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PubMed1998

Metronidazole resistance and microaerophily in Campylobacter species.

Metronidazole is active against most anaerobic organisms and is also used in the treatment of the microaerophilic bacterium Helicobacter pylori. Resistance to metronidazole is uncommon in most anaerobic organisms, but it is increasingly prevalent in H. pylori. Previously we have suggested that metronidazole resistance in H. pylori is inherent in the microaerophilic nature of the organism and therefore would be present in other microaerophiles such as Campylobacter. Short periods of anaerobiosis caused metronidazole-resistant (MtrR) strains of Campylobacter spp. to become sensitive to metronidazole. Under microaerophilic conditions, cultures of the MtrR mutant Campylobacter coli R1 at bacterial cell densities of greater than 10(8) cfu/ml lost viability, whereas no loss in viability was observed in cultures at cell densities of less than 10(8). The MtrS C. coli strain lost viability at all cell densities. Comparisons of NAD(P)H oxidase activity between MtrS and MtrR strains indicated that the MtrS C. coli strain contained fourfold higher NADH oxidase activity and twofold higher NADPH oxidase activity than did the MtrR Campylobacter strains. These results show that MtrR Campylobacter spp. display resistance characteristics similar to those of H. pylori, suggesting that the resistance mechanism is a phenomenon of the microaerophilic nature of these bacteria.

Aerobiosis↗

PubMed2005

Bioadhesive patch-based delivery of 5-aminolevulinic acid to the nail for photodynamic therapy of onychomycosis.

The in vitro penetration of 5-aminolevulinic acid (ALA) across human nail and into neonate porcine hoof when released from a novel bioadhesive patch containing 50 mg cm(-2) ALA is described. ALA is a naturally occurring precursor of the photosensitiser protoporphyrin IX (PpIX). Topical application of excess ALA bypasses negative feedback inhibition and yields photosensitising concentrations of PpIX at the application site. ALA-based photodynamic therapy (PDT) has been extensively investigated in the topical treatment of various skin neoplasias. Recently, its use has been extended to the microbiological field. If sufficient concentrations of ALA could be achieved within the nail matrix, and at the nail bed, PDT may prove to be a useful treatment for onychomycosis. Patch application for 24 h allowed an ALA concentration of 2.8 mM to be achieved on the ventral side of excised human nail. Application for 48 h induced a concentration of 6.9 mM. Application time had no significant effect on the ALA concentration at mean depths of 2.375 mm in neonate porcine, with application times of 24, 48 and 72 h all producing concentrations of 0.1 mM. Incubation of Candida albicans and Trichophyton interdigitale with ALA concentrations of 10.0 mM for 30 min and 6 h, respectively, caused reductions in viability of 87% and 42%, respectively, following irradiation with red light. Incubation with 0.1 mM ALA for 30 min and 6 h, respectively, caused reductions in viability of 32% for Candida albicans and 6% for Trichophyton interdigitale, following irradiation. Drug penetration across nail may be improved using penetration enhancers, or by filing of the impenetrable dorsal surface of the nail. Moreover, iron chelators can be used to increase PpIX production for a given ALA dose. Therefore, with suitable modifications, ALA-PDT may prove to be a viable alternative in the treatment of onychomycosis.

Adhesives↗

PubMed1992

Growth and differentiation of embryonic stem cells that lack an intact c-fos gene.

The c-fos protooncogene encodes a transcription factor that is thought to play a critical role in proliferation and differentiation as well as in the physiological response of mature cells to their environment. To test directly the role of c-fos in growth and differentiation, we generated mouse embryonic stem cell lines in which both copies of the c-fos gene were specifically disrupted by homologous recombination. Remarkably, the disruption of both copies of c-fos in these cells has no detectable effect on embryonic stem cell viability, growth rate, or differentiation potential. Embryonic stem cells lacking c-fos can differentiate into a wide range of cell types in tissue culture and also in chimeric mice. We conclude that despite a large body of literature suggesting an important role for c-fos in cell growth and differentiation, in at least some cell types this gene is not essential for these processes.

Alleles↗

PubMed2000

Use of a lux reporter system for monitoring rapid changes in alpha-toxin gene expression in Clostridium perfringens during growth.

