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Microbial diversity in inactive chimney structures from deep-sea hydrothermal systems.

Massive chimney structures, which are characteristic of many hydrothermally active zones, harbor diverse microbial communities containing both thermophilic and hyperthermophilic microbes. However, vent chimneys ultimately become hydrothermally inactive, and the changes that occur in the microbial communities upon becoming inactive have not been documented. We thus collected inactive chimneys from two geologically and geographically distinct hydrothermal fields, Iheya North in the western Pacific Ocean and the Kairei field in the Indian Ocean. The chimneys displayed easily distinguishable strata, which were analyzed with regard to both mineralogical and microbiological properties. X-ray diffraction pattern and energy-dispersive spectroscopic analyses revealed that the main mineral components of the chimney substructures from Iheya North and the Kairei field were barite (BaSO4) and chalcopyrite (CuFeS2), respectively. Microbial cell densities in the substructures determined by DAPI counting ranged from 1.7 x 10(7) cells g(-1) to 3.0 x 10(8) cells g(-1). The proportions of archaeal rDNA in the whole microbial rDNA assemblages in all substructures were, at most, a few percent as determined by quantitative fluorogenic PCR. The microbial rDNA clone analysis and whole-cell fluorescence in situ hybridization revealed a community that was decidedly different from any communities previously reported in active chimneys. Curiously, both samples revealed the abundant presence of a group of Bacteria related to a magnetosome-bearing bacterium, " Magnetobacterium bavaricum" of the Nitrospirae division. These results suggest that inactive chimneys provide a distinct microbial habitat.

Archaea↗

Microbial generation time during the early phases of supragingival dental plaque formation.

The objective of this investigation was to determine the microbial generation time during early human supragingival plaque formation. On 2 occasions, 13 participants refrained from all oral hygiene measures for periods of 1, 2, 4, 8, 24 and 72 h. Plaque was sampled from defined areas of the teeth 13, 14, 15, 23, 24 and 25. Two independent methods were used to estimate the total number of viable cells: colony-forming units BCVF, total bacterial counts (BC) as enumerated by microscopic examination, and the percentage of vital bacteria using a vital fluorescence technique (VF) were recorded. The total number of vital microorganisms was calculated by BCVF (= BC x VF). The generation time based on either colony-forming units or BCVF increased significantly with plaque age. During the first interval (1-4 h), the microbial multiplication took slightly less than 1 h. After the establishment of a more mature plaque (24-72 h), the replicating time was between 12.5 h (BCVF) and 14.8 h (colony-forming units). It was concluded that the microbial generation time recorded at the initial stages of plaque formation is considerably lower than that found in older plaque.

Adult↗

[Viability and activity of the lactic bacteria (Streptococcus salivarius ssp thermophilus y Lactobacillus delbrueckii ssp bulgaricus) del yogurt en Venezuela].

National and international legislations have agreed that the population of lactic bacteria in yogurt must be viable and not less than 10(6) ufc/g. In Venezuela, during last years, observations indicate that the number of viable cells in some commercial samples show high variations, as low levels. This research attempted to find the origin of this problem in the local industry. For this purpose 105 commercial samples were analyzed during their shelf life and 32 samples of yogurt prepared in the laboratory following the flow diagram of the local industry. The different conditions of freeze dried lactic culture, were also analyzed. These samples were evaluated for viable cell count of lactic bacteria and possible variations of pH and acidity. The absence or low number of lactic bacteria detected in some commercial samples is due to the use of inadequate working cultures that show imbalanced proportions of the two microorganisms, besides a low count below 106 ufc/g. The succesive propagation and storage time of mother culture, and the overacidification of the product, produce subletal injury to the microbial cells of the yogurt starter culture. The data indicate that manufacturing practices significantly affect the survival of the lactic flora.

Colony Count, Microbial↗

Bacterial vaginosis-associated microflora and effects of topical intravaginal clindamycin.

