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PubMed2001

Cytometric approach for a rapid evaluation of susceptibility of Candida strains to antifungals.

OBJECTIVE: To achieve a fast and reliable determination of the susceptibility of Candida strains to amphotericin B (Am B), fluconazole (Flu) and 5-fluorocytosine (5-FC), using cytometric methods as an alternative to the classical dilution method. METHODS: Twenty-three clinical isolates of Candida with different susceptibility patterns were treated for 1 h with two concentrations each of Am B (2 and 8 mg/L), Flu (8 and 64 mg/L) and 5-FC (4 and 32 mg/L), followed by staining with three different fluorochromes, under conditions previously defined through an optimisation study. These were 1 mg/L propidium iodide (PI)/10(6) cells for 30 min at 30 degrees C (a marker that only penetrates cells with severe lesions of the membrane); 0.5 microM FUN-1/10(6) cells for 30 min at 30 degrees C (a fluorescent probe which after entering the yeast cell is converted, by metabolically active yeasts, from a diffuse cytosolic pool with a yellow-green fluorescence into red cylindrical intravacuolar structures) and 0.25 microM of JC-1/10(6) cells for 15 min at 37 degrees C (a monomer that changes reversibly from green to red the J-aggregates, with the increased membrane potential). About 50 000 yeast cells were analysed by flow cytometry (FCM), at FL3 (red, 620 nm) for PI and FL2 (yellow-green, 575 nm) for FUN-1 and the ratio of FL3 to FL1 was determined (red, 620 nm/green, 525 nm) for JC-1; 200 cells of each suspension were also analysed by epifluorescence microscopy (EPM). Viability studies were performed in parallel to count the number of colony forming units. RESULTS: Susceptible (S) strains exposed to Am B and stained with JC-1 showed a dose-dependent decrease in the mitochondrial potential, i.e. a decreased ratio between red/green fluorescence by FCM and a decrease in J-aggregates by EPM. Neither FUN-1 nor PI was useful in the study of Am B activity. Susceptibility to Flu and 5-FC could be detected with FUN-1 staining: metabolic changes were detected by an increase in yellow-green intensity of fluorescence by FCM or a decrease of cylindrical intravacuolar structure formation by EPM, although no decrease in total viability was registered. Staining with JC-1 could predict resistance to both drugs, but did not allow distinction between sensitive dose-dependent strains (S-DD) or intermediate (I) resistance to Flu or 5-FC, respectively, from S strains. PI did not stain Candida cells treated with Flu or 5-FC under our experimental conditions. CONCLUSION: Susceptibility patterns of Candida strains to Am B can be determined by using JC-1, and to Flu and 5-FC by using FUN-1. PI was not a useful probe with which to study the effect of such antifungals under the conditions described here.

Antifungal Agents↗

PubMed1993

Tn5-mediated bleomycin resistance in Escherichia coli requires the expression of host genes.

The transposon Tn5 expresses a gene, ble, whose product increases the viability of Escherichia coli and also confers resistance to the DNA-cleaving antibiotic bleomycin and the DNA-alkylating agent ethylmethanesulphonate. We find that the Ble protein induces expression of an alkylation inducible gene, aidC, and that both the AidC gene product and DNA polymerase I are required for Ble to confer bleomycin resistance. These findings support models in which Ble enhances DNA repair and suggest that Tn5 confers a fitness advantage to the host bacterium by increasing the repair of spontaneous DNA lesions. Such co-operation between a transposon and its host suggests that Tn5 is a symbiotic rather than a selfish DNA element.

Bacterial Proteins↗

PubMed1986

Isolation of smooth muscle cells from swine carotid artery by digestion with papain.

A new method is described for the preparation of viable, elongated smooth muscle cells from the swine carotid artery. Cells were prepared by papain digestion of pressurized arteries in calcium-free solution. After digestion, the arteries were everted, and fine strips were teased from the intimal surface of the media in calcium-free solution, releasing single cells. Viability was assessed by exclusion of trypan blue and by appearance under phase-contrast microscopy. By these criteria, approximately 20% of the isolated cells were viable. The most distinguishing and unexpected characteristic of these cells was their length. Mean length of the relaxed viable cells was 240.4 +/- 47.4 microns (SD, n = 76), which is much longer than previously reported for arterial smooth muscle cells. Calcium (1.6 mM) caused most of the viable cells to contract slightly, and the mean cell length in calcium was 194.4 +/- 57.7 microns. Cells in 1.6 mM calcium contracted substantially in response to 10 microM histamine or the calcium ionophore A23187 (10 microM), demonstrating that histamine receptors and the contractile apparatus were still functional.

