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PubMed2001

A new in vitro model for the study of microbial microleakage around dental restorations: a preliminary qualitative evaluation.

AIM: The aim of this study was to develop an in vitro model to replicate microbial microleakage at a tooth/ restoration interface using a constant depth film fermentor (CDFF). METHODOLOGY: Amalgam restorations were placed in machined bovine dentine cylinders and sealed externally with varnish, leaving a 1-mm perimeter exposed around the tooth/restoration interface. The dentine cylinders were housed in a CDFF and 300-microm thick microcosm dental plaques were grown over their exposed surfaces. The biofilms were maintained with a mucin-containing artificial saliva for up to 8 weeks. Cylinders were aseptically removed from the CDFF (at 1, 2, 4, & 8 weeks) and surface-decontaminated with validated protocols prior to splitting and sampling of apposing amalgam and dentine surfaces. Scanning electron microscopy (SEM) was used to ascertain the position and structure of the bacterial aggregates. Bacterial viability was determined by vital staining of the bacteria in situ. RESULTS: At all sampling times, SEM showed cocci, rods and filaments on both amalgam and dentine surfaces; some originated as cascades from the surface biofilm and extended into the tooth/restoration microspace. Vital staining showed the majority of bacteria from both dentine and amalgam surfaces to be viable. CONCLUSION: This preliminary investigation showed that the CDFF may be a valuable tool for the in vitro study of the dynamics of microbial microleakage around dental restorations.

Animals↗
PubMed2003

Monsel's solution: a potential vector for nosocomial infection?

Monsel's solution is a common topically applied hemostatic agent used in minor dermatologic and gynecologic surgery. Clinically, because it is often stored for long periods and dispensed from a common source for multiple patients, Monsel's solution is a potential vector for transmission of infection. However, microbiologic inoculation studies and contamination surveys indicate that Monsel's solution has properties that prohibit microbial growth, making it an unlikely vector for nosocomial infection.

Bacterial Infections↗
PubMed2006

Test to determine sterile integrity of wrapped medical products at a probability of recontamination of 1 : 1,000,000.

OBJECTIVE: We developed a microbiological test to detect the penetration of airborne microorganisms through the packaging of medical products after sterilization, to meet the requirements of European standard EN 556. We applied this test method to transparent pouches. DESIGN: The microbial-barrier properties of the transparent pouches were determined using the microbial challenge test, in which the package was placed inside an exposure chamber and exposed to a defined aerosol of Saccharomyces cerevisiae. The atmospheric pressure in the chamber was periodically reduced by 0-75 millibars, to simulate weather-dependent pressure changes. Thermoresistant petri dishes filled with nutrient agar were integrated into the transparent pouches before sterilization. The packages were incubated after exposure. They were then opened and examined for colony growth. RESULTS: The number of recontaminated packages per test group (n=50) depended on the microbial bioload (defined as the number of colony-forming units per plate) to which the packages were exposed and on the size and number of decreases in atmospheric pressure. Results of multiple regression analysis showed a significant increase in the number of recontaminated packages in correlation with the product of the values for microbial bioload and the size and number of decreases in atmospheric pressure. When we analyzed the probability of recontamination of wrapped medical devices after 2 reductions in atmospheric pressure (30 millibars each) and with a surface microbial load of 10 colony-forming units per 64 cm(2), we estimated that the frequency of recontamination was 1 : 100,000. CONCLUSION: Multiple regression analysis showed that the proposed microbial challenge test is suitable to determine the probability of package recontamination at the 1 : 1,000,000 level.

Air Microbiology↗
PubMed1997

Potential sources of bacteria that are isolated from contact lenses during wear.

PURPOSE: The aim of this paper was to determine the possible contamination sources of contact lenses during wear. METHODS: Potential sources of the microbiota that colonized hydrogel contact lenses during wear were examined. The microorganisms that colonize contact lenses were grown, identified, and compared to those microorganisms that colonized the lower lid margins, upper bulbar conjunctiva, hands, and contact lens cases of contact lens wearers. In addition, the incidence of contamination of the domestic water supply in the Sydney area was obtained, and this was compared to the incidence of colonization of contact lenses by microorganisms in general and gram-negative bacteria in particular. RESULTS: There was a wide diversity of bacteria that were isolated from each site sampled. Coagulase-negative staphylococci and Propionibacterium spp. were the most common isolates from all ocular sites examined, and constituted the normal ocular microbiota. Other bacteria, including members of the families Enterobacteriaceae and Pseudomonadaceae, were isolated infrequently from all sites, but most frequently from contact lens cases. Statistical analysis revealed that there was a correlation between the isolation of bacteria from the contact lens and the lower lid margin (p < 0.001). Analysis of this correlation revealed that this was true for the normal microbiota. A correlation was also noted between the colonization of contact lenses by gram-negative bacteria and contamination of the domestic water supply. DISCUSSION: This study has demonstrated that the likely route for the normal ocular microbiota colonizing contact lenses is via the lid margins, whereas colonization by gram-negative bacteria, including potential agents of microbial keratitis, is likely to be from the domestic water supply.

