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PubMed1977

Effects of ampicillin on intracellular levels of adenosine triphosphate in bacterial cultures related to antibiotic susceptibility.

Short time effects of ampicillin on viability and levels of intracellular ATP were studied in bacterial cultures and a close relationship between intracellular ATP levels and viability was demonstrated. The connection between the effects observed and MIC values is discussed. The possibility of using the phenomenon for rapid antibioitc susceptibility testing was studied in clinical isolates incubated for 2 hours followed by luciferase assay of intracellular ATP. A positive correlation was demonstrated between ampicillin-induced decreases in intracellular ATP and inhibitory zone diameters, as measured by the agar diffusion technique.

Adenosine Triphosphate↗

PubMed1984

Gentamicin uptake in Staphylococcus aureus possessing plasmid-encoded, aminoglycoside-modifying enzymes.

[3H]gentamicin uptake and killing were studied in three strains of gentamicin-resistant Staphylococcus aureus possessing plasmid-encoded, gentamicin-modifying enzymes and in three isogenic, enzyme-free, gentamicin-susceptible derivatives. At low (less than or equal to 2.0 micrograms/ml) concentrations of gentamicin, uptake by resistant organisms was impaired compared with that of susceptible strains, and no killing was noted. In contrast, at higher (2.5 to 10.0 micrograms/ml) concentrations (which were below the MIC for the resistant strains), rapid gentamicin uptake similar to that seen in susceptible isolates was observed. Although growth inhibition at these concentrations was apparent, there was no loss of viability in resistant strains. Consistently, the membrane H+-ATPase inhibitor N,N'-dicyclohexyl carbodiimide caused resistant strains to take up low concentrations (1.0 microgram/ml) of gentamicin at rates comparable to those seen in susceptible organisms without causing an associated loss of viability. These studies show differences between gentamicin uptake in S. aureus and streptomycin uptake in Escherichia coli (Dickie et al., Antimicrob. Agents Chemother. 14:569-580, 1978) regarding the kinetics of uptake in resistant strains with plasmid-encoded aminoglycoside-modifying enzymes. Specifically, they suggest that for 2-deoxystreptamine compounds such as gentamicin, ribosomal binding followed by accelerated uptake and subsequent interference with cell growth may occur without invariably being associated with lethal effect.

Dicyclohexylcarbodiimide↗

PubMed1991

Microculture screening assay for primary in vitro evaluation of drugs against Pneumocystis carinii.

Pneumocystis carinii inoculated into 96-well filtration plate assemblies was shown to synthesize radiolabeled folates de novo from [para-3H]aminobenzoic acid ([3H]pABA). At the end of each incubation with [3H]pABA, a vacuum manifold was used to remove the medium and wash P. carinii. The membrane at the base of each well was dried and punched out, and the level of 3H retained was determined by direct scintillation counting. High-pressure liquid chromatography analysis of duplicate filters confirmed that direct counting of 3H retained on membranes (after correction for unmetabolized [3H]pABA) was an accurate reflection of total [3H]pABA incorporation by P. carinii. Greater than 95% of the 3H recovered was shown to be present as polyglutamated species. After digestion with rat plasma folic acid gamma-glutamyl hydrolase, para-aminobenzoylglutamate, N10-formyltetrahydrofolate, and tetrahydrofolate were identified as the major 3H-labeled components. para-Aminobenzoylglutamate was presumed to have arisen from folylpolyglutamates synthesized by P. carinii and was therefore included in the calculation of total [3H]pABA incorporation. P. carinii incorporation of [3H]pABA under optimal conditions was used as a selective measure of in vitro viability against which the inhibitory effects of some antipneumocystis agents (pentamidine, sulfamethoxazole, 566C80, and piritrexim) were quantitated. The concentrations of pentamidine, sulfamethoxazole, 566C80, and piritrexim required for 50% inhibition in this assay were 7.3, 0.1, 1.4, and approximately 100 microM, respectively. The results suggest that this 96-well [3H]pABA incorporation assay has considerable potential for objective in vitro drug screening against P. carinii.

4-Aminobenzoic Acid↗

PubMed2004

In vitro activity of caspofungin combined with sulfamethoxazole against clinical isolates of Aspergillus spp.

