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Problems posed by natural environments for monitoring microorganisms.

Microorganisms in natural environments have evolved to withstand fluctuations in physical and chemical conditions. This means that they often manifest very different biochemical and morphological features compared with those seen during laboratory culture. A major limitation in natural ecosystems is nutrient limitation under which microorganisms are exposed to starvation conditions and grow slowly or not at all. This review identifies the role of inimical processes on microbial properties such as the responses to starvation that may result in the adoption of viable but nonculturable (VBNC) states, discusses the problems that altered physiological states pose for detection and identification and highlights novel methods that have been developed to circumvent these difficulties. These factors dictate that to survive and respond to environmental stimuli, a cell must have evolved sophisticated programs of gene expression. These include the sigma factor rpoS that directs RNA polymerase to transcribe genes whose expression aids survival during severe nutrient limitation or cell-cell communication systems that promote a concerted population response termed quorum sensing.

Adaptation, Physiological↗

The antimicrobial effect of a triclosan/copolymer dentifrice on oral microorganisms in vivo.

BACKGROUND: The authors compared the in vivo antimicrobial effects on microorganisms from dental plaque, saliva and the tongue in subjects who used a triclosan/copolymer dentifrice and a fluoride dentifrice (control). METHODS: The authors assigned 15 subjects randomly to the control dentifrice or the triclosan/copolymer dentifrice for twice-daily use for one week. They collected samples of plaque, saliva and tongue scrapings six and 12 hours after the final brushing. They analyzed colony-forming units of Veillonella species, Fusobacteria species, total cultivable anaerobes and hydrogen sulfide (H(2)S)-producing bacteria. A one-week washout followed. The authors repeated the protocol with the second dentifrice. RESULTS: The results showed no differences at baseline. Significant reductions (88 to 96 percent) in oral anaerobic bacteria were observed in the triclosan/copolymer group six and 12 hours after brushing compared with the control group (P = .001). Fusobacteria decreased by 77 to 92 percent and Veillonella decreased by 84 to 89 percent six and 12 hours after brushing in the triclosan/copolymer group versus the control group. The triclosan/copolymer group also demonstrated a significant decrease in H(2)S-producing bacteria six and 12 hours after brushing (74 to 85 percent) (P = .001). CONCLUSIONS: Brushing with the triclosan/copolymer dentifrice resulted in significant reductions in microorganisms from the three sites compared with the control dentifrice. CLINICAL IMPLICATIONS: The triclosan/copolymer dentifrice produced sustained effects on oral bacteria for 12 hours.

Adult↗

Assessment of lead toxicity to the marine bacterium, Vibrio fischeri, and to a heterogeneous population of microorganisms derived from the Pearl River in Jackson, Mississippi, USA.

Microorganisms are known to be excellent test organisms because of the relative ease for handling and suitability for analysis related to their small size, large number and convenient growing conditions. In this research, we tested the toxic effects of lead against a marine bacterium (Vibrio fischeri), and a heterogeneous population of bacteria derived from the Pearl River in Jackson, Mississippi. Using the level of bioluminescence in the Microtox Assay (V. fischeri), and the kinetics of dissolved oxygen uptake and growth (mixed bacterial population) as measures of toxicity, lead concentrations effecting a 50% reduction in these parameters (EC50) were determined as the toxic end-points. The activity quotients were also computed to determine the degrees of toxicity. Optical density (measure of growth) and oxygen uptake were measured over an extended period of time (20 h). EC50 values of 0.34 +/- 0.03, 3.10 +/- 0.01, and 3.80 +/- 0.02 mg/L were recorded for bioluminescence, growth, and oxygen uptake, respectively. As expected, the results indicated that the sensitivity to lead toxicity of V. fischeri was about one order of magnitude (10 times) greater than that of the mixed population of Pearl River microorganisms. Reductions in bioluminescence, growth, and oxygen uptake were directly correlated to lead concentrations, with toxic levels ranging from slightly toxic in lower concentrations to extremely toxic in higher concentrations. Upon 20 h of exposure, the times required to produce 50% reduction in dissolved oxygen uptake were (TD50S) 8.01 +/- 0.44, 9.60 +/- 0.46, 11.29 +/- 0.46, 13.03 +/- 0.57, 17.32 +/- 0.95, and 20.00 +/- 0.00 h in 0, 1, 2, 3, 4, 5, and 6 mg/L of lead, respectively, indicating a time-response relationship with respect to lead toxicity.

Fresh Water↗

Relative frequency of nosocomial microorganisms at UNICAMP University Hospital from 1987 to 1994.