OBJECTIVES: The goals of this study were as follows: (1) to further define the microbiologic characteristics of bacterial vaginosis in nonpregnant women, (2) to evaluate the in vivo activity of topical intravaginal clindamycin and reference this activity to clinical cure, and (3) to evaluate for clindamycin-related emergence of species. STUDY DESIGN: Vaginal microflora was identified and quantified from 33 women with bacterial vaginosis at enrollment and 4 to 7 days after treatment with placebo or three different dosages of clindamycin administered twice daily for 5 days in a double-blind, randomized trial. RESULTS: Clindamycin eradicated and/or decreased counts of major bacterial vaginosis-associated microflora such as Gardnerella, gram-negative and gram-positive anaerobes, and Mycoplasma hominis; this was correlated with cure in 22 of 24 (92%) women. Altered flora (nonlactobacilli) among some of the women who received clindamycin appeared to be transient and without apparent adverse effects. CONCLUSION: Microbial (and clinical) results support use of clindamycin as effective treatment of bacterial vaginosis.

Administration, Intravaginal↗

Control of Listeria monocytogenes in raw-milk cheeses.

The development of Listeria monocytogenes in cheeses made with raw-milk originating from six different farms and according to the Saint-Nectaire cheesemaking technology was studied. Milk was inoculated with two strains of L. monocytogenes at 5 to 10 CFU/25 ml. Microbial and chemical analyses were carried out at appropriate intervals during ripening. L. monocytogenes did not grow in the cores of cheeses prepared with milk originating from three farms. That inhibition could be partially attributed to the pH values and L-lactate content. There was no growth in cheeses with pH below 5.2 and lactate content around 14 mg/g. In all cheeses, L. monocytogenes stopped growing in the cores of cheeses after eight days and some other factors may be involved in the inhibition. No relation was found between L. monocytogenes count and other microbial counts. Growth occurred on cheese surfaces between eight and eighteen days, when the pH significantly increased. The lowest L. monocytogenes growth was found on the surface of cheeses with the lowest pH and without any core growth. Further studies will be performed to clarify the involvement of the microbial community in L. monocytogenes inhibition, in particular during the ripening period.

Cheese↗

Cold and carbon dioxide used as multi-hurdle preservation do not induce appearance of viable but non-culturable Listeria monocytogenes.

AIMS: To study whether the exposure to cold (4 degrees C) and carbon dioxide which results in the elongation of Listeria cells, induces a viable but nonculturable (VBNC) state. METHODS AND RESULTS: When cold and CO2 stressed L. monocytogenes were observed under a fluorescence microscope, using the LIVE/DEAD BacLight bacteria viability kit (Molecular Probes, Eugene, OR, USA), the healthy, mildly injured, and the putative VBNC cells accounted for 31.0% of the stressed cell population. By using the selective plate count, 31.4% of the same stressed cell population was found to be healthy and mildly injured (putative VBNC cells not included). If there were VBNC state cells present, we should have observed a significant difference between the above two numbers. In fact, there was no significant difference between the results obtained from those two methods. CONCLUSIONS: There were no VBNC state cells observed in the stressed cell population. We conclude that cold and CO2 do not induce L. monocytogenes to enter a VBNC state. SIGNIFICANCE AND IMPACT OF THE STUDY: Cold and modified atmospheres are widely used in fresh muscle food and fruit preservation. Whether they would induce L. monocytogenes into a VBNC state is of a great concern for microbial food safety.

Adenosine Triphosphate↗

Microbial quality of commercial 'Golden Delicious' apples throughout production and shelf-life in Lleida (Catalonia, Spain).