Caspofungin (CAS) inhibits fungal cell wall synthesis. Sulfamethoxazole (SMX) inhibits folate biosynthesis and is active in vitro against Aspergillus spp. We studied the activities of the combination of CAS and SMX against 31 Aspergillus isolates and compared them with that of SMX combined with amphotericin B (AMB) or itraconazole (ITC). MICs and minimal effective concentrations (MECs) were determined by the NCCLS broth microdilution method. With MIC endpoints, the combination of SMX and CAS showed synergy or synergy to additivity against 29 of 31 isolates. With MEC endpoints, synergy to additivity was found against 12 of 31 isolates and indifference was displayed against the rest of them. SMX in combination with AMB or ITC was not truly synergistic, while synergy to additivity was found for SMX-AMB and SMX-ITC against 17 of 31 and 3 of 12 isolates, respectively. No antagonism was found with any of the drug combinations. Further analysis of the synergy of CAS and SMX was performed by detailed measurement of hyphal length by microscopy and time-dependent 2,3-bis(2-methoxy-4-nitro-5-[(sulfenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT)-based hyphal damage experiments. With MEC endpoints, the combination of CAS and SMX was characterized by a greater than 50% decrease in hyphal length compared to the hyphal lengths achieved with double the concentration of each drug alone. The XTT-based hyphal damage studies showed a statistically significant (P < 0.05) reduction in viability with CAS and SMX in combination compared to the viabilities achieved with double the concentration of each drug alone. These findings support the synergy results found by using MIC endpoints and suggest that visual MEC measurements may not be sufficient to identify the synergistic interactions seen by more sensitive, quantitative methods. Animal models are required to validate the significance of the synergy of CAS and SMX against Aspergillus spp. observed in vitro.

Amphotericin B↗

PubMed1982

The effect of exposure of Staphylococcus aureus to penicillin on susceptibility to the bactericidal activity of human leukocytes.

The effect of brief exposure of staphylococci to penicillin upon susceptibility to leukocidal activity was studied. Antibiotic pretreated staphylococci were incubated with normal human leukocytes and viability of the bacteria was determined. Penicillin pretreated staphylococci were more susceptible to killing by leukocytes than untreated control bacteria. The extent of sensitization varied between different strains of staphylococci. When staphylococci were exposed simultaneously to leukocytes and penicillin, the bacteria were protected from the lethal action of penicillin. This study demonstrates that phagocytosed staphylococci are protected from penicillin, but prior exposure to the antibiotic increases their susceptibility to the activity of leukocytes.

Adult↗

PubMed2002

[Evaluation of levofloxacin antitubercular activity in vitro and in lung tissue culture].

Levofloxacin in vitro demonstrated bactericidal effect against susceptible and multi-drug resistant Mycobacterium tuberculosis: range of MICs was 0.25-0.5 mcg/mL, MBC-0.5-1.0 mcg/mL. Postantibiotic effect after 24-hour exposition to bactericidal concentrations was 35-39 days. Levofloxacin possesed low toxicity when tested in mice lungs tissue culture--maximum safety concentration was 50 mcg/mL. Bactericidal effect of levofloxacin started three days after exposition and was maximal by 7 days of incubation: by this time mycobacterial microcolonies destruction started with detritus formation. It is emphasized that lung cells kept their viability completely. Combination of levofloxacin with isoniazide or pirazinamide resulted in strong synergistic effect obvious after 5 days of incubation, mycobacterial colonies destruction was registered by 7th day. Combination of levofloxacin with rifampicin resulted in antagonistic effect obvious by 7th day of the contact: the resulting effect was statistically significant and was manifested as microcolonies number and size enlargement when compared to data for single levofloxacin.

Anti-Infective Agents↗

PubMed1991

[The clinical value of Mycobacterium tuberculosis with different biological properties].

The main biological properties (including viability, drug resistance and virulence) of Mycobacterium tuberculosis, identified in 180 newly-diagnosed patients with different forms of destructive pulmonary tuberculosis and living in Leningrad, North-Western and Extreme Northern regions, were studied as compared with those identified in 40 patients with chronic destructive pulmonary tuberculosis. The viability of M. tuberculosis by their growth and mass increase rates has a prognostic importance and should be taken into account in choosing an appropriate chemotherapy. The use of a standard method of absolute concentrations to determine the M. tuberculosis drug resistance is quite sufficient in clinical practice to introduce corrections into antituberculous chemotherapy. No correlation between M. tuberculosis virulence, and the course of tuberculous process and its treatment efficacy was found.

Drug Resistance, Microbial↗

PubMed2004

Osmotic stress response: quantification of cell maintenance and metabolic fluxes in a lysine-overproducing strain of Corynebacterium glutamicum.