The frequency of microorganisms identified in nosocomial infections at Unicamp University Hospital from 1987 to 1994 was analysed. The most common microorganism was S. aureus (20.9%), which was found in surgical wound, bloodstream and arterial-venous infections. In urinary tract infections (UTI), gram-negative rods (56.5%) and yeasts (9%) predominated. A. baumannii isolates were observed to have increased in the last three years. There was a gradual increase in the frequency of coagulase-negative staphylococci and A. baumannii in bloodstream infections but there wasn't any change in Candida sp.

Brazil↗

Control of microorganisms in vitro by endodontic irrigants.

The aim of this study was to determine the minimum inhibitory concentration (MIC) and antimicrobial effectiveness by the direct exposure test of 4 endodontic irrigants [1% sodium hypochlorite (NaOCl), 2% chlorhexidine (CHX), 1% calcium hydroxide (Ca(OH)2; prepared with 1 g of Ca(OH)2 and 100 mL of sterile distilled water), a solution of Ca(OH)2 + detergent (HCT20)] for S. aureus, E. faecalis, P. aeruginosa, B. subtilis, C. albicans and a mixed culture. Microbial growth was analyzed by two methods: turbidity of the culture medium that was confirmed by Gram stain and subculture in a specific nutrient broth. In the dilution test, NaOCl solution showed MIC equal to 0.1% for S. aureus, E. faecalis, P. aeruginosa and C. albicans and equal to 1% for B. subtilis and the mixed culture. CHX (2%) presented MIC equal to 0.000002% for S. aureus, 0.02% for E. faecalis, B. subtilis, C. albicans and the mixed culture and 0.002% for P. aeruginosa. Ca(OH)2 solution (1%) showed MIC greater than 1% for all the microorganisms except P. aeruginosa for which it was equal to 1%. Calcium hydroxide solution + detergent showed MIC equal to 4.5 mL for S. aureus, P. aeruginosa, B. subtilis, C. albicans and the mixed culture and greater than 4.5 mL for E. faecalis. In the direct exposure test, NaOCl had better antimicrobial effectiveness for all microorganisms at all times. CHX (2%) was effective for S. aureus, E. faecalis and C. albicans at all times, and ineffective for P. aeruginosa, B. subtilis and the mixed culture. The other solutions showed the worst results.

Anti-Infective Agents, Local↗

Antimicrobial activity of Arctium lappa constituents against microorganisms commonly found in endodontic infections.

This study evaluated in vitro the antimicrobial activity of rough extracts from leaves of Arctium lappa and their phases. The following microorganisms, commonly found in the oral cavity, specifically in endodontic infections, were used: Enterococcus faecalis, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis and Candida albicans. The agar-diffusion method allowed detection of the hexanic phase as an inhibitor of microbial growth. Bioautographic assays identified antimicrobial substances in the extract. The results showed the existence, in the rough hexanic phase and in its fractions, of constituents that have retention factors (Rf) in three distinct zones, thereby suggesting the presence of active constituents with chemical structures of different polarities that exhibited specificity against the target microorganisms. It may be concluded that the Arctium lappa constituents exhibited a great microbial inhibition potential against the tested endodontic pathogens.

Acetates↗

Retention of oral microorganisms on conventional and resin-modified glass-ionomer cements.

Secondary caries are a worldwide public and socioeconomic problem. The placement of restorations can lead to the development of environmental conditions favorable to microbial colonization, especially on the tooth/restoration interface, which is a predisposing factor for secondary caries. The aim of this study was to evaluate microbial retention on conventional (Chelon-Fil and Vidrion R) and resin-modified (Vitremer and Fuji II LC) glass-ionomer cements, in situ, using a hybrid composite resin (Z100) as a control. Twelve volunteers wore Hawley appliances with specimens made of all tested filling materials for 7 days. The specimens were then removed from the appliances and transferred to tubes containing 2.0 ml of Ringer-PRAS. Microorganisms from the samples were inoculated onto blood agar and Mitis Salivarius Bacitracin agar and incubated under anaerobiosis (90% N2, 10% CO2), at 37 degrees C, for 10 and 2 days, respectively. The resin-modified glass-ionomer cements and the composite resin retained the same levels of microorganisms on their surfaces. The resin-modified glass-ionomers retained less mutans streptococci than the composite resin and conventional glass-ionomer cements. The conventional glass-ionomer cements retained less mutans streptococci than the composite resin, but that difference was not statistically significant.

Adult↗

Effect of a 0.5% chlorhexidine gel on dental plaque superinfecting microorganisms in mentally handicapped patients.

