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PubMed2006

Estimation of microbial contamination of food from prevalence and concentration data: application to Listeria monocytogenes in fresh vegetables.

A normal distribution and a mixture model of two normal distributions in a Bayesian approach using prevalence and concentration data were used to establish the distribution of contamination of the food-borne pathogenic bacteria Listeria monocytogenes in unprocessed and minimally processed fresh vegetables. A total of 165 prevalence studies, including 15 studies with concentration data, were taken from the scientific literature and from technical reports and used for statistical analysis. The predicted mean of the normal distribution of the logarithms of viable L. monocytogenes per gram of fresh vegetables was -2.63 log viable L. monocytogenes organisms/g, and its standard deviation was 1.48 log viable L. monocytogenes organisms/g. These values were determined by considering one contaminated sample in prevalence studies in which samples are in fact negative. This deliberate overestimation is necessary to complete calculations. With the mixture model, the predicted mean of the distribution of the logarithm of viable L. monocytogenes per gram of fresh vegetables was -3.38 log viable L. monocytogenes organisms/g and its standard deviation was 1.46 log viable L. monocytogenes organisms/g. The probabilities of fresh unprocessed and minimally processed vegetables being contaminated with concentrations higher than 1, 2, and 3 log viable L. monocytogenes organisms/g were 1.44, 0.63, and 0.17%, respectively. Introducing a sensitivity rate of 80 or 95% in the mixture model had a small effect on the estimation of the contamination. In contrast, introducing a low sensitivity rate (40%) resulted in marked differences, especially for high percentiles. There was a significantly lower estimation of contamination in the papers and reports of 2000 to 2005 than in those of 1988 to 1999 and a lower estimation of contamination of leafy salads than that of sprouts and other vegetables. The interest of the mixture model for the estimation of microbial contamination is discussed.

Bayes Theorem↗
PubMed2005

Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Tannerella forsythensis are components of a polymicrobial intracellular flora within human buccal cells.

Previously, we used in situ hybridization and confocal microscopy to detect the periodontal pathogens Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Tannerella forsythensis within buccal epithelial cells taken directly from the mouth. This study tested the hypothesis that the intracellular flora of buccal cells is polymicrobial. Mixtures containing a red fluorescent universal probe paired with green fluorescent versions of either A. actinomycetemcomitans-, P. gingivalis-, or T. forsythensis-specific probes were hybridized with buccal cells collected from each of 38 healthy humans. We verified co-localization of probe pairs within cells by generating three-dimensional reconstructions. Intracellular bacteria were detected in every subject. Each cell that was labeled with a species-specific probe also contained bacteria recognized only by the universal probe. Bacteria labeled with specific probes often occupied smaller regions within larger masses of bacteria. Those findings suggest that future studies of invasion by oral bacteria may need to include microbial consortia.

Adult↗
PubMed1998

Estimating distributions of numbers of organisms in food products.

Procedures of sampling and measurement contribute variability and uncertainty to exposure models that predict incidence and levels of organisms in food products. This paper focuses on methods that account for sampling and measurement error in fitting distributions of organisms in food products for use in exposure models for microbial risk assessment. Define y to be a measured density on a sample selected with stipulated probability from a population, and define x to be the "true" density for that sample. Designate the conditional distribution of y given the sample with "true" value x as g(y/x), and let F(x) be the unknown cumulative density of x. The distribution of the observed values y, h(y), can be expressed through the integral equation h(y) = integral of g(y/x) dF(x). Knowledge of g(y/x) and h(y) enables an estimate of the unknown distribution of the organism's F(x). In applications to risk assessment, use of continuous distributions described by a few parameters is desirable. Also desirable may be imputation or assignment of possible nonzero values for nondetect observations--those results below the limit of detection of the methodology--that may not be truly zero. This paper explores the use of the above formulation for estimating distributions of organisms used for microbial risk assessment and presents some simple examples.

Clostridium perfringens↗
PubMed2004

Efficacy of chlorine dioxide gas as a sanitizer of lettuce leaves.