Bacteria↗
PubMed2004

Identification method based on PCR combined with automated ribotyping for tracking probiotic Lactobacillus strains colonizing the human gut and vagina.

AIMS: A molecular methodology based on PCR-associated automated ribotyping was developed to specifically detect the Lactobacillus strains of two probiotic products (an orally administered lyophilized preparation and vaginal tablets) in human faeces and vaginal swabs. METHODS AND RESULTS: The 16S-23S rDNA sequences and the ribotype profiles of the probiotic lactobacilli were characterized and new species-specific primer sets were designed. The identification of faecal and vaginal lactobacilli isolated from subjects administered with the probiotic products was performed by using PCR with species-specific primers followed by strain-specific automated ribotyping. CONCLUSIONS: The PCR-ribotyping identification allowed to study the colonization patterns of the probiotic lactobacilli in the human gut and vagina evidencing the strains with the best survival capability. SIGNIFICANCE AND IMPACT OF THE STUDY: The proposed molecular method represents a powerful tool of strain-specific identification, useful for differentiating exogenous from indigenous strains in any microbial ecosystem and for rationally choosing probiotic bacteria with the best chance of survival in the host.

Colony Count, Microbial↗
PubMed2001

Identification and characterization of elevated microbial counts in bulk tank raw milk.

The bacterial composition of bulk tank milk from 13 farms was examined over a 2-wk period to characterize sudden elevations in the total bacterial count referred to as "spikes." Bulk tank milk samples collected at each pick-up were analyzed for standard plate count, Petrifilm aerobic count, somatic cell count, gram-negative organisms, and streptococci. Twenty standard plate count spikes were observed: 12 associated with streptococci, 4 associated with gram-negative organisms, 2 associated with streptococci and gram-negative organisms, and 2 that were not definitively characterized. Spikes ranged from 14,000 to 600,000 cfu/ml. Streptococcus uberis was isolated as the predominant organism from 11 spikes, and Escherichia coli was isolated from 4 spikes. Statistical analysis of total bacterial counts indicated a high correlation (r = 0.94) between standard plate counts and Petrifilm aerobic count. Regression analysis of standard plate counts and Petrifilm aerobic counts yielded the equation log10 (standard plate count) = 0.73 + 0.85log10 (Petrifilm aerobic count), indicating that the correlation, although strong, is not one to one. In a related pilot study, triplicate bulk tank milk samples were collected and analyzed for total bacterial count and presumptive streptococcus, gram-negative, and staphylococcus counts. Two-way ANOVA of these triplicate data indicated a lack of significant variation among the triplicate samples, suggesting that one sample can reliably gauge the microbial status of the entire bulk tank.

Animals↗
PubMed1999

Bactericidal effect of 0.95-mW helium-neon and 5-mW indium-gallium-aluminum-phosphate laser irradiation at exposure times of 30, 60, and 120 seconds on photosensitized Staphylococcus aureus and Pseudomonas aeruginosa in vitro.

BACKGROUND AND PURPOSE: Studies have demonstrated a bactericidal effect of laser irradiation when lasers with power outputs of (6 mW are directed toward pathogenic or opportunistic bacteria previously treated with a photosensitizing agent. The purpose of this study was to determine the bactericidal capabilities of irradiation from lasers with power outputs of less than 6 mW on photosensitized microorganisms. METHODS: Two bacteria that commonly infect skin lesions, Staphylococcus aureus and Pseudomonas aeruginosa, were used. The 2 lasers used, the 0.95-mW helium -neon laser and the 5-mW indium-gallium-aluminum-phosphate laser, emit light at a wavelength close to the absorption maxima of the sensitizing agent chosen, toluidine blue O. This agent was used because of its proven effectiveness in sensitizing bacteria. For each bacterial strain, toluidine blue O was added to a 108 cells/mL solution until a 0.01% weight/volume ratio was obtained. These mixtures were spread on agar-coated petri dishes, which were then exposed to 1 of the 2 lasers for 30, 60, and 120 seconds. The cultures were then grown overnight and examined for one or more visible zones of inhibition. The areas surrounding the irradiated zone provided a control for the effects of toluidine blue O alone. To determine the effects of laser irradiation without prior toluidine blue O sensitization, separate plates were established using unsensitized bacteria. RESULTS: Although inconsistencies between plates were noted, both lasers produced at least one zone of inhibition in both bacterial species at all 3 time periods. The 5-mW laser, however, produced a greater number of these zones. CONCLUSION AND DISCUSSION: Laser-induced microbial killing of photosensitized organisms could have clinical applications in the treatment of infected skin lesions, pending in vivo studies.