Animals↗

PubMed1987

Kidney proximal tubular cells isolated by collagenase perfusion grow in defined media in the absence of growth factors.

Cells from kidney proximal tubules have been successfully isolated, characterized, and cultured from male Fischer 344 rats between 150-400 g using a two-step collagenase perfusion. The cells undergo high levels of DNA synthesis and mitosis in both serum free media (with an without hormone supplementation) and media containing 10% fetal bovine serum. Confluent monolayers were observed between 5 to 7 days after seeding 2 X 10(5) cell/35 mm collagen-coated plate. Approximately 50% of the total kidney and 70% of the cortex was isolated using this technique. The viability of the isolated tubules was 75 +/- 8% and the estimated number of viable cells was 12 +/- 3 X 10(6) cells. At the time of isolation greater than 90% of the isolated tubules and cells were positive for gamma glutamyltransferase (GGT), periodic acid-schiff (PAS), and glucose-6-phosphatase (G-6-Pase). Both GGT and G-6-Pase decreased rapidly during the first 3 days in primary culture as assessed by histochemistry. Ultrastructurally the isolates consisted of cells with numerous microvilli and mitochondria. The size and number of microvilli decrease rapidly in primary culture. The morphologic and biochemical evidence suggests that the primary isolates and cultures are proximal tubular in origin.

Animals↗

PubMed2006

Effects on Escherichia coli O157:H7 and meat starter cultures of bovine lactoferrin in broth and microencapsulated lactoferrin in dry sausage batters.

The effects of lactoferrin (LF) alone or with various chelating agents on the growth of 5 strains of Escherichia coli O157:H7 and 7 meat starter cultures were evaluated. E.coli O157:H7 and starter cultures were grown at 13 or 26 degrees C in Lauria (LB) or All Purpose Tween (APT) broths, respectively, with both broths being supplemented with 2.9% NaCl. LF alone prevented the growth of E. coli O157:H7 strains 0627 and 0628 but other strains grew. The antimicrobial effectiveness of LF was enhanced by EDTA but LF alone did not affect the growth of meat starter cultures in broth. However, when LF plus EDTA and sodium bicarbonate (SB) were used the growth of all meat starter cultures except Lactobacillus curvatus was reduced. During dry sausage manufacture with L. curvatus and Staphylococcus carnosus starter cultures the effects of LF, unencapsulated or microencapsulated in paste-like and dried powder forms, in sausage batters with or without EDTA and SB, on the viability of E. coli O157:H7 were examined. The reduction of E. coli O157:H7 during sausage manufacture was significantly enhanced (p<0.05) by all LF treatments. The largest reduction (4.2 log units) was obtained with unencapsulated LF. However, some of the apparent reduction in E.coli O157:H7 numbers with all treatments was due to cell injury rather than lethality, since significantly greater numbers were recovered on APT agar overlaid with the selective medium cefixime-tellurite Sorbitol McConkey agar (ct-SMAC) than on ct-SMAC alone. The narrow spectrum of LF activity and induction of injury rather than inactivation of E. coli O157:H7 limit the effectiveness of this agent against the pathogen in fermented meats.

Animals↗

PubMed2001

Selection and characterization of mixed starter cultures for lactic acid fermentation of carrot, cabbage, beet and onion vegetable mixtures.