Aqueous solutions of sodium hypochlorite or hypochlorous acid are typically used to sanitize fresh fruits and vegetables. However, pathogenic organisms occasionally survive aqueous sanitization in sufficient numbers to cause disease outbreaks. Chlorine dioxide (ClO2) gas generated by a dry chemical sachet was tested against foodborne pathogens on lettuce leaves. Lettuce leaves were inoculated with cocktail of three strains each of Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella Typhimurium and treated with CLO2 gas for 30 min, 1 h, and 3 h in a model gas cabinet at room temperature (22 +/- 2 degrees C). After treatment, surviving cells, including injured cells, were enumerated on appropriate selective agar or using the overlay agar method, respectively. Total ClO2, generated by the gas packs was 4.3, 6.7, and 8.7 mg after 30 min, 1 h, and 3 h of treatment, respectively. Inoculated lettuce leaves exposed to ClO2 gas for 30 min experienced a 3.4-log reduction in E. coli, a 4.3-log reduction in Salmonella Typhimurium, and a 5.0-log reduction in L. monocytogenes when compared with the control. After 1 h. the three pathogens were reduced in number of CFU by 4.4. 5.3, and 5.2 log, respectively. After 3 h, the reductions were 6.9, 5.4, and 5.4 log, respectively. A similar pattern emerged when injured cells were enumerated. The ClO2, gas sachet was effective at killing pathogens on lettuce without deteriorating visual quality. Therefore, this product can be used during storage and transport of lettuce to improve its microbial safety.

Chlorine Compounds↗
PubMed2004

Effects of an antimicrobial additive to toothbrushes on residual periodontal pathogens.

OBJECTIVE: Previous studies have reported the link between residual microbial contamination of toothbrushes and periodontal diseases. The goal of this pilot study was to evaluate the effects of an antimicrobial additive (Microban) to toothbrushes on residual retention of periodontal pathogens. METHODOLOGY: Twenty patients had one side of their mouths brushed with a toothbrush containing the antimicrobial agent (experimental side), and the other side with a toothbrush containing no agent (control). Toothbrushes were air-dried (25 degrees C) for four or 24 hours. Toothbrush heads were vortexed and cultured for Prevotella species (Ps), Porphyromonas gingivalis (Pg), Actinobacillus actinomycetemcomitans (Aa), and non-specific colony-forming units (NS). The plates were incubated and counted. Means and standard deviations were calculated, and data were analyzed using a series of t-tests (paired and unpaired) and Wilcoxon matched-pairs signed-rank test. RESULTS: No significant inter- or intra-group differences in mean counts were found; however, when four-hour and 24-hour data for Aa, Pg, or NS were combined, experimental counts were lower than controls in 39/50 (78%) of the matched pairs (Wilcoxon signed-rank test p = 0.01). CONCLUSION: Toothbrushes containing the antimicrobial additive showed lower microbial counts than those without, but between-group means were not statistically significant.

Aggregatibacter actinomycetemcomitans↗
PubMed1998

Shelf life of ground beef patties treated by gamma radiation.

The effects of irradiation on microbial populations in ground beef patties vacuum package and irradiated frozen at target doses of 0.0, 1.0, 3.0, 5.0, and 7.0 kGy were determined. Irradiated samples were stored at 4 or -18 degrees C for 42 days, and mesophilic aerobic plate counts (APCs) were periodically determined. Fresh ground beef (initial APC of 10(2) CFU/g) treated with 3.0, 5.0, and 7.0 kGy was acceptable (< 10(7) CFU/g) for 42 days at 4 degrees C. The 1.0 kGy-treated beef samples were acceptable microbiologically (< 10(7) CFU/g) after 42 days but developed an unacceptable off-odor after 21 days. Shelf life diminished in fresh ground beef patties with an initial APC of 10(4) CFU/g. Only beef patties treated with 7.0 kGy were found to be acceptable at 42 days. Beef patties treated at 1.0 and 3.0 kGy reached spoilage APC levels (> 10(7) CFU/g) by day 14 and 21, respectively, whereas patties treated at 5.0 kGy did not spoil until 42 days. The nonirradiated control samples for both batches of ground beef spoiled within 7 days. Microbial counts in ground beef patties stored at -18 degrees C did not change over the 42-day period. Shelf life of ground beef patties stored at 4 degrees C may be extended with gamma radiation, especially at 5.0 and 7.0 kGy. Initial microbial load in ground beef samples was an important shelf life factor.

Animals↗
PubMed2003

Suitability of the traditional microbial indicators and their enumerating methods in the assessment of fecal pollution of subtropical freshwater environments.

