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PubMed2006

Design and evaluation of 16S rRNA sequence based oligonucleotide probes for the detection and quantification of Comamonas testosteroni in mixed microbial communities.

BACKGROUND: The beta-proteobacterial species Comamonas testosteroni is capable of biotransformation and also biodegradation of a range of chemical compounds and thus potentially useful in chemical manufacturing and bioremediation. The ability to detect and quantify members of this species in mixed microbial communities thus may be desirable. RESULTS: We have designed an oligonucleotide probe for use in fluorescent in situ hybridization (FISH) and two pairs of PCR primers targeting a C. testosteroni subgroup. The FISH probe and one of the PCR primer pairs are suitable for quantification of C. testosteroni in mixed microbial communities using FISH followed by quantitative image analysis or real-time quantitative PCR, respectively. This has been shown by analysis of samples from an enrichment of activated sludge on testosterone resulting in an increase in abundance and finally isolation of C. testosteroni. Additionally, we have successfully used quantitative PCR to follow the C. testosteroni abundance during a laboratory scale wastewater bioaugmentation experiment. CONCLUSION: The oligonucleotides presented here provide a useful tool to study C. testosteroni population dynamics in mixed microbial communities.

Colony Count, Microbial↗
PubMed1995

Microbial vitality of supragingival dental plaque during initial stages of experimental gingivitis in humans.

Although vital plaque micro-organisms are part of the natural ecosystem in the oral cavity they are also the key factor in the development of diseases induced by the human dental plaque. In a previous study (9) the portion of vital bacteria related to the total number of plaque micro-organisms (i.e. the microbial vitality) appeared low in small plaque samples. The objective of this investigation was to determine the exact relationship of microbial vitality and age of supragingival plaque during the early phases of human dental plaque formation. Between intervals of optimal oral hygiene, thirteen participants refrained from all oral hygiene measures for periods of 1, 2, 4, 8, 24 and 72 h. Plaque was completely sampled from a defined area situated on the vestibular surface of the teeth 13, 14, 15, 23, 24 and 25. The pooled plaque from these areas was immediately processed. Total bacterial counts (BC) as enumerated by darkfield microscopy, and colony-forming units (CFU) were recorded. The microbial vitality was calculated indirectly as plating efficiency (PE = CFU per BC) and directly assessed using a vital fluorescence (VF) technique. In the 1 h old plaque samples the median values of PE and VF were 29% and 18%, respectively. Thereafter, the microbial vitality increased significantly with plaque age. The 24 h old plaque samples yielded values of 77% (PE) and 62% (VF). It was concluded that the microbial vitality of the early dental plaque investigated was considerably lower compared to that of a more mature plaque.

Adult↗
PubMed1990

Protective effect of recombinant human granulocyte colony-stimulating factor (rG-CSF) against various microbial infections in neutropenic mice.

Protective effect of recombinant human granulocyte colony-stimulating factor (rG-CSF) on microbial infections was studied in cyclophosphamide (CPA)-induced neutropenic mice. The neutropenic mice showed severely decreased resistance against systemic infections of Pseudomonas aeruginosa, Escherichia coli, Serratia marcescens, Staphylococcus aureus, and Candida albicans. When such mice were injected subcutaneously with rG-CSF on four consecutive days beginning the day after CPA injection, the decreased anti-microbial resistance of the mice was restored to the level of that in normal mice. The anti-infective effect of rG-CSF was dose-dependent and the 50% effective doses (ED50) in various microbial infections tested were 1-10 micrograms/kg/day. The results suggest that rG-CSF is useful for protection of neutropenic patients from microbial infections.

Animals↗
PubMed2004

Effect of saliva on an antimicrobial tissue conditioner containing silver-zeolite.