An evaluation of various lactic acid bacteria (LAB) for the fermentation of cabbage, carrot and beet-based vegetable products was carried out. As part of a screening process, the growth of 15 cultures in a vegetable juice medium (VJM) was characterized by automated spectrophotometry. Acidification patterns as well as viability during storage of the LAB were also established. There were greater differences between the pure cultures than the mixed ones with respect to growth in VJM and viability during storage. Reductions in viable cell counts during storage of the fermented VJM occurred more rapidly with a Leuconostoc strain than for pediococci or lactobacilli. Inoculation of vegetables was carried out with cultures of Lactobacillus plantarum NK-312, Pediococcus acidilactici AFERM 772 and Leuconostoc mesenteroides BLAC which were rehydrated in a brine. This rehydration procedure was not detrimental to viability. During fermentation of a carrot/cabbage vegetable mix, sugar metabolism was characterized by the assimilation of both glucose and fructose, but sugars remained in the fermented vegetables when acidification stopped. The pH in the LAB-inoculated vegetables after 72 h at 20 degrees C was significantly lower (by 0.2 units) than the uninoculated control. Inoculation with LAB designed for silage fermentation resulted in the inhibition of acetic acid production, and reduced the production of ethanol during fermentation. The selection process on VJM enabled the preparation of a mixed culture that was more rapid than the silage inoculants in acidifying the medium and was more effective in reducing the production of gas during the fermentation and storage of the fermented vegetables.

Colony Count, Microbial↗

PubMed1992

Salivary anti-candidal assays.

Inhibition of Candida albicans blastospore viability by parotid, submandibular-sublingual and whole salivas could not be determined by direct assay of yeast cells in each respective saliva. Determination of antifungal activity could, however, be carried out if saliva was first preincubated with Candida cells and this was immediately followed by removal of saliva and resuspension of yeast cells in nonenriched buffers of pH 5-7 for appropriate incubation periods. To attain accurate reproducible quantitative data, parotid, submandibular-sublingual and whole salivas each required different preincubation times with C. albicans as well as prior acidification and boiling. Acidification was also necessary for optimizing the germ tube assay although, in contrast to blastospore viability, inhibition of blastospore-germ tube conversion could be determined directly in saliva. Salivary antifungal effects on blastospore division were negligible at yeast cell concentrations greater than 10(6) colony-forming units per ml and were found to be independent of pH, whereas salivary inhibition of germ tube formation was significant only at pH 5 in the assay systems employed. The requirement for acidification and an observed enhancement of antifungal activity on aqueous dilution of the saliva suggested that only a fraction of the salivary antifungal components present in saliva were available in the free form to exert their biological activity. These results open up the possibility of investigating salivary antifungal activity in human health and disease.

Antifungal Agents↗

PubMed1979

Activity of 2,4-diaminoquinazoline compounds against Candida species.

Forty recent clinical isolates of three different Candida sp. were tested in the microtiter system for susceptibility to two new 2,4-diaminoquinazoline (DAQ) compounds, amphotericin B and flucytosine. The two DAQ preparations showed activity similar to amphotericin B and flucytosine. The geometric mean minimal inhibitory concentrations for these four drugs were as follows: DAQ 1A, 0.64 mug/ml; DAQ 2A, 1.39 mug/ml; amphotericin B, 1.03 mug/ml; and flucytosine, 0.72 mug/ml. An additional seven DAQ compounds were tested but showed less or no activity against 17 Candida isolates. Forty-eight-hour viability studies with DAQ 2A alone or in combination with amphotericin B, flucytosine, or sulfamethoxazole were carried out with one isolate of intermediate susceptibility to each of these agents except sulfamethoxazole. For this isolate the combination of DAQ 2A and sulfamethoxazole was synergistic, and the combination of DAQ 2A and AMB was either synergistic or additive, whereas the combination of DAQ 2A and flucytosine was antagonistic. Although regrowth of cultures exposed to DAQ 2A was noted over a 48-h period, neither degradation of the drug nor development of resistance to the drug could be detected. Swiss white mice receiving DAQ 1A at a dose of 6 mg/kg for 5 days showed no obvious signs of toxicity, including weight loss.

Amphotericin B↗

PubMed1980

Characterization of complement resistance in Escherichia coli conferred by the antibiotic resistance plasmid R100.