Twenty-four freshwater sampling sites (11 river water, 6 spring water, and 7 groundwater) were selected from 4 sampling areas located in the northern and central parts of Taiwan. A total of 125 water samples were collected during a 5-month sampling period, and the numbers of total heterotrophic bacteria, total coliforms, fecal coliforms, enterococci, Aeromonas hydrophila, and Salmonella spp. were enumerated. Besides the traditional membrane filtration method, total coliforms and Escherichia coli were also simultaneously enumerated using the Colilert (Quanti-Tray/2000) method. On average, 94% and 80% of the water samples assessed with the Colilert method had equal or higher total coliform and E. coli counts, respectively, as compared with the membrane filtration method. Furthermore, when m-FC agar was used to enumerate fecal coliforms, 18% of the samples failed to yield the typical bluish colonies, while E. coli were counted in the same samples using the Colilert method. The data indicate that the m-FC agar culture method is inadequate for the enumeration of fecal coliforms in subtropical water samples. Significant correlations were observed between the total number of bacteria and various indicator bacteria in river water samples, but no such correlations were found for groundwater and spring water. This finding suggested that the river water was polluted by anthropogenic sources. The counts of total coliforms, E. coli, and other indicator bacteria were significantly correlated in all river water samples, while in groundwater and spring water, significant (p<0.01) correlation was only observed with enterococcal counts. The presence of total coliforms/E. coli generally implies the presence of fecal pollution possibly including pathogenic enteric bacteria. However, no Salmonella spp. were detected in any of the 107 water samples analyzed. The results of this study suggest that the use of these commonly employed microbial indicators for assessing subtropical water quality, especially in a pristine body of water (ie, mountain spring and groundwater), is highly questionable.

Colony Count, Microbial↗
PubMed2002

Evaluation of the spiral plating method for the enumeration of microorganisms throughout the manufacturing and ripening of a raw goat's milk cheese.

A statistical comparison of the spiral plate count (SPLPC) and the standard plate count (SPC) methods for enumeration of microorganisms in raw goat's milk cheese throughout its manufacturing and ripening was carried out. Enumeration of mesophiles, lactic acid bacteria (presumptive lactococci, presumptive leuconostocs, and presumptive lactobacilli), Micrococcaceae, Enterobacteriaceae, and molds and yeasts was carried out for milk, curd, and 2-, 5-, 10-, 17-, and 27-day-old cheeses. Average counts for the SPLPC and SPC methods differed by less than half of a log cycle for all microbial groups studied (range of difference, -0.1386 [mesophiles] to +0.4397 [presumptive lactobacilli]). The results of the SPLPC method compared favorably with the results of the SPC procedure for mesophiles, presumptive lactococci, presumptive leuconostocs, Enterobacteriaceae, and molds and yeasts (the variance between replicate platings was close to 0.005, and correlation coefficients were >0.9). Correlation coefficients were lower for Micrococcaceae (r = 0.824) and presumptive lactobacilli (r = 0.670). Analysis of variance showed that the plating method was a significant factor (P < 0.05) for presumptive lactobacilli counts. In general, results from the SPLPC method compared favorably with results from SPC procedure in the enumeration of microorganisms in goat cheese throughout its manufacturingand ripening processes. However, the suitability of the SPLPC method depends mainly on the microbial group studied.

Animals↗
PubMed2001

Survival of Listeria monocytogenes during the manufacture and ripening of Turkish white cheese.

Listeria monocytogenes was enumerated during the manufacture and ripening of Turkish White cheese with particular reference to a) pasteurized milk, b) cheese milk after inoculation with L. monocytogenes (0 h), c) after curd formation (2 h), d) curd after pressing (6 h), e) curd after pH was reduced (17 h), f) curd after salting (32 h), and g) cheeses during ripening. Cheeses were also examined periodically for total solids, moisture and salt contents, pH values and aerobic plate count. An increase in the number of L. monocytogenes was observed during manufacture. Following salting and throughout the storage period, numbers of L. monocytogenes decreased at a rate depending on the salt concentration, starter activity and storage time. The initial microbial number had a significant (P < 0.01) effect on the survival of L. monocytogenes during the storage period.

Cheese↗
PubMed1999

Marine bacterial isolates display diverse responses to UV-B radiation.

The molecular and biological consequences of UV-B radiation were investigated by studying five species of marine bacteria and one enteric bacterium. Laboratory cultures were exposed to an artificial UV-B source and subjected to various post-UV irradiation treatments. Significant differences in survival subsequent to UV-B radiation were observed among the isolates, as measured by culturable counts. UV-B-induced DNA photodamage was investigated by using a highly specific radioimmunoassay to measure cyclobutane pyrimidine dimers (CPDs). The CPDs determined following UV-B exposure were comparable for all of the organisms except Sphingomonas sp. strain RB2256, a facultatively oligotrophic ultramicrobacterium. This organism exhibited little DNA damage and a high level of UV-B resistance. Physiological conditioning by growth phase and starvation did not change the UV-B sensitivity of marine bacteria. The rates of photoreactivation following exposure to UV-B were investigated by using different light sources (UV-A and cool white light). The rates of photoreactivation were greatest during UV-A exposure, although diverse responses were observed. The differences in sensitivity to UV-B radiation between strains were reduced after photoreactivation. The survival and CPD data obtained for Vibrio natriegens when we used two UV-B exposure periods interrupted by a repair period (photoreactivation plus dark repair) suggested that photoadaptation could occur. Our results revealed that there are wide variations in marine bacteria in their responses to UV radiation and subsequent repair strategies, suggesting that UV-B radiation may affect the microbial community structure in surface water.