A randomized clinical trial was conducted to investigate the effect of a 0.5% chlorhexidine (CHX) gel on dental plaque superinfecting microorganisms in mentally handicapped patients. Thirty inmates from the institution "Casas André Luiz" were assigned to either test group (CHX gel, n = 15) or control group (placebo gel, n = 15). The gel was administered over a period of 8 weeks. Supragingival plaque samples were collected at baseline, after gel use (8 weeks) and 16 weeks after baseline. The presence of Gram-negative Enterobacteriaceae, Staphylococcus and yeasts was evaluated. No significant growth of any superinfecting microorganism was observed in the CHX group, when compared to the placebo group. The results indicated that the 0.5% chlorhexidine gel did not produce an undesirable shift in these bacterial populations.

Adolescent↗

In vitro minocycline activity on superinfecting microorganisms isolated from chronic periodontitis patients.

Chronic periodontitis is the most common type of periodontitis and it is associated with various species of microorganisms. Enteric rods, Pseudomonas, Staphyloccocus and Candida have been retrieved from periodontal pockets of patients with chronic periodontitis and correlated to cases of superinfection. Local or systemic antibiotic therapy is indicated to reinforce the effects of the conventional mechanical therapy. Minocycline has been suggested as one of the most effective drugs against periodontal pathogens. The aim of this work was to evaluate the minimal inhibitory concentration (MIC) of minocycline on superinfecting microorganisms isolated from the periodontal pocket and the oral cavity of individuals with chronic periodontitis. Isolates of Enterobacteriaceae (n = 25), Staphylococcus spp. (n = 25), Pseudomonas aeruginosa (n = 9) and Candida spp. (n = 25) were included in the study. Minimal inhibitory concentrations (MIC) of minocycline were determined using the Müeller-Hinton agar dilution method. Staphylococcus spp. isolates were the most sensitive to minocycline with a MIC of 8 microg/mL, followed by Enterobacteriaceae with a MIC of 16 microg/mL. The concentration of 16 microg/mL inhibited 96% of Candida spp. isolates. The MIC for 88.8% of the isolates of Pseudomonas aeruginosa was 128 microg/mL. A concentration of 1,000 microg/mL was not enough to inhibit 100% of the tested isolates.

Anti-Bacterial Agents↗

Scanning and transmission electron microscopic study of tissue-invading microorganisms in localized juvenile periodontitis.

This transmission and scanning electron microscope study documents the invasion of microorganisms into the gingiva and along resorbing alveolar bone surfaces in a 15-year-old patient with localized juvenile periodontitis. The invading bacteria were mainly Gram-negative fusiform, coccobacilli and spirochetes. Microorganisms identified as Mycoplasma were also found to invade in some areas. Polymorphonuclear neutrophils (PMNs) were seen in association with the bacteria. The majority of bacteria were covered by PMNs and enclosed by the phagocyte but did not appear to lie within phagosomes. High numbers of PMNs were found in the gingival connective tissue inflammatory infiltrate and in the pocket epithelium. Invasion into the gingiva and alveolar bone by bacteria and Mycoplasma in localized juvenile periodontitis may be importance in the therapy of this infection.

Adolescent↗

Humoral immune response to selected subgingival plaque microorganisms in insulin-dependent diabetic children.

Juvenile diabetics have been shown to have an increased susceptibility to gingivitis and periodontitis following puberty. However, little data are available on changes in the microbial flora that occur at the onset of puberty. This study was performed to determine if antibacterial antibody titers to selected periodontal disease-associated microorganisms might be helpful in revealing changes in plaque flora at the onset and conclusion of puberty. Sera was obtained from 35 subjects (ages 7 to 18 years) selected from a population of insulin-dependent diabetics. The subjects were given a thorough medical examination which included an assessment of sexual maturation and a dental examination which included the recording of onset and magnitude of bleeding according to the papillary bleeding score. Antibody titers to A. naeslundii (AN), B. intermedius (BI), B. gingivalis (BG), F. nucleatum (FN), A. actinomycetemcomitans (AA), C. ochracea (CO) and T. denticola (TD) were determined using the microELISA. Stratification of antibody titers by age groups (less than or equal to 12 years, 12 to 15 years, greater than 15 years) revealed that titers to AN increased significantly (P less than 0.025, ANOVA) and progressively (P less than 0.05, regression analysis) with increasing age. In contrast, the titers to FN were maximal in the under 12 year group and decreased with age (ANOVA, P less than 0.05; regression analysis, P less than 0.05). There were no significant variations in titers observed for the other microorganisms. Stratification by sexual maturity revealed a similar progressive decrease of the titer to FN (ANOVA, P less than 0.05; regression analysis, P less than 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)

Actinomyces↗

E-test: a new technique for antimicrobial susceptibility testing for periodontal microorganisms.