We have examined the effect of the antibiotic resistance plasmid, R100, on the complement (C) mediated killing of Escherichia coli K-12 strains. The viabilities, in dilute normal rabbit serum (NRS), of 5 such strains were compared with the viabilities of the same strains harboring R100. For 1 strain, J6-2, we also measured the effect of R100 on viability in normal human serum (NHS) and in guinea pig serum (GPS); in NRS, NHS,and GPS devoid of classical C pathway activity; and in NHS devoid of alternative pathway activity. Finally, we compared the depletion of individual complement components in sera exposed to J6-2 and J6-2 harboring R100. Our results demonstrate that 1) R100 renders E. coli K-12 resistant to killing by NRS, NHS, and GPS; 2) the level of resistance, which ranges from 30- to 10,000-fold, is strain dependent and serum dependent; 3) R100 inhibits killing by both the classical and alternative C pathway. Resistance appears to involve a disruption on the C pathway subsequent to the activation of C5.

Animals↗

PubMed1998

Lenticules for the control of quantitative methods in food microbiology.

Control dried organisms as lenticules are a dependable and convenient alternative to wet cultures for quality assurance and process controls in routine food microbiology. Lenticules are designed to give a fixed, reproducible inoculum over an extended period of time without loss of cultural characteristics or viability. During a period of 23 months, 596 paired counts were performed by both Miles and Misra and spiral plating techniques on lenticule controls. Correlation between the two methods and within batches was excellent. Only 14 counts (2.5%) fell outside the standard operating limit of 0.5 log10. All were within 1.0 log10. On two separate occasions, replicate runs were performed on five reconstituted lenticules from a batch. The counts obtained showed variation within and between lenticules only slightly in excess of what is expected by chance. Lenticule replicates performed by three other laboratories also produced satisfactory results. It is thought that lenticules could improve the accuracy of total plate counts and lead to a better standardization of quantitative methods in food microbiology within and between laboratories.

Colony Count, Microbial↗

PubMed1985

Inhibition of penicillin-binding protein 3 of Escherichia coli K-12. Effects upon growth, viability and outer membrane barrier function.

A temperature-conditional, cell-division mutant of Escherichia coli K-12 possessing a thermolabile penicillin-binding protein (PBP) 3 was isolated. The mutant phenotype was due to a lesion in the pbpB gene. This mutant, and leu+ pbpB co-transductants of E. coli C600 grew as rods at 30 degrees C but were converted to filaments at 42 degrees C upon denaturation of PBP3 and concomitant cessation of cell division. These strains have been used to study the consequences of the specific inhibition of PBP3 of E. coli K-12 upon growth, viability and outer membrane integrity. Our results indicate that the singular inhibition of PBP3 is bactericidal in E. coli K-12, even though the turbidimetric response of the bacteria in broth culture suggests bacteriostasis. Furthermore, filament formation is accompanied by disruption of outer membrane barrier function, as witnessed by the rapid leakage of periplasmic beta-lactamase. This latter finding was confirmed by observing the lytic effect of a sub-inhibitory concentration of cefsulodin on filaments of E. coli K-12 induced by PBP3-specific beta-lactams. The impact of these results upon the testing of beta-lactam sensitivity of E. coli K-12 is discussed.

Acyltransferases↗

PubMed1972

Temperature-sensitive mutant of Bacillus subtilis that accumulates membrane-associated protein inclusions.

At 47 C, a temperature-sensitive mutant of Bacillus subtilis 168 accumulates membrane-associated protein inclusions and exhibits a pleiotropic phenotype indicative of a defect in lipid synthesis. The mutant bacteria cease growing at 47 C, and the turbidity of the culture gradually declines. The lack of growth is not due to the death or lysis of the cells, since viability does not decrease for about 1 hr and the "lysis" can be delayed for several hours by increasing the osmotic pressure of the medium. Synthesis of deoxyribonucleic acid and ribonucleic acid stops at 47 C although a residual synthesis of protein occurs. When the temperature is raised, the mutant fails to increase the proportion of 17:0 branched-chain fatty acids and to decrease the proportion of 18:0 and 18:1 fatty acids. The membrane-associated inclusions can be seen by phase-contrast or electron microscopy and remain attached to protoplast membranes during isolation. The inclusions are mostly protein and are digested with Pronase.

Bacillus subtilis↗

PubMed1999

The use of multiple indices of physiological activity to access viability in chlorine disinfected Escherichia coli O157:H7.