Colony Count, Microbial↗
PubMed1994

Influence of tooth instrumentation roughness on subgingival microbial colonization.

This study evaluated microbial colonization of periodontal pockets subjected to root instrumentation with a curet or a rotating diamond. Ten maxillary and 10 mandibular subgingival pockets were established in the canines of 5 beagle dogs. The subgingival root surface areas were debrided by a sharp curet or a flame-shaped, fine-grained, rotating diamond point. The dogs were fed a plaque-inducing diet for 70 days. Specimens from both instrumentation groups were then harvested and prepared for stereomicroscopic and scanning electron microscopic evaluation. Grading of the extent of subgingival colonization was performed in coded specimens directly on the fluorescent screen of the scanning electron microscope in a grid-counting system. Error of the method was assessed by duplicate counts. The subgingival root surface areas were divided into 3 zones: cervical, middle, and apical, and statistical differences between these zones as well as between the 2 instrumentation groups were calculated. The results revealed that curet-treated surfaces were smoother and promoted less subgingival colonization than diamond-treated surfaces. The difference in amount of bacterial colonization between the 2 groups was statistically significant (P < 0.05) in all zones. Bacterial colonization decreased in apical direction in both instrumentation groups. For the diamond-treated specimens, this decrease was significant (P < 0.05) between each of the 3 zones. In the curet-treated specimens, the decrease was significant only between the cervical and the apical zone (P < 0.05). The present study has demonstrated that subgingival instrumentation roughness significantly influences the subgingival microbial colonization.

Animals↗
PubMed1990

Colonization resistance of defined bacterial plaques to Streptococcus mutans implantation on teeth in a model mouth.

We investigated the ability of Streptococcus mutans C67-1 to colonize simple bacterial plaques and the effects of age and stability of the pre-formed plaque on colonization resistance. Mixed-plaques of Actinomyces viscosus WVU627, 'Streptococcus mitior' LPA-1, and Veillonella dispar OMZ193 were grown on tooth segments, mounted back to back for simulation of approximal sites in a model mouth for 66 h. S. mutans C67-1 was either included in the original inoculum or super-inoculated onto the developing plaque. Inclusion of S. mutans C67-1 did not alter the total viable counts, but the proportional composition changed due to inter-species interactions. Colonization resistance of the mixed-plaque samples developed within 24 h, although S. mutans C67-1 was always able to colonize these stagnation sites. Colonization resistance of 24-hour plaque against a fresh isolate, S. mutans CP3, was also studied. There was greater colonization resistance by the basic plaque to this organism, compared with S. mutans C67-1, although the reasons for this were not clear. These initial experiments demonstrate the way in which the factors involved in bacterial colonization resistance in microbial films on teeth can be studied under controlled conditions.

Actinomyces↗
PubMedDate not supplied

Quantifying heterogeneity: flow cytometry of bacterial cultures.

Flow cytometry is a technique which permits the characterisation of individual cells in populations, in terms of distributions in their properties such as DNA content, protein content, viability, enzyme activities and so on. We review the technique, and some of its recent applications to microbiological problems. It is concluded that cellular heterogeneity, in both batch and continuous axenic cultures, is far greater than is normally assumed. This has important implications for the quantitative analysis of microbial processes.

Bacteria↗
PubMed2006

Changes in volatile compounds and related biochemical profile during controlled fermentation of cv. Conservolea green olives.