Colony Count, Microbial↗
PubMedDate not supplied

[Seasonal dynamics in a yeast population on the Oxalis acetosella L. leaves].

Analysis of an epiphytic yeast population on the leaves of the evergreen common wood sorrel Oxalis acetosella L. throughout a year showed that the density and the species composition of this population underwent regular seasonal changes. There were almost no yeasts on the young spring leaves. However, the yeast population on the mature leaves tended to increase in the autumn, reaching a maximum after the formation of continuous snow cover. Then the yeast population on the leaves tended to decrease, reaching a minimum in the spring. The species diversity of the yeasts was maximum in the autumn. The population of the epiphytic yeast species Cystofilobasidium capitatum, Rhodotorula fujisanensis, Leucosporium scottii, and Cryptococcus flavus peaked in the autumn. On the other hand, the population of the widespread epiphytic species Cryptococcus laurentii on the wood sorrel leaves peaked in January. The relative abundance of the red-pigmented phytobionts Rhodotorula glutinis and Sporobolomyces roseus virtually did not change throughout the year. The relative abundance of the euribiotic species Cryptococcus albidus showed irregular monthly variations. The data obtained show that the epiphytic microbial population of various plants can be comprehensively studied only by analyzing this population throughout the vegetative period of the plants.

Colony Count, Microbial↗
PubMed1996

Combined effects of packaging atmosphere and lactic acid on growth and survival of Listeria monocytogenes in crayfish tail meat 4 degrees C.

The effect of lactic acid on growth and survival of Listeria monocytogenes in crayfish tail meat stored under refrigeration and various gas environments was investigated. Frozen crayfish tail meat was thawed overnight, autoclaved, cooled, and inoculated with approximately 4 log colony-forming units (CFU) of a mixed-strain (Scott A and F5027) L. monocytogenes culture per gram of meat. Inoculated samples were blended with 0, 0.5, 1.0, 1.5, or 2.0% lactic acid and packaged under air, vacuum, or modified atmosphere (74.8% CO2, 10.4% O2, and 14.8% N2) and stored at 4 degrees C for 20 days. Results demonstrated that modified atmosphere packaging inhibited the growth of L. monocytogenes more than air and vacuum packaging at 0 and 1% lactic acid. Microbial counts declined steadily in crayfish tail meat treated with 2% lactic acid, with no differences among the packaging atmospheres. The lag phase was extended by 8 days in samples treated with 1% lactic acid and modified atmosphere compared to that in air or vacuum packaging. Overall, the combination of lactic acid and modified atmosphere had the greatest potential to prevent growth of L. monocytogeines.

Animals↗
PubMed2003

Bacterial community structure and location in Stilton cheese.

The microbial diversity occurring in Stilton cheese was evaluated by 16S ribosomal DNA analysis with PCR-denaturing gradient gel electrophoresis. DNA templates for PCR experiments were directly extracted from the cheese as well as bulk cells harvested from a variety of viable-count media. The variable V3 and V4-V5 regions of the 16S genes were analyzed. Closest relatives of Lactococcus lactis, Enterococcus faecalis, Lactobacillus plantarum, Lactobacillus curvatus, Leuconostoc mesenteroides, Staphylococcus equorum, and Staphylococcus sp. were identified by sequencing of the DGGE fragments. Fluorescently labeled oligonucleotide probes were developed to detect Lactococcus lactis, Lactobacillus plantarum, and Leuconostoc mesenteroides in fluorescence in situ hybridization (FISH) experiments, and their specificity for the species occurring in the community of Stilton cheese was checked in FISH experiments carried out with reference cultures. The combined use of these probes and the bacterial probe Eub338 in FISH experiments on Stilton cheese sections allowed the assessment of the spatial distribution of the different microbial species in the dairy matrix. Microbial colonies of bacteria showed a differential location in the different parts of the cheese examined: the core, the veins, and the crust. Lactococci were found in the internal part of the veins as mixed colonies and as single colonies within the core. Lactobacillus plantarum was detected only underneath the surface, while Leuconostoc microcolonies were homogeneously distributed in all parts observed. The combined molecular approach is shown to be useful to simultaneously describe the structure and location of the bacterial flora in cheese. The differential distribution of species found suggests specific ecological reasons for the establishment of sites of actual microbial growth in the cheese, with implications of significance in understanding the ecology of food systems and with the aim of achieving optimization of the fermentation technologies as well as preservation of traditional products.

