteriocin-like inhibitory substance (BLIS) until it reached the late log or stationary phase. The JL5 BLIS did not cause the lysis of B. fibrisolvens 49, but the intracellular potassium level, the ATP level, the electrical potential, and the viability decreased rapidly. The JL5 BLIS also caused marked decreases in the viability and cellular potassium level of C. sticklandii SR. The membrane potential and intracellular ATP level also declined. The BLIS was degraded very slowly by pronase E, but it could be precipitated with 60% ammonium sulfate and dialyzed (3,500-Da cutoff). The BLIS could be separated from other peptides by polyacrylamide gel electrophoresis, and C. sticklandii SR overlays indicated that the molecular size of this compound was approximately 3,600 Da. Based on these results, it appeared that the JL5 BLIS was a pore-forming peptide. Because carbohydrate-fermenting ruminal bacteria could inhibit the growth of obligate amino acid-fermenting bacteria, BLIS may play a role in regulating ammonia production in vivo.

Amino Acids↗

PubMed2000

Salmonella enterica serovar typhimurium waaP mutants show increased susceptibility to polymyxin and loss of virulence In vivo.

In Escherichia coli, the waaP (rfaP) gene product was recently shown to be responsible for phosphorylation of the first heptose residue of the lipopolysaccharide (LPS) inner core region. WaaP was also shown to be necessary for the formation of a stable outer membrane. These earlier studies were performed with an avirulent rough strain of E. coli (to facilitate the structural chemistry required to properly define waaP function); therefore, we undertook the creation of a waaP mutant of Salmonella enterica serovar Typhimurium to assess the contribution of WaaP and LPS core phosphorylation to the biology of an intracellular pathogen. The S. enterica waaP mutant described here is the first to be both genetically and structurally characterized, and its creation refutes an earlier claim that waaP mutations in S. enterica must be leaky to maintain viability. The mutant was shown to exhibit characteristics of the deep-rough phenotype, despite its ability to produce a full-length core capped with O antigen. Further, phosphoryl modifications in the LPS core region were shown to be required for resistance to polycationic antimicrobials. The waaP mutant was significantly more sensitive to polymyxin in both wild-type and polymyxin-resistant backgrounds, despite the decreased negative charge of the mutant LPSs. In addition, the waaP mutation was shown to cause a complete loss of virulence in mouse infection models. Taken together, these data indicate that WaaP is a potential target for the development of novel therapeutic agents.

Animals↗

PubMed1994

Chemostat flow cell system: an in vitro model for the evaluation of antiplaque agents.

We developed an experimental in vitro model of dental plaque to assess the potential efficacy of antiplaque agents. The model used a chemostat, which provided a continuous source of 5 species of oral bacteria grown in an artificial "saliva-like" medium. This mixture was pumped through six flow cells, each containing two types of surfaces on which plaque formed and was subsequently measured. Formation of bacterial plaque on hydroxyapatite surfaces was assessed by measurement of the DNA and protein content of the plaque film. The amount of bacterial plaque formed on germanium surfaces was measured by attenuated total reflectance (ATR/FT-IR) spectroscopy. Plaque viability was also assessed by a fluorescent staining technique. The quantity of plaque formed on both types of surfaces gradually increased with the duration of flow (from 24 to 72 h) through the cells during a 72-hour experimental period. The flow cells were then pulsed with experimental treatment solutions for 30 s, twice daily. Parallel to results of human clinical studies, the model was capable of discriminating among water, a placebo mouthrinse, and an active antimicrobial mouthrinse formulation containing 0.03% triclosan. It therefore offers a valuable alternative to animal model testing and allows for more rapid evaluations under well-controlled experimental conditions.

Actinomyces viscosus↗

PubMed1988

[Comparative bactericidal activity of imipenem and other antibiotics against Pseudomonas aeruginosa].

The bactericidal activity of imipenem (IM), ticarcillin (TIC), ceftazidime (CEZ), amikacin (AMK) or tobramycin (TOB), ciprofloxacin (CIP), was compared on 6 P. aeruginosa: 1 ticarcillin, susceptible strain (TICs), 5 constitutive beta-lactamases producing strains (PSE, TEM, OXA1, OXA2, cephalosporinase (CEP). The time-kill-curves method was performed. Bacteria were incubated with antibiotics at M.I.C. X2 and at concentrations obtained in vivo with usual therapeutic doses: IM 4-8 mg/l, TIC 64-128, CEZ 4-32, TOB 2-8, AMK 4-16, CIP 1-4. The bactericidal activity of IM was independent of the concentration. A 5 Log10 reduction in viability was observed at 3 h for strains TICs, TEM and PSE, 3 Log10 for strains OXA and CEP. This bactericidal activity was also observed in a non growing system. The bactericidal activity of TIC (strain TICs) and CEZ was slower and weaker: 1-2 Log10 at 6 h, 2-3 Log10 at 24 h. It is not observed in a non growing system. On the other hand, the bactericidal activity of aminoglycosides and ciprofloxacin was rapid: 3-4 Log10 at 3 h, 5 Log10 at 6 h for all strains in growing and non growing systems. IM is the only beta-lactam antibiotic bactericidal on growing and non growing cells of P. aeruginosa like aminoglycosides and new quinolones.

