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Bacteria and Archaea physically associated with Gulf of Mexico gas hydrates.

Although there is significant interest in the potential interactions of microbes with gas hydrate, no direct physical association between them has been demonstrated. We examined several intact samples of naturally occurring gas hydrate from the Gulf of Mexico for evidence of microbes. All samples were collected from anaerobic hemipelagic mud within the gas hydrate stability zone, at water depths in the ca. 540- to 2,000-m range. The delta(13)C of hydrate-bound methane varied from -45.1 per thousand Peedee belemnite (PDB) to -74.7 per thousand PDB, reflecting different gas origins. Stable isotope composition data indicated microbial consumption of methane or propane in some of the samples. Evidence of the presence of microbes was initially determined by 4,6-diamidino 2-phenylindole dihydrochloride (DAPI) total direct counts of hydrate-associated sediments (mean = 1.5 x 10(9) cells g(-1)) and gas hydrate (mean = 1.0 x 10(6) cells ml(-1)). Small-subunit rRNA phylogenetic characterization was performed to assess the composition of the microbial community in one gas hydrate sample (AT425) that had no detectable associated sediment and showed evidence of microbial methane consumption. Bacteria were moderately diverse within AT425 and were dominated by gene sequences related to several groups of Proteobacteria, as well as Actinobacteria and low-G + C Firmicutes. In contrast, there was low diversity of Archaea, nearly all of which were related to methanogenic Archaea, with the majority specifically related to Methanosaeta spp. The results of this study suggest that there is a direct association between microbes and gas hydrate, a finding that may have significance for hydrocarbon flux into the Gulf of Mexico and for life in extreme environments.

Archaea↗

Pharmaceutical container/closure integrity. III: Validation of the helium leak rate method for rigid pharmaceutical containers.

Validation of a helium leak rate method for pharmaceutical container/closure integrity quality assurance required the demonstration that this physical testing method was as good or better than microbial immersion challenge testing in detecting potential integrity failures. One lot of rubber-stoppered, broth-filled glass vials also containing defective vials with known leaks were subjected to both helium leak rate and microbial challenge testing. The defective vials were prepared by affixing glass micropipettes (0.1 to 10 microns) into the vial side walls. The validation lot included a 10% seeded defect rate of which about 50% contained leaks with a predicted probability of failing a microbial challenge (> 10%). Helium tracer was placed in the test units by charging them for 4 hours under a 40 psi helium pressure. The critical leak rate after charging was determined to be 10(-7) standard cc/second, and test units with measured leak rates greater than this value were considered helium leak rate failures. Microbial immersion challenge was conducted by exposing the test units in a bath inoculated with 10(9-10) viable E. coli and B. diminuta organisms for 24 hours followed by a 13 day (35 degrees C) incubation. Microbial failures were determined visually. The helium and microbial leak test methods were compared statistically using mean failure rates. The mean helium failure rate was 6.9%, whereas the mean microbial failure rate was 2.8%. The difference between helium and microbial failure rates was significantly greater than zero. Thus, helium leak rate testing was demonstrated to be a suitable pharmaceutical container/closure integrity method for microbial quality assurance of rigid containers.

Drug Packaging↗

Isolation of a Bacillus sp. capable of transforming isoeugenol to vanillin.

Natural aroma compounds are of major interest to the flavor and fragrance industry. Due to the limited sources for natural aromas, there is a growing interest in developing alternative sources for natural aroma compounds, and in particular aromatic aldehydes. In several microbial species aromatic aldehydes are detected as intermediates in the degradation pathway of phenylpropanoids. Thus, bioconversion of phenylpropanoids is one possible route for the production of these aroma compounds. The present work describes the isolation of microbial strains, capable of producing vanillin from isoeugenol. Bacterial strains isolated from soil, were screened for their ability to transform isoeugenol to vanillin. One of these strains, strain B2, was found to produce high amounts of vanillin when grown in the presence of isoeugenol, and was also capable of growing on isoeugenol as the sole carbon source. Based on its fatty acids profile, strain B2 was identified as a Bacillus subtilis sp. The bioconversion capabilities of strain B2 were tested in growing cultures and cell free extracts. In the presence of isoeugenol, a growing cultures of B. subtilis B2 produced 0.61 g l-1 vanillin (molar yield of 12.4%), whereas cell free extracts resulted in 0.9 g l-1 vanillin (molar yield of 14%).