To determine whether the luxA-luxB reporter system is suitable as a sensitive reporter for rapid real-time measurements of alpha-toxin gene (cpa) expression in Clostridium perfringens, and to widen the range of alpha-toxin-producing C. perfringens strains examined with respect to cpa expression during growth, the reporter plasmid pPS14 (possessing the alpha-toxin promoter region plus 0.7 kb of upstream region linked to the luxA-luxB genes), was used in batch growth experiments of C. perfringens P90.2.2, an alpha-toxin-producing strain with no known association with disease. Levels of in vivo bioluminescence obtained during growth were broadly in agreement with previous mRNA and reporter studies of cpa expression (Bullifent et al., FEMS Microbiol. Lett. 131 (1995) 99-105), confirming the suitability of lux as an accurate reporter system in this organism, but the sensitive nature of the lux reporter permitted the in vivo detection of a very rapid reduction in expression during late-exponential phase that was not attributable to loss in cell viability or limiting bioluminescence assay substrates. There was also a small peak in cpa expression in early- to mid-exponential phase cells, that was not detected in previous studies with other reporters. This may be indicative of the exquisite sensitivity of the lux reporter, or this may be a difference in cpa expression that occurs specifically in this C. perfringens strain. Whichever is the case, these results confirm the complexity of alpha-toxin gene expression in different strains of this pathogenic bacterium.

Clostridium perfringens↗

PubMed1993

Lethal photosensitization of bacteria in subgingival plaque from patients with chronic periodontitis.

Subgingival plaque samples from patients with chronic periodontitis were exposed to light from a 7.3 mW Helium/Neon laser for 30 s in the presence and absence of 50 micrograms/ml toluidine blue O as a photosensitizer. Viable counts of various groups and species of bacteria were carried out before and after irradiation. The median numbers of viable bacteria initially present in the 30-microliters aliquots irradiated were 1.13 x 10(5) cfu (aerobes), 4.08 x 10(5) cfu (anaerobes), 4.92 x 10(3) cfu (black-pigmented anaerobes), 4.75 x 10(2) cfu (Porphyromonas gingivalis), 6.15 x 10(3) cfu (Fusobacterium nucleatum) and 1.7 x 10(4) cfu (streptococci). The dye/laser combination achieved significant reductions in the viability of these organisms, the median reductions in the viable counts being 91.1% for aerobes, 96.6% for anaerobes, 100% for black-pigmented anaerobes, P. gingivalis and F. nucleatum and 94.2% for streptococci. Overall, the viability of bacteria in the 20 plaque samples was not significantly decreased by the dye alone. However, in a small minority of samples there were indications of light-independent, dye-induced toxicity. Low-power lasers, in conjunction with appropriate photosensitizers, may be a useful adjunct to mechanical debridement in the treatment of inflammatory periodontal diseases if a similar effectiveness against subgingival plaque bacteria can be achieved in vivo.

Adult↗

PubMed2002

Broad-spectrum bactericidal activity of Ag(2)O-doped bioactive glass.

Bioactive glass has found extensive application as an orthopedic and dental graft material and most recently also as a tissue engineering scaffold. Here we report an initial investigation of the in vitro antibacterial properties of AgBG, a novel bioactive glass composition doped with Ag(2)O. The bacteriostatic and bactericidal properties of this new material and of two other bioactive glass compositions, 45S5 Bioglass and BG, have been studied by using Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus as test microorganisms. Concentrations of AgBG in the range of 0.05 to 0.20 mg of AgBG per ml of culture medium were found to inhibit the growth of these bacteria. Not only was AgBG bacteriostatic, but it also elicited a rapid bactericidal action. A complete bactericidal effect was elicited within the first hours of incubation at AgBG concentrations of 10 mg ml(-1). 45S5 Bioglass and BG had no effect on bacterial growth or viability. The antibacterial action of AgBG is attributed exclusively to the leaching of Ag(+) ions from the glass matrix. Analytical measurements rule out any contribution to AgBG-mediated bacterial killing by changes in pH or ionic strength or the dissolution of other ionic species from the biomaterials. Our observations of the dissolution profiles of Ag(+) from AgBG in the presence and absence of bacteria are consistent with silver accumulation by the bacteria.

Bacteria↗

PubMed1992

Eradication of biofilm cells of Staphylococcus aureus with tobramycin and cephalexin.