The purpose of this study was to evaluate the influence of human saliva on the antimicrobial effect of a tissue conditioner containing an antibiotic agent, silver-zeolite. Samples of each tissue conditioner with or without silver-zeolite were prepared and a plastic disk was used as a control. Candida albicans and nosocomial respiratory infection-causing bacteria, Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), Pseudomonas aeruginosa and the Streptococcus milleri group (S. constellatus and S. intermedius), were selected as test microorganisms. Antimicrobial effects of samples after water or saliva immersion for 28 days were evaluated by counting the number of viable cells [colony forming unit (CFU)] in each microbial suspension (100 microL). All data were statistically analysed by one-way anova and Bonferroni's test (P < 0.05). The antimicrobial effects of samples with silver-zeolite immersed in saliva against C. albicans, S. aureus and MRSA were observed while CFU of P. aeruginosa indicated no significant difference from that of the control. As for the S. milleri group, its CFU after saliva immersion showed the significantly smaller value than that of the control. It is concluded that the antimicrobial effects of samples containing silver-zeolite against all tested microbes except for P. aeruginosa and the S. milleri group are not influenced by saliva immersion for 28 days.

Anti-Bacterial Agents↗
PubMed1995

Production of tumor necrosis factor-alpha in granulocytopenic mice with pulmonary candidiasis and its modification with granulocyte colony-stimulating factor.

In the present study, a lethal model of pulmonary candidiasis was established using granulocytopenic mice with cyclophosphamide. These mice started to die 1 day after infection and had all died within the next 48 hr. The counts of live C. albicans in the lung gradually increased with time, while the organisms were quickly eliminated in the normal mice. From the histology and measurements on bronchoalveolar lavage fluid (BALF), polymorphonuclear cells (PMN) response was almost zero up to 24 hr; and then a weak but significant response was observed at 48 hr, while a marked accumulation of PMN was detected from as early as 6 hr in normal mice. In contrast, macrophages had accumulated in BALF by 48 hr in granulocytopenic mice, but not in normal mice. Both in serum and BALF, a considerable level of tumor necrosis factor-alpha (TNF-alpha) was detected from 6 hr, peaking at 24 to 48 hr, while in normal mice the quantity was under the detection limit in serum and very low in BALF. The effects of administering granulocyte colony-stimulating factor (G-CSF) on these parameters were next examined. G-CSF significantly prolonged the survival time of granulocytopenic mice, promoted the clearance of organisms through increasing the counts of PMN in the lung, and strongly inhibited the production of TNF-alpha both in BALF and serum. These results suggest that this cytokine does not protect them, but plays some role in their death due to candidial infection in granulocytopenic mice.

Agranulocytosis↗
PubMed1997

Vital microorganisms in early supragingival dental plaque and in stimulated human saliva.

The aim of this study was to compare the percentage of vital microorganisms (= microbial vitality) of saliva with that of supragingival plaque both collected at various times during the early phases of de novo plaque formation. Between intervals of optimal oral hygiene, 14 healthy participants refrained from all oral hygiene measures for periods of 1, 4, 8 and 72 h. Stimulated whole saliva was collected at the beginning (= baseline) and the end of each period. Vestibular plaque was removed from teeth 13-16, and 23-26. Analysis of the pooled plaque (p) and saliva (s) samples comprised the total number of bacterial counts and colony-forming units to estimate the percentage of viable microorganisms (PEp; PEs). The microbial vitality (VFp; VFs) was determined by using a fluorescence staining to differentiate vital from dead bacterial cells. The bulk of the PEs values reached 5-30%. At baseline VFs ranged between 70% and 90%. The VFs values recorded at baseline or in the presence of 1 h and 4 h-old plaque, were significantly (alpha = 0.05) higher than the corresponding VFp values ranging from 5% to 30%. It was concluded that there is a considerable discrepancy between the microbial vitality of a very early dental plaque and that of whole surrounding saliva sampled at the same time. Unfavourable local environmental conditions prevailing at cervical tooth surfaces are suggested to restrain the survival of the majority of the first bacteria adhering to a particular tooth area during the early phases of supragingival plaque formation.

Adult↗
PubMed2002

Flow cytometric techniques for the detection of microorganisms.