The effect of controlled fermentation processes on the profile of volatile and other biochemical compounds of cv. Conservolea green olives processed by the Spanish method was studied. The different treatments included: (a) inoculation with a commercial starter culture of Lactobacillus pentosus, (b) inoculation with a wild strain of Lactobacillus plantarum isolated from a previous fermentation, (c) uninoculated spontaneous process (control). Microbial growth, pH, titratable acidity, reducing sugars, organic acids and volatile compounds were monitored. Starter cultures were effective in establishing an accelerated fermentation process. Both were able to reduce the survival period of Enterobacteria by 7 days, minimizing thus the likelihood of spoilage. Higher acidification of the brines and faster pH drop was observed in inoculated processes, with L. pentosus presenting better performance than the wild strain of L. plantarum. Lactic and acetic were the major organic acids detected by HPLC, the concentration of which increased in the course of fermentation. Citric and malic acids were also present in the brines but they were degraded completely within the first 2 weeks of fermentation. Ethanol, methanol, acetaldehyde, ethyl acetate, isobutyric acid were the major volatile compounds identified by GC. Their concentration varied greatly among the fermentation processes, reflecting varying degrees of microbial activity in the brines.

Chromatography, High Pressure Liquid↗
PubMed2002

Microbial composition, including the incidence of pathogens, of goat milk from the bergamo region of italy during a lactation year.

Sixty samples of raw goat milk intended for Caprino cheese-making were collected from ten farms in the Bergamo area over a 6-month period. Analyses of main microbial groups, somatic cell count (SCC) and pH were performed to determine the effect of origin (farm) and lactation period (April - September) on microbial composition and the incidence of pathogens in milk. Overall mean values were: standard plate count (SPC), 5.0 x 10(4) cfu/ml; yeasts, 2.5 x 10(2) cfu/ml; coliforms, 91 x 10(2) cfu/ml; Escherichia coli, 2.9 cells/ml: enterococci, 1.1 x 10(2) cfu/ ml; lactococci, 3 4 x 10(3) cfu/ml; lactobacilli, 3.0 x 10(3) cfu/ml; halotolerant bacteria, 8.2 x 10(3) cfu/ml; spores of mesophilic aerobic bacteria, 11 cfu/ml; SSC, 9.9 x 10(5) cells/ml; pH, 6.63. Moulds and spores of sulphite-reducing clostridia were found intermittently. Neither Salmonella spp. nor Listeria monocytogenes was detected, while Esch. coli O157: H7 was isolated from one milk sample (an incidence of 1.7%). Staphylococcus aureus was discovered at a level > 10(2) cfu/ml in 26 samples (43%) with an overall mean of 12 x 10(3) cfu/ml, whereas coagulase-negative staphylococci were found in 54 samples (90%) with an overall mean of 1.3 x 10(3) cfu/ml. Of Staph. aureus strains, 23% proved to be enterotoxinogenic with a prevalence of enterotoxin C producers. Staph. caprae was the coagulase-negative species most frequently isolated; none of the coagulase-negative staphylococci strains synthesized any of the enterotoxins tested for. Sample source was the major factor affecting the microbial composition of goat milk: significant differences (P < 0.01) were observed among samples from different farms for SPC, coliforms, lactococci, lactobacilli and halotolerant bacteria. Period of lactation had a significant effect (P < 0.025) on SCC and pH. SPC correlated well with coliforms, lactococci and lactobacilli; SSC did not reveal positive interactions with any microbial groups or pH.

Animals↗
PubMed2004

PCR-DGGE fingerprints of microbial succession during a manufacture of traditional water buffalo mozzarella cheese.

AIMS: To monitor the process and the starter effectiveness recording a series of fingerprints of the microbial diversity occurring at different steps of mozzarella cheese manufacture and to investigate the involvement of the natural starter to the achievement of the final product. METHODS AND RESULTS: Samples of raw milk, natural whey culture (NWC) used as starter, curd after ripening and final product were collected during a mozzarella cheese manufacture. Total microbial DNA was directly extracted from the dairy samples as well as bulk colonies collected from the plates of appropriate culture media generally used for viable counts of mesophilic and thermophilic lactic acid bacteria (LAB) and used in polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) experiments. The analysis of the DGGE profiles showed a strong influence of the microflora of the NWC on the whole process because after the starter addition, the profile of all the dairy samples was identical to the one shown by the NWC. Simple indexes were calculated for the DGGE profiles to have an objective estimation of biodiversity and of technological importance of specific groups of organisms. LAB grown on Man Rogosa Sharp (MRS) and Rogosa agar at 30 degrees C showed high viable counts and the highest diversity in species indicating their importance in the cheese making, which had not been considered so far. Moreover, the NWC profiles were shown to be the most similar to the curd profile suggesting to be effective in manufacture. CONCLUSIONS: The PCR-DGGE analysis showed that in premium quality manufacture the NWC used as starter had a strong influence on the microflora responsible for process development. SIGNIFICANCE AND IMPACT OF THE STUDY: The molecular approach appeared to be valid as a tool to control process development, starter effectiveness and product identity as well as to rank cheese quality.

Animals↗