Bacillus subtilis↗

[Detection of low lipase activities in food].

A spectrofluorimetric procedure using 4-methylumbelliferyl esters as substrates was developed for the determination of lipase activity in foods. It was tested with among others whey and egg powders, almonds, pepper, paprika and cinnamon. In addition, some lipase preparations of microbial origin were analyzed. The detection limit of the method is one to two orders of magnitude lower than that of a test paper which contains indoxyl acetate as the substrate.

Dietary Proteins↗

Microbiological status of commercially available medicinal herbal drugs--a screening study.

One hundred and thirty-eight medicinal herbal drugs obtained from different suppliers were examined for microbial contaminants and for the detectability of pathogenic microorganisms. For this purpose, several microbiological standard parameters (total aerobic mesophilic count, enterobacteria, coliforms, aerobic sporeformers, yeasts and moulds, enterococci, lactobacilli, pseudomonades and aeromades) and selective methods for the detection of indicator microorganisms and pathogens (E. coli, enterohaemorrhagic E. coli [EHEC], Salmonella, Campylobacter jejuni, Pseudomonas aeruginosa, Bacillus cereus, Clostridium perfringens, Listeria, coagulase-positive staphylococci, Candida albicans, potentially aflatoxigenic moulds) were applied. The microbial load of the samples varied considerably. While none of the samples contained EHEC, Salmonellae, Pseudomonas aeruginosa, Listeriae, Staphylococcus aureus or Candida albicans, four samples were E. coli positive, two samples were presumptively Campylobacter jejuni positive and nine herbal drugs contained a potentially aflatoxigenic mould flora. Further details regarding different viable count classes as well as preparation techniques are discussed.

Bacteria↗

Microbial contamination potential of sterile disposable plastic syringes.

The contamination potential of sterile disposable plastic syringes was evaluated after subjecting the syringes to both simulated in-use conditions and an intentional microbial challenge. Lots of 20 Luer-lock syringes in 10 or 12-cu cm and 20- or 30-cu cm sizes from three manufacturers were tested. Sampling was conducted using 30-ml vials of sterile aerobic culture media containing 14C-labeled substrates. Microbial contamination was confirmed by both visual observation of the turbidity caused by colonization and instrumental detection of 14CO2 caused by the microbial metabolism of 14C-substrates. No contamination of 120 samples was found after the ribbed plunger shaft was grasped by a bare, unprepared dry hand during five cycles of filling and injecting the medium into the vials without removing the needles from the stoppers. When this sampling technique was applied to the syringes inoculated on the upper piston surface with Bacillus subtilis suspension, a 100% contamination rate was observed in 120 samples each under both positive and negative in-vial pressure. Grasping the ribbed plunger shaft of disposable plastic syringes with a dry bare hand did not compromise the sterility of the syringe contents in this study; however, this practice should be avoided when possible. Personnel should absolutely avoid introducing fluid-borne microbial contaminants into the distal barrel end of these syringes because the contents are readily labile to contamination under these conditions.

Bacillus subtilis↗

Microbial transaminase activities and their relationship with bovine rumen metabolites.