'Golden Delicious' apples taken from different points throughout the production chain and shelf-life were analysed for Enterobacteriaceae, thermotolerant coliforms, E. coli, E. coli O157:H7 and Salmonella spp. A total of 36 samples picked up in the orchard, 36 after storage and handling in the packinghouses and 144 from different retail stores were analysed using standardised techniques. Enterobacteriaceae counts were very variable. Pantoea spp. was the main genera isolated. The percentage of samples with thermotolerant coliforms was 16.6%, 22.2% and 10.4% after harvest, after handling in the packinghouse and in the stores, respectively. Strains belonging to Citrobacter, Enterobacter, Klebsiella and Escherichia were isolated. Three samples coming from orchards, five from the packinghouse and two from retailers harboured E. coli. However, none of the E. coli strains isolated had the virulence genes that are pathogenic for humans. None of the samples was Salmonella positive. This is the first study regarding microbial safety of fresh apples in the European Union (EU).

Colony Count, Microbial↗

Distribution of indicator bacteria in Canyon Lake, California.

The spatial and temporal distributions of indicator bacteria in a small, multiple-use source drinking water reservoir in Southern California, USA were quantified over the period August 2001-July 2002. High levels of total and fecal coliform bacteria were present in Canyon Lake (annual geometric mean concentrations+/-SEM of 3.93+/-0.02 and 3.02+/-0.03 log cfu/100mL, respectively), while comparatively low levels of enterococci and E. coli were found (1.16+/-0.02 log cfu/100mL and 0.30+/-0.03 log MPN/100mL, respectively). As a result, these different indicator bacteria yielded quite divergent indices of water quality, with 72.1% of all surface samples (n=294) exceeding the USEPA single-sample limit of 400 cfu/100mL fecal coliform bacteria, while none (0%) of the samples exceeded the single-sample limit for E. coli (n=194). Regression analyses found a positive correlation between total and fecal coliform bacteria (R=0.50, significant at p<0.001) and between enterococci and E. coli (R=0.51, significant at p<0.001), but no correlation or inverse correlations were found between coliform concentrations and enterococci and E. coli levels. External sources responsible for the high total and fecal coliform bacteria were not identified, although laboratory studies demonstrated growth of the coliform bacteria in lake water samples. Enterococci and E. coli were not observed to grow, however. Bacteria concentrations varied relatively little laterally across the lake, although strong vertical gradients in fecal coliform and enterococcus bacteria concentrations were present during summer stratification, with concentrations about 10x higher above the thermocline when compared with surface concentrations. In contrast, total bacteria, total virus and total coliform bacteria levels were unchanged with depth. Seasonal trends in bacteria concentrations were also present. This study shows that the choice of indicator bacteria and sampling depth can both strongly affect the apparent microbial water quality of a lake or reservoir.

California↗

Differential responses of benthic microbes and meiofauna to fish-farm disturbance in coastal sediments.

Bacterial and meiofaunal abundance and biomass and their response to the disturbance induced by fish-farm biodeposition were investigated from March to October 1997 on a monthly basis at two stations of the Gaeta Gulf (Tyrrhenian Sea, Mediterranean Sea). The biopolymeric fraction of the organic matter was characterized by high concentrations which was similar at both fish-farming-impacted and control stations. Similarly, bacteria accounted for a small fraction of the biopolymeric organic carbon (< 1%), while the contribution due to auto-fluorescent cell biomass (i.e. prokaryotic and eukaryotic cells displaying auto-fluorescence) to the total biopolymeric carbon was quantitatively negligible (< 0.1%). Benthic bacteria appear to be sensitive to organic enrichment as their abundance increased significantly beneath the cage, whilst numbers of meiofauna was lower than in the control. Changes occurred also in terms of individual nematode biomass that increased as result of the biodeposition. A particularly useful tool appeared to be represented by the ratio of benthic auto-fluorescent cells to bacterial abundance, bacteria to meiofaunal biomass and auto-fluorescent cells to meiofauna biomass. All these parameters described well the impact due to biodeposition on the benthic environment as their ratios displayed significantly higher values in farm sediments, but recovered rapidly (15 days) to values observed in the control (i.e. undisturbed conditions) immediately after cage removal. Changes observed in the present study highlight that the increased organic loading determined a shift of the relative contribution of the different benthic components to the total biopolymeric carbon, so that in highly impacted systems total benthic biomass becomes increasingly dominated by microbial components.