Cheese↗
PubMed2001

Bacterial contamination associated with electronic faucets: a new risk for healthcare facilities.

OBJECTIVE: To investigate the safety of the hospital water supply following a major flood. DESIGN: Surveillance was conducted of the hospital water supply as it entered the hospital and at randomly selected water faucets throughout the facility. SETTING: A newly constructed surgical critical-care unit in a 265-bed community hospital that had to be evacuated and was out of operation for 6 weeks following a major flood of the city. METHODS: Random water samples throughout the facility were analyzed for heterotrophic plate counts (HPCs), chlorine, and coliforms utilizing standard methods. RESULTS: Water samples entering the hospital met appropriate standards, indicating the city water distribution system was not contaminated. Of 169 faucets tested, 13 (22%) of 59 electronic faucets exceeded the HPC threshold, and 12 (11%) of 110 manual faucets exceeded the HPC threshold (P<.14). A comparison of two brands of electronic faucets with manual faucets and with each other revealed that the HPC threshold was exceeded by 11 (32%) of 34 brand A faucets as compared to 12 (11%) of 110 manual faucets (P<.006). The HPC threshold was exceeded by 2 (8%) of 25 brand B faucets compared to 12 (11%) of 110 manual faucets (P<.94). Contamination rates of brand A and brand B faucets differed significantly (P<.003). Similar testing 2 months after hyperchlorination of the water supply indicated that the HPC threshold was exceeded by 16 (52%) of 31 brand A faucets compared to 10 (9.%) of 110 manual faucets (P<.0000003) and by 2 (18%) of 25 brand B faucets compared to 10 (9%) of 110 manual faucets (P=1.0). CONCLUSIONS: A certain brand of electronic water faucet used in the hospital was associated with unacceptable levels of microbial growth in water and was a continuing source of bacteria potentially hazardous to patients.

Chlorine↗
PubMed2002

Effects of fructooligosaccharide on conversion of L-tryptophan to skatole and indole by mixed populations of pig fecal bacteria.

An in vitro study was conducted to examine the effects of fructooligosaccharide (FOS) at levels of 0.5, 1.0, and 1.5% on conversion of L-tryptophan to skatole and indole by a mixed bacterial population from the large intestines of pigs. Microbial suspensions were anaerobically incubated at 38 degrees C for 24 h. Samples were periodically removed for determination of pH and indole compounds. After 24 h incubation, microbial populations in each culture media were analyzed. Addition of 0.5, 1.0, and 1.5% FOS to the slurries with L-tryptophan significantly decreased the skatole concentration, the peak value of indole-3-acetic acid and the medium pH. The viable counts of Bifidobacterium were significantly higher as compared with the control. Addition of 1.0 and 1.5% FOS significantly decreased the rate of tryptophan degradation and the relative rate of skatole production. The relative rate of indole production was significantly increased. The viable counts of Clostridium and Escherichia coli were significantly reduced. The total viable counts of anaerobes were significantly increased. These results suggest that the reduced concentration of skatole observed in the presence of FOS may be caused by the decreased tryptophan degradation due to the increased need for amino acids in the synthesis of bacterial cellular protein, and by shifting microbial metabolism of tryptophan toward indole production at the expense of skatole, which might result from the changed microbial ecosystem and pH. Our observations open the possibility of inhibiting microbial production of skatole and decreasing the skatole concentration in backfat by feeding pigs diets containing FOS, but it remains to be demonstrated in vivo.

Anaerobiosis↗
PubMedDate not supplied

Rapid methods for the microbiological surveillance of pharmaceuticals.