Two each adult male crossbred cattle and murrah buffalo were fed a diet of alfalfa hay, chopped wheat straw, and concentrate mixture. Total rumen transaminase activity of cattle was higher than that of buffalo. Rumen protozoal fractions showed higher total transaminase activity than bacterial fractions in both ruminant species. Besides generally studied glutamate oxalacetate transaminase and glutamate pyruvate transaminase, a large number of other microbial transaminases also have been detected in the rumen of both the ruminant species. Bacterial fractions of rumen liquor were devoid of transaminases utilizing tryptophan, threonine, and lysine as their substrates. Ruminal ammonia and nonprotein nitrogen were correlated positively with microbial transaminases in both species. Transamination reactions may be important for assimilation of ruminal ammonia to cellular proteins.

Alanine Transaminase↗

Use of viral pathogens and indicators to differentiate between human and non-human fecal contamination in a microbial source tracking comparison study.

Assays for the detection and typing of adenoviruses, enteroviruses and F+ specific coliphages were performed on samples created as part of a national microbial source tracking methods comparison study. The samples were created blind to the researchers, and were inoculated with a variety of types of fecal contamination source (human, sewage, dog, seagull and cow) and mixtures of sources. Viral tracer and pathogen assays demonstrated a general ability to discriminate human from non-human fecal contamination. For example, samples inoculated with sewage were correctly identified as containing human fecal contamination because they contained human adenovirus or human enterovirus. In samples containing fecal material from individual humans, human pathogen analysis yielded negative results probably because the stool samples were taken from healthy individuals. False positive rates for the virus-based methods (0-8%) were among the lowest observed during the methods comparison study. It is suggested that virus-based source tracking methods are useful for identification of sewage contamination, and that these methods may also be useful as an indication of the public health risk associated with viral pathogens. Overall, virus-based source tracking methods are an important approach to include in the microbial source tracking 'toolbox'.

Adenoviridae↗

A rapid method for screening for Salmonella typhimurium in a chicken cecal microbial consortium using gene amplification.

A rapid sample processing method has been developed to detect low numbers of Salmonella typhimurium in a chicken cecal microbial consortium. Using phoP-specific primers under stringent amplification conditions and gene probe analysis, fewer than 100 colony-forming units (CFUs) were detectable when pure cultures were employed. When the polymerase chain reaction assay was run on cecal contents from birds infected with S. typhimurium, only positive cecal samples containing as few as 700 CFUs reacted to the assay, and the negative bird samples reacted only when the samples were spiked with S. typhimurium cells. The method employed for sample processing is simple and provides a sensitive means of detecting S. typhimurium-specific sequences in trace amounts in the presence of mixed chicken cecal microbial populations.

Animals↗

Microbial associations of 4 putative periodontal pathogens in Sudanese adult periodontitis patients determined by DNA probe analysis.

The few previous cultivation studies on the in vivo associations between various periodontal microbial species have shown several positive and negative associations. The present investigation utilized DNA probe analysis to examine possible in vivo associations between the periodontal pathogens Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, and Bacteroides forsythus in subgingival plaque samples obtained from 25 Sudanese untreated adult periodontitis patients. The standard paper point technique was used to sample 99 sites with a mean probing depth of 6.8 mm (range 6.0 to 10.0). Microbial associations were determined by detecting the effect of the presence or absence of one species (effector) on the occurrence of the other 3 species (target). The Wilcoxon signed rank test was used to examine variations in occurrence of each target bacteria in the presence or absence of the effector. In addition, the Spearman's rank correlation test was used to assess the relationship between the level of each bacteria to that of the other 3. Results showed bacterial associations with the following effector-on-target effects: F. nucleatum (P < 0.01 Wilcoxon; P < 0.001 Spearman) >> P. gingivalis (P < 0.01 Wilcoxon; P < 0.001 Spearman), and B. forsythus (P < 0.05 Wilcoxon; P < 0.001 Spearman) > P. intermedia (P < 0.01 Spearman). The study demonstrated positive associations between the 4 species investigated, while no neutral or negative associations were revealed. The most striking finding was the effect exerted by F. nucleatum on the colonization of P. intermedia; P. intermedia was never detected in a site unless F. nucleatum was also present.