Osmotic stress diminishes cell productivity and may cause cell inactivation in industrial fermentations. The quantification of metabolic changes under such conditions is fundamental for understanding and describing microbial behavior during bioprocesses. We quantified the gradual changes that take place when a lysine-overproducing strain of Corynebacterium glutamicum is grown in continuous culture with saline gradients at different dilution rates. The use of compatible solutes depended on environmental conditions; certain osmolites predominated at different dilution rates and extracellular osmolalities. A metabolic flux analysis showed that at high dilution rates C. glutamicum redistributed its metabolic fluxes, favoring energy formation over growth. At low dilution rates, cell metabolism accelerated as the osmolality was steadily increased. Flexibility in the oxaloacetate node proved to be key for the energetic redistribution that occurred when cells were grown at high dilution rates. Substrate and ATP maintenance coefficients increased 30- and 5-fold, respectively, when the osmolality increased, which demonstrates that energy pool management is fundamental for sustaining viability.

Biomass↗

PubMed1988

Phospholipase activation and arachidonic acid release in isolated intestinal epithelial cells.

A novel method for studying the mobilization of free arachidonic acid (AA) in isolated intestinal epithelial cells is described. The method is based on labeling the cellular phospholipids with 14C-AA and studying the release of this 14C-AA on subsequent phospholipase activation. Cells of high viability were isolated from the small intestine of guinea pigs and incubated with 14C-AA for 2 h; most of the incorporated 14C-AA was then esterified into phosphatidylethanolamine and phosphatidylcholine. When the labeled cells were stimulated with the calcium ionophore A23187 in the presence of external calcium, they released significant amounts of AA. In contrast, the cells released no AA when stimulated with A23187 in the absence of external calcium or in the presence of chlorpromazine or 4-bromophenacyl bromide, both of which are known to inhibit phospholipase A2 activity. On the other hand, the cells released significant AA in response to exogenous phospholipase C from Clostridium perfringens. These findings indicate that AA release in intestinal cells may be caused by calcium-mediated phospholipase A2 activation or by products of microbial phospholipase C activity. They also suggest the further use of 14C-AA-labeled cells for studying agents and mechanisms that may influence the release of AA in the gastrointestinal tract.

Animals↗

PubMed1988

Monitoring of bacterial drug response by mass spectrometry of single cells.

The application of the laser microprobe mass analyser LAMMA 500 to the solution of problems in the field of microbiology is reported. The special features of this instrument allow the analysis of single bacterial cells, and questions can be answered which are not accessible to the normally applied integral methods. Thus it is possible to establish distributions of, for instance, elemental concentrations within a bacterial population and of correlations between measured characteristics of a bacterium with its morphology. The mass spectrum of a single bacterial cell comprises information on its intracellular cation contents as well as on the organic matrix. The relation between the sodium and potassium contents can serve as a criterion of the physiological state of a cell and of its viability. The information from the organic matrix can be extracted from the complex spectra of fragment ions produced by the interaction of the laser with the cell by applying multivariate data analysis, thus rendering additional information. Examples will be given for in vitro drug screening and in vivo therapy control in leprosy, an infection which is caused by a mycobacterial species, Mycobacterium leprae; this organism does not multiply in artificial growth media so that only limited numbers of organisms are available for microbiological investigations.

Mass Spectrometry↗

PubMed1985

Effects of polymyxin B sulfate and polymyxin B nonapeptide on growth and permeability of the yeast Saccharomyces cerevisiae.

Polymyxin B, a toxic, membrane-affecting antibiotic, can be rendered harmless to yeast cells by enzymatic removal of its fatty acyl moiety. The remaining cyclic peptide portion, polymyxin B nonapeptide, has no significant effect on growth and viability but it drastically reduces mating efficiency. In addition, the cyclic peptide enhances sensitivity of cells to several drugs, presumably by increasing membrane permeability. Mutants resistant to polymyxin B are simultaneously less responsive to the combination of the nonapeptide and the drugs. This indicates that the peptide portion of polymyxin B is the moiety responsible for the permeability changes. The resistance is inherited as a simple recessive trait. The mutation has been mapped to chromosome XV of Saccharomyces cerevisiae.

Anti-Bacterial Agents↗

PubMed1994

Disruption of the transmembrane pH gradient--a possible mechanism for the antibacterial action of azelaic acid in Propionibacterium acnes and Staphylococcus epidermidis.