A suite of fluorescent intracellular stains and probes was used, in conjunction with viable plate counts, to assess the effect of chlorine disinfection on membrane potential (rhodamine 123; Rh123 and bis-(1,3-dibutylbarbituric acid) trimethine oxonol; DiBAC4(3)), membrane integrity (LIVE/DEAD BacLight kit), respiratory activity (5-cyano-2,3-ditolyl tetrazolium chloride; CTC) and substrate responsiveness (direct viable counts; DVC) in the commensal pathogen Escherichia coli O157:H7. After a 5 min exposure to the disinfectant, physiological indices were affected in the following order: viable plate counts > substrate responsiveness > membrane potential > respiratory activity > membrane integrity. In situ assessment of physiological activity by examining multiple targets, as demonstrated in this study, permits a more comprehensive determination of the site and extent of injury in bacterial cells following sublethal disinfection with chlorine. This approach to assessing altered bacterial physiology has application in various fields where detection of stressed bacteria is of interest.

Chlorine↗

PubMed2006

Viability of commercial probiotic cultures (L. acidophilus, Bifidobacterium sp., L. casei, L. paracasei and L. rhamnosus) in cheddar cheese.

Six batches of cheddar cheese were manufactured containing different combinations of commercially available probiotic cultures from three suppliers. Duplicate cheeses contained the organisms of each supplier, a Bifidobacterium spp. (each supplier), a Lactobacillus acidophilus (2 suppliers), and either Lactobacillus casei, Lactobacillus paracasei, or Lactobacillus rhamnosus. Using selective media, the different strains were assessed for viability during cheddar cheese maturation over 32 weeks. The Bifidobacterium sp. remained at high numbers with the three strains being present in cheese at 4 x 10(7), 1.4 x 10(8), and 5 x 10(8) CFU/g after 32 weeks. Similarly the L. casei (2 x 10(7) CFU/g), L. paracasei (1.6 x 10(7) CFU/g), and L. rhamnosus (9 x 10(8) CFU/g) strains survived well; however, the L. acidophilus strains performed poorly with both decreasing in a similar manner to be present at 3.6 x 10(3) CFU/g and 4.9 x 10(3) CFU/g after 32 weeks. This study indicates that cheddar cheese is a good vehicle for a variety of commercial probiotics but survival of L. acidophilus strains will need to be improved.

Bifidobacterium↗

PubMed1982

Some in-vitro characters of the subspecies of Mycoplasma mycoides.

So-called LC strains of Mycoplasma mycoides subsp. mycoides and, where appropriate, SC strains, were examined, together with M. mycoides subsp. capri strains, by growth, fermentation, and antibiotic-sensitivity tests. Growth curves in BVF-OS medium showed that, from the 6th until at least the 19th day of incubation, strain Mankefår 2833 had a viable count strikingly higher than that of any other LC strain. Its robust growth properties may explain its ability--unusual among LC strains--to produce mycoplasmaemia readily in mice. Strain 143-A66 Conn, also shown by earlier experiments in mice to possess unusual properties, lost viability more rapidly than any other LC strain between the 13th and 19th days of incubation. The viable count of a subsp. capri strain was considerably lower than that of any LC strain for much of the period between days 6 and 19. In fermentation tests with 27 substrates and sensitivity tests with 11 antibiotics the strains gave results that, in all but the following respects, were uniform. Sorbitol was fermented to varying degrees by all the LC and subsp. capri strains tested but was unaffected by the SC strains. The LC and subsp. capri strains were in general more resistant than the SC strains to streptomycin. The growth of the LC strains, much more than that of the other strains, was greatly stimulated by the presence of fermentable substrate.

Fermentation↗

PubMed1996

Direct evidence for antipseudomonal activity of macrolides: exposure-dependent bactericidal activity and inhibition of protein synthesis by erythromycin, clarithromycin, and azithromycin.