Adult↗

Enzymatic conversion-based method for screening cyclic dipeptide-producing microbes.

We developed a method for screening cyclic dipeptide-producing microbes by enzymatic conversion. In this method, cyclic dipeptides are detected by the combination of: (i) conversion of cyclic dipeptides to dehydro cyclic dipeptides by cyclo(Leu-Phe) oxidase and (ii) detection of the dehydro derivative by UV spectrophotometry using TLC or HPLC analysis based on the absorbance change caused by the conversion. Using this method, the actinomycete strain A8 was isolated as a cyclic dipeptide-producing strain. The cyclic dipeptides were purified from the microbial extract by enzymatic detection-guided fractionation, and their structures were determined to be cyclo(L-Phe-L-Pro) and cyclo(L-Pro-L-Tyr) by spectroscopic methods.

Actinobacteria↗

Identification of oral spirochetes at the species level and their association with other bacteria in endodontic infections.

OBJECTIVES: Recent molecular approaches have revealed that fastidious organisms such as Bacteroides forsythus and oral treponemes were frequently found in root canals with apical periodontitis. The purpose of this study was to identify the isolates of oral spirochetes at the species level in endodontic infections and to determine their association with B forsythus and Porphyromonas gingivalis. STUDY DESIGN: Seventy-nine teeth with apical periodontitis were selected for this study. After sampling from the root canals aseptically, polymerase chain reaction amplification for the 16S rRNA gene was performed with eubacterial universal primers. Subsequently, dot-blot hybridization was performed with 8 species-specific oligonucleotide probes. The microbial associations were analyzed by using the odds ratio. RESULTS: The most frequently found species was P gingivalis (27.4%), followed by Treponema maltophilum (26%), B forsythus (16.4%), and Treponema socranskii (2.7%). Other treponemes, including Treponema denticola, were not detected in our samples. Significant microbial associations were identified between T maltophilum, B forsythus, and P gingivalis by performing analysis with the odds ratio. CONCLUSION: Our results indicate that T maltophilum should be included in etiologic studies of endodontic diseases.

Adolescent↗

Determination of fluoroquinolone residues in animal tissues using Escherichia coli as indicator organism.

Three strains of Escherichia coli (ATCC 128, 10536, and 25922) and one strain of Bacillus subtilis (ATCC 3491) were compared as indicator microorganisms in microbial inhibition tests for their ability to detect fluoroquinolone residues. E. coli strains 128 and 10536 were most susceptible to fluoroquinolone residues, with detection limits of 35-50 micrograms/kg for enrofloxacin. Of the 2 strains, E. coli 10536 was slightly less susceptible. Ciprofloxacin was detected consistently by E. coli 128 at 30 micrograms/kg. Other fluoroquinolone drugs of veterinary interest detected by E. coli 128 were sarafloxacin and difloxacin at 100-250 micrograms/kg concentration. E. coli 25922 yielded 100% sensitivity in detection of enrofloxacin only at the 250 micrograms/kg concentration, and ciprofloxacin and sarafloxacin at 200 micrograms/kg. B. subtilis detected only enrofloxacin 100% of the time at 250 micrograms/kg. The E. coli strains tested were insensitive to other antibacterials commonly used in animals, with the exception of ceftiofur which was detected by E. coli 128 and 10536 at 500 micrograms/kg. The B. subtilis strain was not effective in detecting the fluoroquinolone drugs, whereas the E. coli strains were selective for the fluoroquinolones. E. coli 128 was 100% effective in detecting enrofloxacin and ciprofloxacin in spiked diaphragm homogenate samples at 50 micrograms/kg. Of the microorganisms tested, E. coli strain ATCC 128 was highly suitable as an indicator microorganism in a microbial inhibition assay for selective detection of fluoroquinolone antibacterial residues in animal tissues.

Animals↗

Microbial activity in soils following steam treatment.