The kinetics of growth and formation of biofilm by Staphylococcus aureus were investigated under iron-limited conditions in the chemostat. The population of planktonic cells reached 5.5 x 10(9) cells/mL 24 h after inoculation (D = 0.05 h-1) and remained constant throughout. The number of biofilm cells of S. aureus colonizing the silicone tubing increased exponentially from 6 x 10(4) to 2.7 x 10(7) cells/cm2 (6 days later) and continued to increase at a reduced rate to 2.7 x 10(8) cells/cm2 on day 13. Planktonic cells of S. aureus were susceptible to tobramycin and cephalexin. The planktonic cells could be successfully eradicated with a combination of 5 micrograms tobramycin plus 100 micrograms cephalexin per millilitre. Exposure of young biofilm cells of S. aureus to 5 micrograms tobramycin plus 100 micrograms cephalexin per millilitre resulted in a rapid loss of cell viability. The percentage of survival dropped to less than 0.0001% after exposure to these concentrations of antibiotics for 3 h. Old biofilm cells of S. aureus were found to be extremely resistant to these antibiotics. The cell viability was reduced to 0.09% after exposure to 10 micrograms tobramycin plus 100 micrograms cephalexin per millilitre. The results suggest that it is possible to eradicate S. aureus infection at the early stage with tobramycin plus cephalexin. Any delay in implementing antibiotic therapy is likely to result in the failure of the treatment. It is important to note that the concentrations of antibiotics required for the eradication of young biofilm cells must be determined for the treatment of device-associated infections.

Bacterial Adhesion↗

PubMed1985

Influence of dietary carrageenans on microbial biotransformation activities in the cecum of rodents and on gastrointestinal immune status in the rat.

Rats, mice, and hamsters were fed iota-carrageenan incorporated in a fiber-free, purified diet for 30 days, and the activities of a number of cecal microbial enzymes were determined in vitro. Carrageenan treatment produced cecal enlargement in all species, yet significantly decreased the concentration of bacteria per gram of cecal content. Azoreductase, beta-glucosidase, beta-glucuronidase, nitrate reductase, and nitroreductase activities per gram of cecal content were significantly decreased in the rat, although less consistent effects were found in these enzymes in the mouse and hamster. beta-Glucuronidase and nitrate reductase functions were increased per gram of cecal contents in the hamster. The total activity per cecum of certain of these enzymes was modified by the concomitant cecal enlargement, yet total nitroreductase activity was significantly decreased in all three rodent species. iota-Carrageenan significantly decreased the concentration of enterobacteria, staphylococci, streptococci, lactobacilli, facultative anaerobes, and the total microscopic count in the rat cecum, but did not exert any effect on bacterial viability in vitro. Although having no effect on biliary IgA antibody concentration, iota- and kappa- carrageenan when present at 50 g/kg diet increased the agglutination response of the IgA specific for the hindgut microflora.

Administration, Oral↗

PubMed1989

Isolation and enrichment of Ca2+-tolerant myocytes for biochemical experiments from guinea-pig heart.

Isolated myocytes for biochemical experiments must be homogeneous and highly enriched in viable cells. For cardiac myocytes, isolation of Ca2+-tolerant cells in high yield and with good viability has been possible from rat. This paper describes myocyte isolation and enrichment procedures which are effective for several species including guinea-pig and rat. New methods for selection of collagenase and viable cells are presented. Using Ca2+-tolerant myocytes obtained from guinea-pig heart and enriched in viable cells, dihydropyridine binding sites are shown to be accessible only in depolarized cells.

Animals↗

PubMed1993

An in-vitro study of the comparative viability on different swab types of simulated specimens of bacteria commonly present in oro-facial infections.

An in-vitro study was undertaken to investigate the survival on bacteriological swabs of three potentially pathogenic organisms found in oro-facial infections, by using simulated clinical specimens incubated in the presence or absence of Amies transport medium. Standard inocula of pure cultures of Streptococcus milleri, Prevotella melaninogenica and Peptostreptococcus anaerobius were soaked on to swabs and plated out on to solid media at various intervals up to 3 days, enabling an estimate of their viability to be made following further incubation. Differential survival of the three test organisms was observed, with Streptococcus milleri being generally the hardiest and Peptostreptococcus anaerobius the least hardy, whilst survival was consistently enhanced by the presence of a transport medium. Recovery of Prevotella melaninogenica was improved by incubation for 5 days. We conclude that transport swabs should be used in preference to plain swabs whenever immediate laboratory culture cannot be assured, to avoid loss of anaerobic bacteria.