Amikacin↗

PubMed1991

Fluorescein diacetate and ethidium bromide staining to determine the viability of Mycobacterium smegmatis and Escherichia coli.

The ability of the fluorescein diacetate and ethidium bromide fluorescent staining method to assess the percentage of viable bacterial cells in suspension was compared with the plate counting method. Mycobacterium smegmatis and Escherichia coli bacterial cell suspensions were incubated at 60 degrees C. At different time intervals samples were taken and the percentage of viable cells in each sample was assessed by the fluorescent staining method and compared with the plate counting method. The fluorescent staining method showed a positive correlation with the plate counting method. However, the viable counts by the plate counting method were lower than the staining method when incubated at 60 degrees C, indicating a lag period in the decay of enzymes after bacterial death. Hence, the fluorescent staining technique can be used to assess the trend of bacterial death rather than to assess to exact number of viable bacilli.

Colony Count, Microbial↗

PubMed1989

Influence of media supplements on growth and survival of Campylobacter pylori.

Experiments were designed to determine the role of heme and the importance of other factors in the growth of Campylobacter pylori. Campylobacter pylori strains were tested for their ability to synthesize porphyrin, for their ability to grow and be maintained on basal medium and basal medium supplemented with blood or blood products, and for the influence of bovine serum albumin and catalase on viability. Results indicated that Campylobacter pylori does not require heme as a source of porphyrin. Growth of Campylobacter pylori could not be sustained on media containing starch or hemoglobin, but was sustained on media containing erythrocytes, serum, bovine serum albumin or catalase. The ability to grow on media containing bovine serum albumin and catalase suggests that protection from toxic fatty acids and the prevention of toxic product formation may be important factors in the growth and survival of Campylobacter pylori in vitro. Both bovine serum albumin and catalase combined provide the minimum requirements which allow the spectrum of Campylobacter pylori present in a single culture to grow on blood-free media.

Bacteriological Techniques↗

PubMed1985

Isolation of rat hepatocytes with EDTA and their metabolic functions in primary culture.

Isolated hepatocytes from adult rat liver were prepared after dissociation of the liver with EDTA. The morphological appearance, viability (94.5%) and yield (1.76.10(7) cells/g liver) compare well with those of previously described methods using collagenase. Differentiated functions of the hepatocytes in primary culture such as albumin secretion (10.9 micrograms/mg cell protein/d) and triglyceride synthesis and secretion are maintained. Induction of triglyceride synthesis and secretion by oleic acid takes place to an extent similar to that observed in vivo and liver perfusion. Particles with a lipid composition resembling circulating very low density lipoproteins are secreted into the medium. These characteristics demonstrate the ability of hepatocytes isolated with EDTA and subsequently used in primary culture to retain complex and highly differentiated functions of the intact liver.

Albumins↗

PubMed2005

[Pt(HPxSC)Cl(3)], a novel platinum(IV) compound with anticancer properties.

There has been a continuing effort for the discovery of novel platinum(IV)-based antitumor compounds with better therapeutic performances than cisplatin. In the present work, the anticancer action of recently synthesized Pt(IV)-based complex [Pt(HPxSC)Cl(3)] was investigated using rat and human astrocytoma cell lines C6 and U251. [Pt(HPxSC)Cl(3)] markedly reduced the number of cultured astrocytoma cells (IC(50), 80 microM), as determined by crystal violet assay. The Pt(IV) complex induced apoptotic death of tumor cells, as flow cytometry analysis of the propidium iodide-stained cellular DNA revealed approx. 30% of hypodiploid cells in [Pt(HPxSC)Cl(3)]-treated astrocytoma cell cultures. On the other hand, [Pt(HPxSC)Cl(3)] at 200 microM did not affect the viability of rat primary astrocytes, unlike the established anticancer drug cisplatin, which displayed high toxicity toward both astrocytoma cells (IC(50), 15 microM) and primary astrocytes (IC(50), 20 microM). Moreover, [Pt(HPxSC)Cl(3)] at 100 microM did not interfere with the ability of rat peritoneal macrophages to produce important antitumor molecules nitric oxide and tumor necrosis factor-alpha. Finally, we assessed the ability of [Pt(HPxSC)Cl(3)] to restrain growth of some bacterial and yeast strains, but it showed rather limited antimicrobial activity.

Animals↗

PubMed2004

A protocol for quantifying the birth, death and emigration rates of produce-associated bacteria, illustrated by its application to Salmonella enterica serovar Typhimurium on cultivated mushrooms.

Colony counts, counts of immunostained cells, fluorescent assays for cell viability and titration of a superinfecting bacteriophage were incorporated into a protocol for studying the growth kinetics of produce-associated bacteria in vivo. A set of equations was assembled for measuring the true rates of birth, death and emigration of the bacteria within the frame of a "transit growth" model, thus allowing the independent measurement of the carrying capacity of the substrate and of the overall productivity of the system. Implementation of the protocol on two species of cultivated mushrooms inoculated with Salmonella enterica serovar Typhimurium showed that large bacterial populations developed on Agaricus bisporus A15 but emigration was not detected, whereas resident populations on Agrocybe aegerita FAR142 amounted to 79.7%, 65.1% and 80.7% of the cultivable, dead and total bacterial cells produced, owing to emigration.

Agaricales↗