The purpose of the study was to validate the Epsilometer test (E-test) method for antimicrobial susceptibility testing of selected periodontopathic microorganisms using the agar dilution method as a standard. The E-test has been developed to provide a direct quantification of antimicrobial susceptibility of microorganisms. The device consists of a predefined, continuous, and exponential gradient of antibiotic concentrations immobilized along a rectangular plastic test strip. After 48 hours incubation a drop-shaped inhibition zone intersects the graded test strip at the inhibitory concentration (IC) of the antibiotic. Twenty-two subgingival plaque samples from periodontitis sites were plated on trypticase soy agar supplemented with 5% rabbit blood or 5% sheep blood and trypticase soy agar supplemented with vancomycin and bacitracin. A total of 60 strains of key periodontal pathogens (Prevotella intermedia, Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Eikenella corrodens, Campylobacter rectus, and Capnocytophaga) isolated from 22 sites of 12 patients were studied. The antibiotics tested were clindamycin, metronidazole, tetracycline, ciprofloxacin, cefoxitin, and ampicillin at concentrations above and below the achieved blood or gingival crevicular fluid levels. As a standard reference the minimal inhibitory concentrations (MICs) were determined using the agar dilution method. MICs were compared with ICs determined using the E-test method. The results showed an agreement ranging from 67% to 100%; sensitivity ranging from 75% to 100%; predictability ranging from 56% to 100% and specificity ranging from 33% to 96%. The E-test ICs for ampicillin, cefoxitin, and metronidazole against the Gram-negative capnophilic and microaerophilic rods and the black-pigmented anaerobic rods ICs for ampicillin, clindamycin, metronidazole, and tetracycline showed a high percentage of agreement with the agar dilution MICS.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

Rapid separation of microorganisms by quartz microchip capillary electrophoresis.

We developed and optimized a system coupling microchip capillary electrophoresis (MCE) and laser-induced fluorescence (LIF) detection for the analysis of microorganisms. The MCE-LIF system successfully separated pure cultures of lactic acid bacteria and Saccharomyces cerevisiae within 200 s. The results indicate that the MCE system can be conveniently used for the rapid and highly sensitive detection of microorganisms. Thus, MCE can provide a cheap and simple method for the on-line detection of microbial contamination.

Bacteria↗

Fingerprinting method for phylogenetic classification and identification of microorganisms based on variation in 16S rRNA gene sequences.

The paper describes a method for the classification and identification of microorganisms based on variations in 16S rRNA sequences. The 16S rRNA is one of the most conserved molecules within a cell. The nature of the variable and spacer regions has been found to be specific to a given organism. Thus, the method presented here can be very useful for the classification and identification of microorganisms for which very little information is available. To automate the method, a comprehensive computer program called FPMAP has been developed for the analysis of restriction fragment pattern data. The method involves the restriction digestion of genomic DNA, preferably using four-cutters that may recognize 6-9 sites within the 16S rDNA. The fragments are separated on a polyacrylamide gel along with a suitable marker, then transferred into a nylon membrane and hybridized with a radiolabeled 16S rDNA probe. After autoradiography, the fragment sizes are calculated, and the data are analyzed using the FPMAP software. We demonstrate that the method can be used for identification of strains of Streptomyces and mycobacteria. The software is available from our ftp site ftp:¿imtech.chd.nic.in/pub/com/fpmap/unix/.

Autoanalysis↗

Determination of growth rate of microorganisms in broth from oxygen-sensitive fluorescence plate reader measurements.

A novel method utilizing the BD Oxygen Biosensor System has been developed to rapidly, simply, and accurately determine the growth rate of microorganisms in broth, with no needfor plate counts, standardized inocula, or technically difficult manipulations. The BD Oxygen Biosensor System incorporates an oxygen-sensitive material into the wells of standard Falcon microplates. The time response of this sensor monitored in afluorescence plate reader can be used to quantitate microbe growth. The method entails seeding a dilution series of microorganism onto the plate and reading at regular intervals for 3-10 h. As the organisms grow and consume oxygen, thefluorescence intensities increase over time to form a family of sigmoidal growth curves. A simple mathematical analysis of the time intervals between the curves yields the doubling time, which is independent of the initial concentration of organism. The method is ideally suited as a screening tool for assessing the impact of culture conditions, media composition, or added compounds on growth kinetics.