Animals↗

Soil microbial population dynamics following bioaugmentation with a 3-chlorobenzoate-degrading bacterial culture. Bioaugmentation effects on soil microorganisms.

Changes in microbial populations were evaluated following inoculation of contaminated soil with a 3-chlorobenzoate degrader. Madera sandy loam was amended with 0, 500, or 1,000 microg 3-chlorobenzoate g(-1) dry soil. Selected microcosms were inoculated with the degrader Comamonas testosteroni BR60. Culturable bacterial degraders were enumerated on minimal salts media containing 3-chlorobenzoate. Culturable heterotrophic bacteria were enumerated on R2A. Isolated degraders were grouped by enterobacterial repetitive intergenic consensus sequence-polymerase chain reaction fingerprints and identified based on 16S ribosomal-DNA sequences. Bioaugmentation increased the rate of degradation at both levels of 3-chlorobenzoate. In both the 500 and 1,000 microg 3-chlorobenzoate g(-1) dry soil inoculated microcosms, degraders increased from the initial inoculum and decreased following degradation of 3-CB. Inoculation delayed the development of indigenous 3-chlorobenzoate degrading populations. It is unclear if inoculation altered the composition of indigenous degrader populations. In the uninoculated soil, degraders increased from undetectable levels to 6.6 x 10(7) colony-forming-units g(-1) dry soil in the 500 microg 3-chlorobenzoate g(-1) dry soil microcosms, but none were detected in the 1,000 microg 3-chlorobenzoate g(-1) dry soil microcosms. Degraders isolated from uninoculated soil were identified as one of two distinct Burkholderia species. In the uninoculated soil, numbers of culturable heterotrophic bacteria initially decreased following addition of 1,000 microg 3-chlorobenzoate g(-1) dry soil. Inoculation with C. testosteroni reduced this negative impact on culturable bacterial numbers. The results indicate that bioaugmentation may not only increase the rate of 3-chlorobenzoate degradation but also reduce the deleterious effects of 3-chlorbenzoate on indigenous soil microbial populations.

Biodegradation, Environmental↗

In situ quantification of biocide efficacy using GFP transformed Aureobasidium pullulans.

AIMS: To develop a real-time in situ method to quantify loss of viability of Aureobasidium pullulans PRAFS8 cells attached to plasticized polyvinyl chloride (pPVC) with incorporated biocides, and to use the method to compare biocide efficacy in situ. METHODS AND RESULTS: A. pullulans PRAFS8, transformed with green fluorescent protein (GFP), was used to quantify the efficacy of a range of biocides incorporated into pPVC. Experimentally, it was found that a density of 1.53 x 10(6) yeast cells per cm(2) of pPVC was optimal as increasing the density of the yeast cells to 6.12 x 10(6) cm(-2) attached to pPVC containing the biocide 2-n-octyl-4-isothiazolin-3-one (OIT) decreased the rate of fluorescence loss. A strong positive correlation between fluorescence and viable yeast cell number was observed and fluorescence was used as a direct indicator of cell viability. The effectiveness of five commercial biocides, commonly incorporated into pPVC at their in-use concentrations, was tested against yeast cells attached to the pPVC surface. The loss of fluorescence and hence viability in situ was quantified using image analysis. The biocides N-(trichloromethylthio) phthalimide (NCMP), 10,10'-oxybisphenoxarsine (OBPA), OIT and 2,3,5,6-tetrachloro-4-(methylsulphonyl) pyridine (TCMP) caused complete loss of fluorescence within 30-50 h. In contrast the biocide dichloro-octyl-isothiazoline caused only 55 +/- 15% fluorescence loss after 50 h. Starvation of the yeast cells in suspension for 24 h prior to attachment reduced their initial sensitivity to OBPA, NCMP, OIT and TCMP by 15-20%, but eventually the fluorescence was also completely lost. CONCLUSIONS: The use of A. pullulans expressing cytosolic GFP enables the in situ quantification of loss of viability when cells are attached to pPVC with incorporated biocides. SIGNIFICANCE AND IMPACT OF THE STUDY: GFP fluorescence was used as a real-time indicator of cell viability and thus can be applied for direct quantification of the effectiveness of a broad range of biocides, incorporated into the polymer mass and used to protect a variety of plastics or other materials from microbial growth.