Several previous investigators have reported that long-term administration of certain macrolides is efficacious in patients with persistent pulmonary Pseudomonas aeruginosa infections, even though the clinically achievable concentrations of these medications are far below their MICs. In the present study, we examined how sub-MICs of macrolide antibiotics affect the viability of and protein synthesis in several strains of P. aeruginosa. We report that 48 h, but not 12 or 24 h, of growth on agar containing a clinically achievable concentration of azithromycin (0.5 microgram/ml, 1/128 the MIC) significantly reduces the viability of strain PAO-1. Similar effects were seen with erythromycin and clarithromycin at 2 micrograms/ml (1/128 and 1/64 the respective MICs), whereas josamycin, oleandomycin, ceftazidime, tobramycin, minocycline, and ofloxacin had no effect on viability, even following 48 h of incubation with concentrations representing relatively high fractions of their MICs. The bactericidal activity of azithromycin seen following 48 h of incubation was not limited to strain PAO-1 but was also seen against 13 of 14 clinical isolates, including both mucoid and nonmucoid strains. Although viability was not decreased prior to 48 h, we found that 4 micrograms of azithromycin per ml inhibits protein synthesis after as little as 12 h and that protein synthesis continues to decrease in a time-dependent manner. We likewise found that P. aeruginosa accumulates azithromycin intracellulary over the period from 12 to 36 h. These results suggested that sub-MICs of certain macrolides are bactericidal to P. aeruginosa when the bacteria are exposed to these antibiotics for longer periods. Exposure-dependent intracellular accumulation of the antibiotic and inhibition of protein synthesis may partially account for the antipseudomonal activity of macrolides over relatively prolonged incubation periods.

Anti-Bacterial Agents↗

PubMed2000

Cellular responses to postsegregational killing by restriction-modification genes.

Plasmids that carry one of several type II restriction modification gene complexes are known to show increased stability. The underlying mechanism was proposed to be the lethal attack by restriction enzyme at chromosomal recognition sites in cells that had lost the restriction modification gene complex. In order to examine bacterial responses to this postsegregational cell killing, we analyzed the cellular processes following loss of the EcoRI restriction modification gene complex carried by a temperature-sensitive plasmid in an Escherichia coli strain that is wild type with respect to DNA repair. A shift to the nonpermissive temperature blocked plasmid replication, reduced the increase in viable cell counts and resulted in loss of cell viability. Many cells formed long filaments, some of which were multinucleated and others anucleated. In a mutant defective in RecBCD exonuclease/recombinase, these cell death symptoms were more severe and cleaved chromosomes accumulated. Growth inhibition was also more severe in recA, ruvAB, ruvC, recG, and recN mutants. The cells induced the SOS response in a RecBC-dependent manner. These observations strongly suggest that bacterial cells die as a result of chromosome cleavage after loss of a restriction modification gene complex and that the bacterial RecBCD/RecA machinery helps the cells to survive, at least to some extent, by repairing the cleaved chromosomes. These and previous results have led us to hypothesize that the RecBCD/Chi/RecA system serves to destroy restricted "nonself" DNA and repair restricted "self" DNA.

Chromosomes, Bacterial↗

PubMed2004

Study of bacterial viability within human supragingival dental calculus.

BACKGROUND: There is evidence that supragingival calculus contains unmineralized channels and lacunae. The purpose of this study was to investigate the viability of bacteria within these areas. METHODS: Supragingival calculus harvested from patients with moderate to severe chronic periodontitis was immediately frozen to -70 degrees C. Six samples were cryosectioned, stained with a bacterial viability kit, and examined with fluorescence microscopy. Controls comprised heat treatment of cryosections prior to staining. Four additional samples were stained and examined whole in a confocal laser scanning microscope (CLSM). Nine additional samples were prepared for bacterial culture, after initial irradiation with ultraviolet light to kill viable organisms on the covering plaque layer. Test samples were crushed to expose internal bacteria, while two controls were used without crushing. RESULTS: Viable bacteria, as identified using the bacterial viability stain, were found within cavities/lacunae in supragingival calculus cryosections. Similar results were obtained from whole calculus samples using CLSM. Of the nine experimental samples where bacterial culture was attempted, five provided positive bacterial culture under both aerobic and anaerobic conditions; one showed positive growth under aerobic conditions only; while one showed no bacterial growth. The controls showed no bacterial growth. CONCLUSIONS: From this study, it appears that viable aerobic and anaerobic bacteria may be present within supragingival calculus, specifically within the internal channels and lacunae. Clinically, this may be important, since incomplete removal of supragingival calculus may expose these reservoirs of possible pathogenic bacteria and be a factor in the recurrence of periodontal diseases after treatment.

Bacteria, Aerobic↗