Steam enhanced extraction (SEE) is an aquifer remediation technique that can be effective at removing the bulk of non-aqueous phase liquid (NAPL) contamination from the subsurface, particularly highly volatile contaminants. However, low volatility compounds such as polynuclear aromatic hydrocarbons (PAHs) are less efficiently removed by this process. This research evaluated the effects of steam injection on soil microbial activity, community structure, and the potential for biodegradation of contaminants following steam treatment. Three different soils were evaluated: a laboratory-prepared microbially-enriched soil, soil from a creosote contaminated field site, and soil from a chlorinated solvent and waste oil contaminated field site. Results from field-scale steaming are also presented. Microbial activity before and after steam treatment was evaluated using direct epifluorescent microscopy (DEM) using the respiratory activity dye 5-cyano-2,3, ditolyl tetrazolium chloride (CTC) in conjunction with the fluorochrome 5-(4,6-dichlorotriazinyl) aminofluorescein (DTAF) to yield a quantitative assessment of active and total microbial numbers. DEM results indicate that steamed soils that were analyzed while still hot exhibited microbial activity levels that were below detection. However, soil samples that were slowly cooled, more closely reflecting the conditions of applied SEE, exhibited microbial activity levels that were comparable to presteamed soils. Samples from a field-site where steam was applied continuously for 6 weeks also showed high levels of microbial activity following cooling. The metabolic capabilities of the steamed communities were investigated by measuring cell growth in enrichment cultures on various substrates. These studies provided evidence that organisms capable of biodegradation were among the mesophilic populations that survived steam treatment. Fluorescent in situ hybridization (FISH) analysis of the soils with domain-level rRNA probes suggest that both Archaea and Bacteria survived steam exposure.

Archaea↗

Biomass and porosity profiles in microbial granules used for aerobic wastewater treatment.

AIMS: To obtain biomass and porosity profiles for aerobically grown granules of different diameters and to determine a suitable range of granule diameters for application in wastewater treatment. METHODS AND RESULTS: Microbial granules were cultivated in an aerobic granulated sludge reactor with model wastewaters containing acetate, or ethanol plus acetate, or glucose as the main carbon source. Granules were formed by retaining microbial aggregates using a settling time of 2 min. Sampled granules had diameters ranging from 0.45 to 3 mm. Microbial biomass in the granules was detected with the nucleic acid stain SYTO 9 and confocal laser scanning microscopy. The thickness of the microbial biomass layer was proportional to the granule diameter, and had a maximum value of 0.8 mm. The thickness of the microbial biomass layer correlated with the penetration depth of 0.1 microm fluorescent beads into the granule. CONCLUSIONS: The microbial biomass and porosity studies suggest that aerobically grown microbial granules should have diameters less than a critical diameter of 0.5 mm, if deployed for wastewater treatment applications. This critical diameter is based on the assumption that whole granules should have a porous biomass-filled matrix. SIGNIFICANCE AND IMPACT OF THE STUDY: This work could contribute to the development of aerobic granulation technology for effective biological wastewater treatment.

Aerobiosis↗

Supplemental protein sources for steers fed corn-based diets: I. Ruminal characteristics and intestinal amino acid flows.