The use of rapid microbiological methods in pharmaceutical laboratories has improved the quality control analysis of water, products, raw materials, and enhanced the antimicrobial effectiveness testing of pharmaceutical finished products. Rapid release of samples has resulted in the optimization of manufacturing, product testing, and release allowing high throughput and simultaneous analysis of pharmaceutical formulations. ATP Bioluminescence, Impedance, Direct Viable Counts, and Flow Cytometry determine the total microbial content in a given pharmaceutical sample while PCR and Immunoassays detect the presence or absence of specific microbial species. Rapid methods provide reliable and cost effective analysis for the microbiological evaluation of pharmaceutical environments. The dramatic reduction in detection times and analysis, e.g., from 30 hours to 90 minutes, by using rapid methods will ultimately lead the pharmaceutical industry closer to real time monitoring of processes and samples.

Colony Count, Microbial↗
PubMed2001

Bioluminescence ATP assay for estimating total plate counts of surface microflora of whole cantaloupe and determining efficacy of washing treatments.

The surface microflora of cantaloupes were estimated using a bioluminescence ATP assay, and results were compared to plate count data. Cantaloupes were treated as follows: (i) water washed, or (ii) washed in solutions of sodium hypochlorite (1,000 mg/liter) or hydrogen peroxide (5%) for 5 min. Bioluminescence ATP assay results showed differences in ATP level/cm2 of cantaloupes dipped in chlorine or hydrogen peroxide solution; ATP levels in these washed samples were lower than in controls due to antimicrobial action of the treatments on the cantaloupe surface. Linear correlations were found between the bioluminescence ATP assay and aerobic plate counts of unwashed cantaloupe (r2 = 0.995) and those washed with water (r2 = 0.990) determined before storage. Lower correlations between the bioluminescence ATP assay and the aerobic plate counts were observed on cantaloupes stored for 120 h at 20 degrees C (r2 = 0.751) than at 4 degrees C (r2 = 0.980) without washing treatment. Lower correlation at 20 degrees C may be the result of clusters or growth that occurred in chains. ATP levels of washed cantaloupes correlated well with bacterial plate counts (r2 = 0.999). A reliable minimum detectable threshold using the bioluminescence ATP assay was established at 3 log10 fg/cm2 corresponding to 4 log10 CFU/cm2. Bioluminescence ATP assay is not recommended for washed samples where the microbial load is near or below the threshold. Therefore, the bioluminescence ATP assay will be recommended for quick estimation of total microbial load on cantaloupe surfaces where the population is expected to exceed this threshold. The assay can save the industry time by eliminating the required incubation required by the conventional methods.

Adenosine Triphosphate↗
PubMed2002

A comparison among different methods for evaluating the biomass activity in activated sludge systems: preliminary results.

In order to improve activated sludge plant operation (achieving higher efficiency and cost savings) beside influent and effluent characteristics and working parameters (e.g. dissolved oxygen, total and volatile suspended solids, pH, recirculation flow rate, etc.), the biomass activity should be monitored, the bacteria being responsible for the pollutant degradation. Since conventional cultivation based methods are inadequate to quantify environmental microorganisms (due to scarce number of cultivable microorganisms and time-consuming procedures) several "non-conventional" techniques were applied in this study, in order to compare the obtainable information and their routine feasibility. Different measurements (VSS concentration, Oxygen Uptake Rate, microbial counts by cultural and biomolecular methods--MPN-PCR, ATP content, dehydrogenase activity, microbial cell viability and enzymatic activity) were carried out on mixed liquor samples, taken from a municipal activated sludge plant (440,000 p. e.). The preliminary results of the research are presented in this paper.

Adenosine Triphosphate↗
PubMed2006

Secondary effluent disinfection: PAA long term efficiency.

The paper summarizes the results of a bench-scale study to evaluate the long-term disinfection efficiency of peracetic acid (PAA). Bacterial counts were repeated 5, 24, and 29 h after the end of the disinfection test, to simulate real re-growth conditions (no residual quenching) and, for the 5 h interval, the potential re-growth (quenching of residual PAA). Fecal coliforms, Escherichia coli, and total heterotrophic bacteria (THB) were enumerated by traditional plate count technique; THB were also enumerated by cytometry. After disinfection, the residual PAA concentration became negligible in about 5 to 11 h, depending on the tested doses. Microbial counts showed that no appreciable re-growth took place after 29 h for coliform group bacteria. For THB, the previously cited enumeration techniques gave different results in re-growth tests, especially for the lowest PAA doses. Indeed plate count technique evaluates the ability to form colonies, while cytometry enumerates intact membrane cells. No regrowth took place, even when no residual disinfectant was present, suggesting that bacteria are unable, even at the lowest doses, to repair damage caused by the PAA disinfecting action. PAA was found to be an efficient disinfecting agent, not only as a bacteriostatic, but also as a bactericide.

Bacteria↗