Algorithms↗

Microorganisms and plant of Autonomous Biological Systems (ABS) samples.

Distribution of microorganisms and cellular structure of an Autonomous Biological Systems (ABS) were studied with a special attention to the effect of space environments. Viable cell densities measured by the direct fluorescence microscopic method were in the order of 10(5) cells/ml for fractions 1 (upper suspension) and 2 (lower suspension), and 10(6) cells/ml for fraction 3 (sediments). These values were 10 to 100 times larger than the values obtained by the classical colony forming unit method. No difference between flight and ground samples was observed in the vertical distribution of viable microorganisms when fractionation and analysis were carried out after recovery. Intracellular distribution of chloroplasts in higher green plants, Ceratophyllum demersum, of flight samples was disturbed after 10 days of flight (24hrs/day light on). After 4 months of flight (Mir/STS-79/81) with 24 hrs light on, Ceratophyllum demersum was completely disintegrated. On the other hand, in the second 4-months-flight experiment with 16 hrs/day light on, Ceratophyllum demersum was only slightly deteriorated.

Aquaculture↗

Theoretical and experimental dissection of gravity-dependent mechanical orientation in gravitactic microorganisms.

Mechanisms of gravitactic behaviors of aquatic microorganisms were investigated in terms of their mechanical basis of gravity-dependent orientation. Two mechanical mechanisms have been considered as possible sources of the orientation torque generated on the inert body. One results from the differential density within an organism (the gravity-buoyancy model) and the other from the geometrical asymmetry of an organism (the drag-gravity model). We first introduced a simple theory that distinguishes between these models by measuring sedimentation of immobilized organisms in a medium of higher density than that of the organisms. Ni2+-immobilized cells of Paramecium caudatum oriented downwards while floating upwards in the Percoll-containing hyper-density medium but oriented upwards while sinking in the hypo-density control medium. This means that the orientation of Paramecium is mechanically biased by the torque generated mainly due to the anterior location of the reaction center of hydrodynamic stress relative to those of buoyancy and gravity; thus the torque results from the geometrical fore-aft asymmetry and is described by the drag-gravity model. The same mechanical property was demonstrated in gastrula larvae of the sea urchin by observing the orientation during sedimentation of the KCN-immobilized larvae in media of different density: like the paramecia, the gastrulae oriented upwards in hypo-density medium and downwards in hyper-density medium. Immobilized pluteus larvae, however, oriented upwards regardless of the density of the medium. This indicates that the orientation of the pluteus is biased by the torque generated mainly due to the posterior location of the reaction center of gravity relative to those of buoyancy and hydrodynamic stress; thus the torque results from the fore-aft asymmetry of the density distribution and is described by the gravity-buoyancy model. These observations indicate that, during development, sea urchin larvae change the mechanical mechanism for the gravitactic orientation. Evidence presented in the present paper demonstrates a definite relationship between the morphology and the gravitactic behavior of microorganisms.

Animals↗

Effects of Aspergillus oryzae fermentation extract on fermentation of amino acids, bermudagrass and starch by mixed ruminal microorganisms in vitro.

The objective of this study was to examine the effects of Aspergillus oryzae fermentation extract (Amaferm) on the in vitro ruminal fermentation of coastal bermudagrass, soluble starch and amino acids. Mixed ruminal microorganisms were incubated in anaerobic media for either 24 h (Amaferm alone, soluble starch, amino acids) or 48 h (bermudagrass). Amaferm was added to the incubation bottles (n = 4) at concentrations of 0, .4 or 1.0 g/liter. When mixed ruminal microorganisms were incubated with only Amaferm, the 1.0 g/liter concentration increased the production of hydrogen (H2; P less than .001), methane (CH4; P less than .01), acetate (P less than .05), butyrate (P less than .01), total VFA (P less than .05) and NH3 (P less than .05). Addition of both levels of Amaferm to soluble-starch fermentations tended to enhance the production of H2 (P less than .11), CH4 (P less than .15), acetate (P less than .29) and total VFA (P less than .19); propionate production was increased (P less than .10) by 1.0 g/liter Amaferm, resulting in a decrease (P less than .05) in the acetate:propionate ratio. Fermentation of amino acids plus 1.0 g/liter Amaferm enhanced the production of acetate (P less than .05), propionate (P less than .05), valerate (P less than .01) and total VFA (P less than .10) and decreased the acetate:propionate ratio (P less than .05). In addition, NH3 production tended (P less than .19) to increase with both levels of Amaferm. When bermudagrass was the substrate, few changes in fermentation products were observed with Amaferm treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