Five cannulated Holstein steers (302 +/- 23 kg) were used in a 4 x 4 Latin square design experiment with extra observations to evaluate the effect of supplemental protein source on postruminal flow and intestinal disappearance of N and amino acids (AA). Diets were formulated to contain 12.5% CP using cracked corn (70%), ground corn cobs (15%), and supplement (15%). Except for an all-urea supplemented control diet, 40% of the total dietary CP was supplied by soybean meal (SBM), a high ruminal escape soybean meal (SoyPLUS; SP), or a corn gluten meal/blood meal combination (CB; 50:50 on a CP basis). The steers were fed twice daily and DMI was restricted to 2.0% of initial BW. Supplementation with CB increased (P < .10) non-microbial N flow at the duodenum but tended to decrease microbial N flow such that no differences (P > .10) in total N flows at the duodenum were detected among treatments. The efficiency of microbial protein synthesis was not affected by treatment but was greatest for urea/and tended to decrease when SP and CB were fed (21.8, 20.5, 19.5, and 15.7 g of N/kg of OM truly digested for urea, SBM, SP, and CB, respectively). A possible shortage of ruminally degradable N, as evidenced by low ruminal NH3 N concentrations (3.6, 4.2, 3.9, and 2.1 mg/dL for urea, SBM, SP, and CB respectively), or other factors, may have limited microbial protein synthesis. However, microbial N flows averaged 60.0% of duodenal N flow across treatments. Duodenal flow of essential, nonessential, and total AA were similar (P > .10) among treatments and averaged 293, 361, and 653 g/d, respectively). Lysine and methionine flows did not differ (P > .10) and averaged 41.6 and 13.3 g/d, respectively. The source of supplemental protein had no impact (P > .10) on small intestinal disappearance of AA or total tract N disappearance. Feeding proteins resistant to ruminal degradation may have limited microbial protein production, resulting in a shift in the proportion of metabolizable protein arriving at the small intestine from dietary and microbial origins. These results suggest that corn-based diets may be limiting in ruminally degradable N, especially when high ruminal escape protein sources are fed as supplemental CP.

Amino Acids↗

Development of steady-state diffusion gradients for the cultivation of degradative microbial consortia.

A diffusion gradient plate was constructed and evaluated for its potential use in the isolation of degradative microbial consortia from natural habitats. In this model, a steady-state concentration gradient of diclofop methyl, established by diffusion through an agarose gel, provided the carbon for microbial growth. Colonization of the gel surface was observed with epifluorescence and scanning confocal laser microscopy to determine microbial responses to the diclofop gradient. A detectable gradient developed over a narrow band (<10 mm). Consequently, quantitative analyses of the microbial response to the gradient were difficult to obtain. A two-dimensional, finite-element numerical transport model for advective-diffusive transport was used to simulate concentration and flux profiles in the physical model. The simulated profiles were correlated with the measured concentration gradient (R = 0.89) and the cell numbers on the gel surface (R = 0.85). The numerical model was subsequently used to redesign the physical model. The detectable concentration gradient in the modified physical model extended over the length of the gel (38 mm). The simulated profile again showed a good correlation with the measured profile (R = 0.96) and the microbial responses to the concentration gradient (R = 0.99). It was concluded that these gradients provide the steady-state environments needed to sustain steady-state consortia. They also provide a physical pathway for the development of degradative biofilms from low to high concentrations of toxicants and simulate conditions under which low concentrations of toxicant are supplied at a constant flux over long periods of time, such as the conditions that could occur in natural environments.

Journal Article↗

Development of an agar lift-DNA/DNA hybridization technique for use in visualization of the spatial distribution of Eubacteria on soil surfaces.

While microbial growth is well-understood in pure culture systems, less is known about growth in intact soil systems. The objective of this work was to develop a technique to allow visualization of the two-dimensional spatial distribution of bacterial growth on a homogenous soil surface. This technique is a two-step process wherein an agar lift is taken and analyzed using a universal gene probe. An agar lift is comprised of a thin layer of soil that is removed from a soil surface using an agar slab. The agar is incubated to allow for microbial growth, after which, colonies are transferred to a membrane for conventional bacterial colony DNA/DNA hybridization analysis. In this study, a eubacterial specific probe was used to demonstrate that growing bacterial populations on soil surfaces could be visualized. Results show that microbial growth and distribution was nonuniform across the soil surface. Spot supplementation of the soil with benzoate or glucose resulted in a localized microbial growth response. Since only growing colonies are detected, this technique should facilitate a greater understanding of the microbial distribution and its response to substrate addition in more heterogenous soil systems.

Agar↗