Antifungal Agents↗

Scanning electron microscopy of bacteria in the apical part of root canals in permanent teeth with periapical lesions.

The most apical 2 mm of the root canals of periapically diseased roots were examined for microorganisms by scanning electron microscopy (SEM). Bacteria in this area were observed in 10 out of 12 (83.3%) cases. The two remaining cases exhibited bacteria more coronally, with tissue remnants between the bacterial front and the apical foramen. Rod-shaped bacteria dominated, but filaments, spirochetes and cocci were also seen. Cocci and rods sometimes formed micro-colonies. Occasionally, cocci were seen attached to filaments forming "corn-cob"-like structures. Deposits resembling bacterial plaque were also found inside the root canal. SEM is useful for studying microbial topography of the apical root canal.

Bacteria↗

Inactivation of Salmonella enterica serovar enteritidis by ultrasonic waves under pressure at different water activities.

The inactivation of Salmonella enterica serovar Enteritidis by ultrasonic waves (20 kHz; 117- microm wavelength) under pressure (175 kPa) at nonlethal temperatures (manosonication [MS]) and lethal temperatures (manothermosonication [MTS]) in media of different water activities has been investigated. Heat decimal reduction time values increased 30 times when the water activity was decreased from nearly 1 to 0.96, but the MS resistance was increased only twofold. The inactivation of Salmonella serovar Enteritidis by ultrasound under pressure at low water activities was a phenomenon of the "all-or-nothing" type. A synergistic lethal effect was observed between heat and ultrasound in media with reduced water activity; the lower the water activity, the greater the synergistic effect. This work could be useful for improving sanitation and preservation treatments of foods, especially those which are sensitive to temperature and those in which components protect microorganisms to heat. It also contributes to our knowledge of microbial inactivation mechanisms by MS and MTS treatments.

Colony Count, Microbial↗

Prediction of microbial growth in fresh-cut vegetables treated with acidic electrolyzed water during storage under various temperature conditions.

Effects of storage temperature (1, 5, and 10 degrees C) on growth of microbial populations (total aerobic bacteria, coliform bacteria, Bacillus cereus, and psychrotrophic bacteria) on acidic electrolyzed water (AcEW)-treated fresh-cut lettuce and cabbage were determined. A modified Gompertz function was used to describe the kinetics of microbial growth. Growth data were analyzed using regression analysis to generate "best-fit" modified Gompertz equations, which were subsequently used to calculate lag time, exponential growth rate, and generation time. The data indicated that the growth kinetics of each bacterium were dependent on storage temperature, except at 1 degrees C storage. At 1 degrees C storage, no increases were observed in bacterial populations. Treatment of vegetables with AcEW produced a decrease in initial microbial populations. However, subsequent growth rates were higher than on nontreated vegetables. The recovery time required by the reduced microbial population to reach the initial (treated with tap water [TW]) population was also determined in this study, with the recovery time of the microbial population at 10 degrees C being <3 days. The benefits of reducing the initial microbial populations on fresh-cut vegetables were greatly affected by storage temperature. Results from this study could be used to predict microbial quality of fresh-cut lettuce and cabbage throughout their distribution.

Bacteria↗

Distribution patterns of Escherichia coli O157:H7 in ground beef produced by a laboratory-scale grinder.