Bacteria↗

PubMed1993

Control of intracellular growth of Mycobacterium fortuitum by human monocytes in vitro.

The ability of human monocytes to phagocytize and respond to infection by Mycobacterium fortuitum was tested using the method of Crowle and May. The monocytes were obtained from heparinized donor blood samples and separated from lymphocytes by adherence to plastic surfaces. M. fortuitum infection was performed immediately. Intracellular viability was indicated by colony forming units (CFU) done at 2 hour intervals. Monocytes were found to cause a rapid reduction in CFU during the first 2 hours of incubation. The rate of killing of M. fortuitum decreased thereafter but continued for the entire 8-hour study period. In parallel tests, ciprofloxacin and ofloxacin were added to the culture medium. Increased intracellular killing was observed with drug concentrations above 2 x MIC in both cases. Ofloxacin showed better antimycobacterial effects than ciprofloxacin.

Ciprofloxacin↗

PubMed1997

Differential effects of cytolytic T cell subsets on intracellular infection.

In analyzing mechanisms of protection against intracellular infections, a series of human CD1-restricted T cell lines of two distinct phenotypes were derived. Both CD4(-)CD8(-) (double-negative) T cells and CD8(+) T cells efficiently lysed macrophages infected with Mycobacterium tuberculosis. The cytotoxicity of CD4(-)CD8(-) T cells was mediated by Fas-FasL interaction and had no effect on the viability of the mycobacteria. The CD8(+) T cells lysed infected macrophages by a Fas-independent, granule-dependent mechanism that resulted in killing of bacteria. These data indicate that two phenotypically distinct subsets of human cytolytic T lymphocytes use different mechanisms to kill infected cells and contribute in different ways to host defense against intracellular infection.

Antigens, CD1↗

PubMed1991

Quantitation of postantibiotic effect by measuring CO2 generation of bacteria with the BACTEC blood culture system.

The duration of the postantibiotic effect (PAE) determined by bacterial CO2 production measured by using the BACTEC NR 730 blood culture system was compared with PAEs determined by standard viability counting. PAEs for Staphylococcus aureus after exposure to dicloxacillin, vancomycin, rifampin, gentamicin, and ciprofloxacin and for Escherichia coli after exposure to ampicillin, gentamicin, and ciprofloxacin were quantitated by the two methods, and an excellent correlation (r = 0.93) was demonstrated. The difference in the PAE durations determined by the two methods was 0.1 +/- 0.4 (mean +/- standard deviation) h. Thus, the BACTEC CO2 generation method provides a simple, alternate way of determining the PAE in vitro.

Anti-Bacterial Agents↗

PubMed1992

[The sensitivity of bacterial strains of Yersinia pestis isolated from voles to normal human serum].

The work deals with the results of the study of the viability of Y. pestis strains, isolated from voles in different natural foci of plague, in human normal serum (HNS) and its dependence on the assimilation of ions of exogenic iron. The cultures isolated at the Transcaucasian mountain focus of infection and having no small plasmid pYP were found highly sensitive to HNS. The introduction of the sources of iron, such as hemoglobin or ferritin, into the serum decreased its bactericidal effect.

Animals↗

PubMed2005

Differential inactivation of glucose- and glutamate dependent acid resistance of Escherichia coli TMW 2.497 by high-pressure treatments.

The inactivation by 200-400 MPa and post-pressure survival at acid conditions of E. coli TMW 2.497 was characterized by the measurement of intracellular pH (pHin), viable cell counts, glutamate (Glu) and arginine (Arg) consumption, and the influence of mild adaptation to mild acid stress prior to pressure treatment. Glutamate and arginine did not affect viable cell counts or the pHin during pressure application but improved the ability to maintain a high pHin after pressure treatment. In pH 4.0 buffer without arg and glu, a 3 log reduction of cell counts occurred after 24 h of incubation, whereas little or no loss of viability was observed after 24 h incubation in the presence of glu and arg. During post-pressure incubation at pH 4.0, 10 mM glutamate were metabolized but only 2 mM arginine were used, indicating that glutamate rather than arginine was responsible for the protective effect on pHin and survival. In conclusion, the pressure induced, irreversible loss of the transmembrane deltapH correlates to cell death and glu stabilizes the pHin of E. coli during post-pressure incubation.

Acids↗

PubMed2002

Influence of composition of diluent on populations of yeasts and moulds recovered from raw fruits.