Flow cytometry (FCM) is a technique, which allows one to analyse cells rapidly and individually, and permits the quantitative analysis of distributions of a property or properties in a population. It therefore offers many advantages over conventional measurements for the analysis of biological cells. Historically the technique has been widely applied for the study of mammalian cells, but its use in microbiology has been more limited; this is mainly a consequence of the smaller size of microbes, which results in the smaller optical signals that can be obtained from them. Developments in light sources and optics, together with brighter, spectrally-diverse dyes have reduced this barrier over recent years and the flow cytometer is now an essential tool in many microbiological research establishments. FCM has an increasing role to play in the detection of microbes in both industrial and clinical settings. Environmental monitoring to prevent outbreaks of human diseases such as cryptosporidiosis and Legionnaires' disease and to detect acts of biowarfare or bioterrorism are all amenable to flow cytometric study. This review seeks to highlight the role of the flow cytometer in the detection of microbial cells.

Animals↗
PubMed1994

Cardiopulmonary effects of granulocyte colony-stimulating factor in a canine model of bacterial sepsis.

We investigated the effects of recombinant granulocyte colony-stimulating factor (G-CSF) in a canine model of septic shock. Awake 2-yr-old beagles were studied before and after intraperitoneal placement of an Escherichia coli-infected clot. Nine days before and until 3 days after clot placement, animals received daily high-dose (G-CSF (5 microgram/kg body wt; n = 17), low-dose G-CSF (0.1 microgram/kg body wt; n = 17), or a control protein (5 micrograms/kg body wt; n = 20). Survival rate was greater (P < 0.04, Wilcoxon test) in the high-dose G-CSF group (14/17) than in the low-dose G-CSF (10/17) and control (12/20) groups. High-dose G-CSF improved cardiovascular function, as evidenced by increased left ventricular ejection fraction (day 1 after clot; P < 0.001) and mean arterial pressure (day 2; P < 0.02) compared with low-dose G-CSF and control groups. High-dose G-CSF increased (P < 0.001) mean peripheral neutrophils before (-3 days) and after (2 h to 4 days) clot and produced a more rapid (P < 0.001) rise (day 2) and fall (day 4) in mean alveolar neutrophil numbers compared with the low-dose G-CSF and control groups. High-dose G-CSF decreased mean serum endotoxin (2-8 h; P < 0.002) and tumor necrosis factor (2 h; P < 0.02) levels and lowered blood bacteria counts (2-6 h; P < 0.04) compared with the low-dose G-CSF and control groups. Thus, in this canine model, G-CSF sufficient to increase peripheral neutrophils before and during peritonitis and septic shock enhances host defense, reduces cytokine (tumor necrosis factor) levels, and improves cardiovascular function and survival.

Animals↗
PubMed2000

Effect of cytokines on antifungal activity of human polymorphonuclear leucocytes against yeast cells of Paracoccidioides brasiliensis.

In our previous study, it was observed that human peripheral blood polymorphonuclear leucocytes (PMNs) exhibited a fungistatic effect on yeast cells of Paracoccidioides brasiliensis, and that interferon-gamma (IFN-gamma), but not tumor necrosis factor-alpha or interleukin-8 (IL-8), enhanced the antifungal activity of PMNs. In the present study, granulocyte-macrophage colony-stimulating factor (GM-CSF) also enhanced the PMN activity. GM-CSF-activated PMNs exhibited a killing effect on P. brasiliensis isolate Bt-4 and an enhanced fungistatic effect on isolate Aoki. IL-1beta activated PMNs to kill isolate Bt-4. Granulocyte colony-stimulating factor had no effect. Combinations of IFN-gamma with GM-CSF or IL-1beta, but not a combination of GM-CSF and IL-1beta, exhibited a synergistic effect in enhancing the antifungal activity of PMNs. These results strongly suggest that PMNs activated with IFN-gamma, GM-CSF and/or IL-1beta might play an important role in host defense in early infection with P. brasiliensis due to their enhanced antifungal activity.

Colony Count, Microbial↗
PubMed1991

Augmented production of granulocyte-macrophage colony-stimulating factor and alpha/beta interferon in mice inoculated with heat-killed Corynebacterium liquefaciens.