This study determined the distribution patterns of Escherichia coli O157:1H7 in ground beef when a contaminated beef trim was introduced into a batch of uncontaminated beef trims prior to grinding in a small-scale laboratory grinder. A beef trim (15.3 +/- 2 g) was inoculated with a rifampicin-resistant strain of E. coli O157:H7 (E. coli O157:H7rif) and introduced into a stream of noncontaminated beef (322 +/- 33 g) prior to grinding. Seven inoculum levels (6, 5, and 4 total log CFU [high]; and 3, 2, 1, and 0 total log CFU [low]) were studied in triplicate. E. coli O157:H7rif was not detected in 3.1 to 43% of the ground beef inoculated with the high levels or in 3.4 to 96.9% of the ground beef inoculated with the low levels. For all inoculum levels studied, the five ground beef fractions (each 7.8 +/- 0.6 g) with the highest pathogen levels accounted for 59 to 100% of the total pathogens detected. For all inoculum levels, there was a linear relationship between the quantity of ground beef containing E. coli O157:H7rif and the inoculum level. The quantity of E. coli O157:H7rif in the beef remaining in the grinder was proportional to the inoculum level and was related to the location in the grinder. Different components of the grinder accumulated E. coli O157:H7rif in different quantities, with the most significant accumulation being in the nut (collar) that attaches the die to the blade. This study determined specific distribution patterns of E. coli O157:H7rif after the grinding of a contaminated beef trim along with uncontaminated trims, and the results indicate that the grinding operation should be regarded as a means of distribution of microbial contamination in risk analyses of ground beef operations.

Animals↗

The effect of raw milk microbial flora on the sensory characteristics of Salers-type cheeses.

The sensory characteristics of Salers Protected Denomination of Origin raw-milk cheeses are linked to the biochemical composition of the raw material (milk) and to the resultant microbial community. To evaluate the influence of the microbial community on sensory characteristics, Salers-type cheeses were manufactured with the same pasteurized milk, reinoculated with 3 different microbial communities from 3 different filtrates from microfiltered milks. Each cheese was subjected to microbial counts (on selective media), biochemical tests, and volatile and sensory component analyses at different times of ripening. Adding different microbial communities to specimens of the same (biochemically identical) pasteurized milk lead to different sensory characteristics of the cheeses. Cheeses with fresh cream, hazelnut, and caramel attributes were opposed to those with fermented cream, chemical, and garlic flavors. The aromatic compounds identified (esters, acids, alcohols, and aldehydes) in these cheeses were quite similar. Nevertheless, one milk was distinguished by a higher content of acetoin, and lower 2-butanone and 3-methylpentanone concentrations. Over the production period of 1 mo, the different cheeses were characterized by the same balance of the microbial population assessed by microbial counts on different media. This was associated with the stability of some sensory attributes describing these cheeses. Nevertheless, there was no linear correlation between microbial flora data and sensory characteristics as measured in this study.

Acetoin↗

Using real-time PCR to assess changes in the hydrocarbon-degrading microbial community in Antarctic soil during bioremediation.

A real-time polymerase chain reaction (PCR) method to quantify the proportion of microorganisms containing alkane monooxygenase was developed and used to follow changes in the microbial community in hydrocarbon-contaminated Antarctic soil during a bioremediation field trial. Assays for the alkB and rpoB genes were validated and found to be both sensitive and reproducible (less than 2% intrarun variation and 25-38% interrun variation). Results from the real-time PCR analysis were compared to analysis of the microbial population by a culture-based technique [most probable number (MPN) counts]. Both types of analysis indicated that fertilizer addition to hydrocarbon-contaminated soil stimulated the indigenous bacterial population within 1 year. The proportion of alkB containing microorganisms was positively correlated to the concentration of n-alkanes in the soil. After the concentration of n-alkanes in the soil decreased, the proportion of alkane-degrading microorganisms decreased, but the proportion of total hydrocarbon-degrading microorganisms increased, indicating another shift in the microbial community structure and ongoing biodegradation.

Antarctic Regions↗