AIMS: The aims of this study were (i) to determine the retention of viability of mycoflora removed from raw fruits, and how this affected diluents used to prepare samples for enumeration of propagules, and (ii) to evaluate the performance of recovery media for supporting colony development. METHODS AND RESULTS: Yeasts and moulds removed from seven types of raw fruit were held in seven diluents for 1 h before plating on dichloran rose bengal chloramphenicol (DRBC) agar and plate count agar supplemented with chloramphenicol (100 micro g ml-1) (PCAC). Significant reductions (P=0.05) in populations of yeasts, moulds, and yeasts plus moulds occurred within the 1 h holding period, regardless of diluent composition. Overall, retention of viability was not influenced by diluent composition, and neither DRBC agar nor PCAC were superior in supporting colony development. CONCLUSIONS: The composition of diluents used to prepare food samples for mycological analysis has little affect on the number of yeasts and moulds recovered from seven types of naturally contaminated raw fruit. Both DRBC agar and PCAC are suitable as enumeration media. SIGNIFICANCE AND IMPACT OF THE STUDY: Diluents and media most often recommended for enumerating yeasts and moulds in foods are appropriate for raw fruits.

Colony Count, Microbial↗

PubMed1994

Calcineurin is essential in cyclosporin A- and FK506-sensitive yeast strains.

The immunophilin-immunosuppressant complexes cyclophilin-cyclosporin A (CsA) and FKBP12-FK506 inhibit the phosphatase calcineurin to block T-cell activation. Although cyclophilin A, FKBP12, and calcineurin are highly conserved from yeast to man, none had previously been shown to be essential for viability. We find that CsA-sensitive yeast strains are FK506 hypersensitive and demonstrate that calcineurin is required for viability in these strains. Mutants lacking cyclophilin A or FKBP12 are resistant to CsA or FK506, respectively. Thus, both the immunosuppressive and the antifungal actions of CsA and FK506 result from calcineurin inhibition by immunophilin-drug complexes. In yeast strains in which calcineurin is not essential, calcineurin inhibition or mutation of calcineurin confers hypersensitivity to LiCl or NaCl, suggesting that calcineurin regulates cation transport.

Amino Acid Isomerases↗

PubMed1991

Improvement in islet yield from a cold-preserved pancreas by pancreatic ductal collagenase distention at the time of harvesting.

This study tried to improve the number of viable islets isolated from a pancreas because a sufficient number cannot be obtained when the organ is preserved in the manner used for pancreas transplantation. The mechanism involved in the decrease in islet yield during preservation was studied to try to develop a better method for islet preparation. First, the integrity of the ductal system was compared between fresh and 6-hr simply preserved (in Hanks' balanced salt solution) rat pancreases. The ductal pressure after ductal injection of HBSS reached a plateau earlier and was significantly lower for the preserved pancreases (0.073 +/- 0.026 min, 410 +/- 17 mmHg, n = 5) than for the fresh ones (0.176 +/- 0.086 min, 561 +/- 103 mmHg, n = 7, P less than 0.05). Second, the extent of pancreatic distention was examined following ductal injection of barium gelatin solution. Solution leakage occurred earlier and distention was less in the preserved pancreas. In addition, the gelatin was found in the capillaries within some islets of the preserved pancreas. These results indicated that the preservation led to a rapid loss of integrity of the ductal system before collagenase injection. We therefore tested the efficacy of ductal collagenase injection at the time of harvesting: 15 ml of 1.0 mg/ml collagenase HBSS was intraductally injected and the pancreas was preserved at 4 degrees C for 2, 4, 6, and 24 hr. The isolation procedure was similar to that used for the fresh pancreas. The yield was significantly better than that of the simply preserved pancreas at 4 hr (241 +/- 22, n = 3, vs. 140 +/- 58, n = 3, P less than 0.05) and at 6 hr (171 +/- 58, n = 14, vs. 32 +/- 33, n = 6, P less than 0.01). These isolated islets were spherical-oval and their viability was confirmed by the ability to reverse STZ-induced diabetes in mice. These results indicated that the integrity of the ductal system, which is necessary for distention of the whole pancreas, was lost during preservation. To solve this problem, ductal collagenase injection should be done at the time of pancreas harvesting and then followed by simple preservation. This method is recommended to obtain viable islets from a preserved pancreas.

Animals↗