The effect of the topical acne treatment azelaic acid on the transmembrane proton gradient (delta pH) of Propionibacterium acnes and Staphylococcus epidermidis was studied in vitro at external pH values found on human skin (pH 4.0-6.0). Bacteria were grown in defined media using continuous culture and delta pH was estimated by measuring the accumulation of [14C] benzoic by the cells using flow dialysis. In both P. acnes and S. epidermidis the addition of 30 mM azelaic acid and the membrane active inhibitors nigericin (150 microM) and CCCP (150 microM) resulted in a rapid release of [14C] label into the dialysate indicating the dissipation of delta pH between external pH values of 4.0-6.0. The addition of 60 mM NaCl as an iso-osmotic control and 150 microM valinomycin did not induce the release of [14C] label. The addition of 30 mM azelaic acid reduced the delta pH of P. acnes by 44% at external pH 4.0 and 28% at external pH 6.0. In S. epidermidis 30 mM azelaic acid reduced delta pH by 88% at external pH 5.0 and 20% at external pH 6.0. Rapid loss of viability occurred in suspensions of P. acnes and S. epidermidis containing 30 mM azelaic acid at pH 4.0 with no viable cells recovered after 60 min incubation. At pH 6.0 little change in viable numbers of P. acnes and S. epidermidis were observed over a 2 h incubation period. The results indicate that the antibacterial activity of azelaic acid is associated with the perturbation of intracellular pH.

Anti-Bacterial Agents↗

PubMed1997

Effects of tetracycline hydrochloride and chlorhexidine gluconate on Candida albicans. An in vitro study.

This study examined the effects of tetracycline hydrochloride (TCN) and chlorhexidine gluconate (CHX) on the growth and viability of Candida albicans. Subcultures of Candida albicans on Sabouraud's agar, were divided into 5 treatment groups: group 1, untreated control; group 2, 0.12% CHX; group 3, 3.0 mg/ml TCN adjusted to pH 4.5; groups 4 and 5, sodium azide free Tris buffer adjusted to pH 4.5 and pH 7.4, respectively. All groups were incubated for 10 days, and sampled and subcultured daily to determine the viability of each group. Additional samples from group 2 (day 4), group 4 (day 7) and all groups at day 10 were selected for SEM and TEM examination. Visual, SEM and TEM results showed that for groups 1, 3, 4, and 5 there was a heavy and constant uniform growth of Candida albicans throughout the period of the study. However, group 2 (CHX), showed decreasing viability and attachment from day 3 to day 10, with SEM and TEM revealing decreased blastospores and profound changes in the ultrastructural morphology, indicating inhibition of normal cell growth and replication. These results show that TCN even when used at high concentrations, in vitro, will allow uninhibited growth of Candida albicans whereas CHX inhibits cell growth and replication.

Adhesiveness↗

PubMed2000

Rdp1, a novel zinc finger protein, regulates the DNA damage response of rhp51(+) from Schizosaccharomyces pombe.

The Schizosaccharomyces pombe DNA repair gene rhp51(+) encodes a RecA-like protein with the DNA-dependent ATPase activity required for homologous recombination. The level of the rhp51(+) transcript is increased by a variety of DNA-damaging agents. Its promoter has two cis-acting DNA damage-responsive elements (DREs) responsible for DNA damage inducibility. Here we report identification of Rdp1, which regulates rhp51(+) expression through the DRE of rhp51(+). The protein contains a zinc finger and a polyalanine tract similar to ones previously implicated in DNA binding and transactivation or repression, respectively. In vitro footprinting and competitive binding assays indicate that the core consensus sequences (NGG/TTG/A) of DRE are crucial for the binding of Rdp1. Mutations of both DRE1 and DRE2 affected the damage-induced expression of rhp51(+), indicating that both DREs are required for transcriptional activation. In addition, mutations in the DREs significantly reduced survival rates after exposure to DNA-damaging agents, demonstrating that the damage response of rhp51(+) enhances the cellular repair capacity. Surprisingly, haploid cells containing a complete rdp1 deletion could not be recovered, indicating that rdp1(+) is essential for cell viability and implying the existence of other target genes. Furthermore, the DNA damage-dependent expression of rhp51(+) was significantly reduced in checkpoint mutants, raising the possibility that Rdp1 may mediate damage checkpoint-dependent transcription of rhp51(+).

Adenosine Triphosphatases↗

PubMed2006

Using Ecosan sludge for crop production.