We demonstrated that heat-killed Corynebacterium liquefaciens bacteria, as a known potent host immune activity modulator, stimulate spleen cells to produce granulocyte-macrophage (GM) colony-stimulating factor (CSF) and another CSF with similar activity, as well as alpha/beta interferon, when injected intravenously into mice. Alpha/beta interferon was shown to be produced by C. liquefaciens-activated plastic-G-10 column-adherent cells (A cells) in a thymus-independent manner. In contrast, augmented production of GM-CSF required the action of C. liquefaciens-activated T lymphocytes that collaborated with normal A cells. Non-T spleen cells from C. liquefaciens-stimulated athymic mice, however, produced an alternative CSF that partially replaced GM-CSF. Correspondingly, the numbers of GM-producing CFU developing in cultures of spleen cells from C. liquefaciens-treated euthymic or athymic mice were 10 to 30 times higher than those in cultures of spleen cells from untreated mice. These results suggest that gram-positive rods such as C. liquefaciens activate T and A cells for production of multiple cytokines and that potential cooperative actions of these cytokines underlie the known immunomodulatory action of coryneforms.

Animals↗
PubMedDate not supplied

Comparison of antibody-direct epifluorescent filter technique with the most probable number procedure for rapid enumeration of Listeria in fresh vegetables.

The antibody-direct epifluorescent filter (Ab-DEFT) technique was evaluated as a rapid alternative to the most probable number (MPN) method for enumeration of artificially inoculated Listeria monocytogenes in ready-to-eat packaged salads and other fresh vegetables. Ab-DEFT was performed by homogenization of food in mesh-lined Stomacher bags, followed by prefiltration of homogenate through a 5 microns pore nylon filter, and passage of filtrate through a 0.4 micron pore black polycarbonate filter to collect and concentrate Listeria cells. After cells were stained with a fluorochrome-labeled polyclonal antibody to Listeria, the filter surface was examined by epifluorescence microscopy, and fluorescent cells were counted. A 3-tube MPN procedure was performed by successive enrichments of homogenized foods in Listeria enrichment and Fraser broths, followed by selective plating. Ab-DEFT provided quantitative determinations of Listeria cells that correlated with plate counts and MPN estimates in a linear response over a range of cell concentrations from 10 to 10(7) colony forming units (CFU)/mL. Microbial backgrounds as high as 10(8) CFU/mL did not affect performance of Ab-DEFT. In contrast to the MPN method, which required 5 days to perform, quantitation by Ab-DEFT could be completed in less than 1 h. Despite cross-reactivities demonstrated by the polyclonal fluorescent antibody, the potential of Ab-DEFT as a rapid alternative to MPN for microbial cell enumeration was evident.

Antibody Specificity↗
PubMed2000

A pilot study of 2 methods for control of dental unit biofilms.

OBJECTIVE: This pilot study was conducted to clinically evaluate 2 different concentrations of sodium hypochlorite for the control of dental unit biofilms and to evaluate the efficacy of pasteurizing dental treatment water for patient care. METHOD AND MATERIALS: Two dental units with no prior chemical treatment were retrofitted with self-contained water systems for this study. One dental unit was treated with 5,000 ppm of sodium hypochlorite and the other with 1,500 ppm. Treatment consisted of a 10-minute contact with the dental unit water lines, followed by a flush with a buffer solution. A pasteurizer was equipped with autoclavable spigots to provide dental treatment water. Heterotrophic Plate Count Samplers (Millipore) water sampler kits were used to quantify microbial contamination as absolute colony-forming units per millimeter. Scanning electron micrographs were taken of water line lumens to compare pretreatment and posttreatment biofilms. RESULTS: Pasteurized water was significantly less contaminated than was tap water. No significant difference in contamination was found between the 5,000 ppm and 1,500 ppm treatment chairs, either in the reservoir water or effluent water. Scanning electron micrographs demonstrated removal of the biofilms after sodium hypochlorite treatments, regardless of the concentration used. CONCLUSION: There was no significant difference between the abilities of 5,000- and 1,500-ppm concentrations of sodium hypochlorite to control contamination of dental treatment water and biofilms. Pasteurization of tap water can reduce contamination; this water can be used as acceptable dental treatment water (< 200 CFU/mL).

Biofilms↗
PubMed1998

Evaluation of the levels of oral Candida in patients with Sjögren's syndrome.

OBJECTIVE: The purpose of this study was to investigate the levels of oral Candida in patients with Sjögren's syndrome METHODS: The Candida count and salivary flow rate of patients with Sjögren's syndrome were compared with those of healthy control subjects. Candida cultures were obtained from oral rinses. The numbers of colony-forming units were determined through use of the Spiral System. RESULTS: The mean Candida count of patients with Sjögren's syndrome was 1672 +/- 1455 colony-forming units per mL; the count of healthy control subjects was 0.00 colony-forming units per mL. The mean salivary flow rate of patients with Sjögren's syndrome was significantly lower than that of healthy control subjects (0.16 +/- 0.13 mL/min/gland vs 0.55 +/- 0.24 mL/min/gland, respectively; p = 0.0001). However, Spearman rank correlation analyses did not reveal a significant correlation between salivary flow rate and Candida count (in colony-forming units per mL) among patients with Sjögren's syndrome. CONCLUSIONS: Alteration in the oral microbial flora in patients with Sjögren's syndrome may be enhanced by the reduction in salivary output.

Candida↗
PubMed1994

Granulocyte macrophage colony-stimulating factor improves survival in two models of gut-derived sepsis by improving gut barrier function and modulating bacterial clearance.

OBJECTIVE: The effect of recombinant murine granulocyte macrophage colony-stimulating factor (rmGM-CSF) on survival and host defense was studied using two clinically relevant models of infection that included transfusion-induced immunosuppression. SUMMARY BACKGROUND DATA: Granulocyte macrophage colony-stimulating factor improves resistance in several models of infection, but its role in transfusion-induced immunosuppression and bacterial translocation (gut-derived sepsis) has not been defined. METHODS: Balb/c mice were treated with 100 ng of rmGM-CSF or placebo for 6 days in a model of transfusion, burn, and gavage, or cecal ligation and puncture (CLP). Translocation was studied in the first model. RESULTS: Survival after transfusion, burn, and gavage was 90% in rmGM-CSF-treated animals versus 35% in the control group (p < 0.001). After CLP, survival was 75% in the rmGM-CSF group versus 30% in the control group (p = 0.01). Less translocation and better killing of bacteria was observed in the tissues in animals treated with rmGM-CSF. CONCLUSION: The ability of rmGM-CSF to improve gut barrier function and enhance killing of translocated organisms after burn injury-induced gut origin sepsis was associated with improved outcome. Granulocyte macrophage colony-stimulating factor also improved survival after CLP.

Animals↗
PubMed2004

Clinical and microbiological efficacy of chlorine dioxide in the management of chronic atrophic candidiasis: an open study.

OBJECTIVE: To assess the clinical and microbiological efficacy of chlorine dioxide (ClO2) as a topical antiseptic for the treatment of chronic atrophic candidiasis in geriatric patients. PARTICIPANTS: Thirty patients with chronic atrophic candidiasis. METHODS: Patients were instructed to rinse the mouth with 0.8% ClO2 mouth rinse (DioxiDent) twice daily for one minute and to soak their dentures overnight in the ClO2 for 10 days. Patients were evaluated both clinically and microbiologically at baseline and after 10 days, and any significant side effects were recorded. The clinical appearance of the oral soft tissues was scored on a scale of 0-3 (0 indicating no clinical signs, 1 indicating involvement of < 25% of the palatal mucosa, 2 indicating involvement of 25-50% of the palatal mucosa, and 3 indicating marked erythema involving > 50% of the palatal mucosa). Microbiological testing was undertaken to determine the number of colony forming units (CFUs) of Candida albicans. RESULTS: ClO2 significantly improved the clinical appearance and microbial count (p < 0.001) after treatment, without significant side effects. Results showed marked improvement in the clinical appearance of the tissues after 10 days, with total resolution in the majority of cases. The total CFU/ml ranged from 15,000-53,000 at baseline and was reduced to < or = 500 after 10 days of treatment (p < 0.001). The mean clinical score was 2.50 at baseline, and was reduced to 0.17 after 10 days of treatment (p < 0.001). CONCLUSIONS: Within the limitations of this pilot study, the effectiveness of topical chlorine dioxide (0.8%) in the management of chronic atrophic candidiasis was demonstrated. ClO2 provided a safe and clinically effective option in the management of chronic atrophic candidiasis.

Aged↗