This paper presents the microbial risks associated with the reuse of Ecosan sludge in agriculture. Sludge from KwaZulu Natal Province in South Africa with a helminth ova content of 30 HO/g TS, faecal coliforms of 10(6) CFU/g TS, faecal streptococci of 10(6) CFU/g TS and Salmonella spp. of 10(5) CFU/g TS was used for this purpose. Applying different rates of sludge to spinach and carrots resulted in an increase in bacterial and helminth ova counts in crops as the quantity of sludge increased. The helminth ova content in crops was always greater in leaves than in stems, with a content varying from 2 to 15 HO/g TS for spinach with sludge application rates of 0-37.5 ton/ha and from 2-8 HO/g TS for carrot crops with sludge application rates varying from 0-35 ton/ha. Nevertheless, viability in crops was 20-25%, reducing the risk of disease. Consequently, the development of an Ecosan sludge revalorisation standard is considered important. Some recommendations are made taking into account the characteristics of the sludge as well as conditions in rural areas.

Animals↗

PubMed2003

Direct microscopic observation and viability determination of Campylobacter jejuni on chicken skin.

A method was developed to determine the survival of Campylobacter jejuni at specific sites on chicken skin, and this method was used to observe the survival of C. jejuni at various locations on the skin during storage. This method uses confocal scanning laser microscopy to visualize C. jejuni transformed with P(c)gfp plasmid (GFP-Campylobacter) and stained with 5-cyano-2,3-ditolyl tetrazolium chloride (CTC). The green fluorescence of dead C. jejuni cells and the red fluorescent CTC-formazan in viable Campylobacter cells were clearly visible on chicken skin. The GFP-Campylobacter remaining on the chicken skin surface after rinsing was mostly located in crevices, entrapped inside feather follicles with water, and entrapped in the surface water layer. Most viable cells were entrapped with water in the skin crevices and feather follicles. These sites provide a suitable microenvironment for GFP-Campylobacter to survive. The population of C. jejuni on chicken skin decreased by 1 log unit during storage at 25 degrees C for 24 h. C. jejuni located in sites 20 to 30 microm beneath the chicken skin surface maintained viability during incubation at 25 degrees C. C. jejuni on chicken skin stored at 4 degrees C maintained constant numbers during a 72-h incubation with no significant changes in population feather follicles or crevices. Live and dead cells were initially retained with water on the skin and penetrated into the skin follicles and channels during storage. Microscopic observations of GFP-producing cells allowed the identification of survival niches for C. jejuni present on chicken skin.

Animals↗

PubMed1993

[Contribution of flow cytometry in virology].

The development of commercial multichannel air-cooled laser flow cytometers which allow measurement of one or more characteristic parameters of particles or cells has been furthered due to advances in probes and fluorochromes. FITC-conjugated antibodies specific for selected cell surface molecules or for viral antigen expression in infected cells, fluorescein diacetate for labeling cells as a parameter of viability and propidium iodide as a DNA stain are generally used. Flow cytometric assays have been developed in virology to study the interactions of viruses with the immune system (Hantan Virus), to evaluate the diagnosis of secondary immunodeficiency syndromes (HIV), to analyze the protein and DNA content of virally infected cells during the cell cycle (SV40), to detect immediate early, early or late antigens in HCMV-infected cells, to assess the HCMV persistence in mononuclear cells, for use as a model for studying virus attachment to cellular receptors (EBV-echovirus), to screen and evaluate antiviral agents (Influenza C, HCMV, HSV, HIV) and to quantify antigens or antibodies simultaneously against various viruses (HCMV and HSV) or against different antigenic glycoproteins (HIV). This technology has thus become an important tool for the clinical virology and microbial serology laboratories.

Antibodies, Viral↗

PubMedDate not supplied

Molecular approaches to the investigation of viable dinoflagellate cysts in natural sediments from estuarine environments.

Molecular methods offer an efficient alternative to microscopic identification of dinoflagellate cysts in natural sediments. Unfortunately, amplification of DNA also detects the presence of dead cells and is not a reliable indication of cyst viability. Because mRNA transcripts are more labile than DNA, the presence of specific transcripts may be used as a proxy for cyst viability. Here, we evaluate mRNA detection capabilities for identification of viable cysts of the dinoflagellate, Pfiesteria piscicida, in natural sediment samples. We targeted transcripts for cytochrome c oxidase subunit 1, cytochrome b (COB), and Tags 343 and 277, recently identified by serial analysis of gene expression. Expression was confirmed in laboratory cultures and compared with natural sediment samples. Three of the transcripts were detected in sediments by RT-PCR. The fourth transcript, for COB, was not detected in sediments, perhaps because of down-regulation of the gene in anoxic conditions. Our results suggest that methods targeting specific mRNA transcripts may be useful for detection of viable cysts in natural sediment samples. In addition, dinoflagellate cysts, which sustain extended periods of anoxia, may provide an important source of data for studies of anoxia tolerance by microbial eukaryotes.

Animals↗