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PubMed1997

Codon-anticodon assignment and detection of codon usage trends in seven microbial genomes.

We have assigned codon-anticodon recognition patterns for the whole set of transfer RNAs of Haemophilus influenzae Rd, Methanococcus jannaschii, and Synechocystis sp. PCC6803 using sequence information derived from the complete genome sequence of these organisms and have tabulated them along with those previously reported for Escherichia coli, Mycoplasma genitalium, Mycoplasma pneumoniae, and Saccharomyces cerevisiae. Using the resulting codon-anticodon tables, the bias in codon usage of genes encoding the entire protein and ribosomal protein complement of each of the seven microbial genomes was analyzed. Then, the codon adaptation index (CAIrp) for each protein gene was calculated using the codon usage preference of the ribosomal protein genes of the corresponding organism. Of the seven genomes examined, six showed CAIrp scores that roughly coincided with the expected level of gene expression. The result demonstrates that CAIrp analysis may be useful for prediction of the expression level of unknown genes when all or at least considerable portions of the genome sequence are available.

Codon↗
PubMed2000

Expression of nifH genes in natural microbial assemblages in Lake George, New York, detected by reverse transcriptase PCR.

A modified nested reverse transcriptase PCR (RT-PCR) method was used to detect the expression of nitrogenase genes in meso-oligotrophic Lake George, New York. Net (>20-microm pore size) plankton samples collected from two sites (Dome Island and Hague Marina) were extracted for total RNA and genomic DNA to determine the identity of diazotrophic organisms that were present and those that were actively expressing nitrogenase genes. Phylogenetic analysis of individual sequences cloned from PCR amplifications showed that there were phylogenetically diverse groups of bacteria that possessed a nifH gene, including representatives of unicellular and filamentous cyanobacteria, the alpha- and gamma-subdivisions of the division Proteobacteria (alpha- and gamma-proteobacteria), and a previously undefined group of bacteria. The phylotypes cloned from RT-PCR amplifications, which were actively expressing nifH transcripts, clustered with the unicellular and filamentous cyanobacteria, alpha-proteobacteria, and the novel bacterial cluster. No bacterial sequences were found which clustered with sequences from cluster II (alternative nitrogenases), III (nitrogenases in strict anaerobes), or IV (nifH-like sequences). These results indicate that there were several distinct groups of nitrogen-fixing microorganisms in the net plankton from both sampling sites and that most of the groups had representative phylotypes that were actively expressing nitrogenase genes.

Alphaproteobacteria↗
PubMed2002

Phylogenetic characterization of microbial mats and streamers from a Japanese alkaline hot spring with a thermal gradient.

Dense microbial mats and streamers of various colors developed in an alkaline-hot spring water at 48-76 degrees C and ~0.077 mm sulfide in Nakabusa, Japan. The microbial community structures with a thermal gradient were compared by denaturing gradient gel electrophoresis (DGGE) analysis of the PCR-amplified 16S ribosomal RNA gene fragments. The sequence analysis revealed that a predominant cyanobacterial DGGE band phylogenetically related to Synechococcus elongatus was detected only from green mats at 48 degrees C. Four DGGE bands were detected commonly from green mats at 48 degrees C, orange mats at 58 degrees C and brown mats at 60 degrees C. The sequence analysis revealed that these were phylogenetically related to Chloroflexaceae group, Rhodothermus group, a candidate division OP10, and an unclassified bacterium. On the other hand, Aquificae-, Thermodesulfobacteria-, Thermus group-, and Crenarchaeota-like sequences were detected as a predominant component of DGGE profiling from the streamers only at temperatures over 66 degrees C, but no phototrophic bacterial bands were detected. Thus, the microbial community structure above 60 degrees C was drastically different from that at the lower temperatures. After the addition of hydrogen into in vitro gray streamers with in situ spring water, sulfide production markedly occurred in the presence of ambient sulfate at 66 degrees C. This result suggests that in situ sulfide is partly produced by Thermodesulfobacteria-like sulfate-reducing bacteria in the streamers.

Bacteria↗
PubMed1999

Inhibition of potassium transport and growth of mycobacteria exposed to clofazimine and B669 is associated with a calcium-independent increase in microbial phospholipase A2 activity.

Altered phospholipase A2 (PLA2) activity and its relationship to cation (K+, Ca2+) uptake and growth were investigated in mycobacteria exposed to the riminophenazine antimicrobial agents, clofazimine and B669 (0.15-2.5 mg/L). Microbial PLA2 activity was measured using a radiometric thin-layer chromatography procedure, whereas K+ and Ca2+ transport were measured using 86Rb+ or 42K+ and 45Ca2+, respectively. Short-term exposure (15-30 min) of Mycobacterium aurum A+ or the virulent and avirulent isolates of Mycobacterium tuberculosis H37R to the riminophenazines resulted in dose-related enhancement of microbial PLA2 activity, which was associated with inhibition of K+ influx and growth. Uptake of Ca2+ by mycobacteria was unaffected, or minimally affected, by the riminophenazines at concentrations of < or = 0.6 mg/L, whereas higher concentrations resulted in increased uptake of the cation in the setting of decreased microbial ATP concentrations. The results of kinetic studies using a fixed concentration (2.5 mg/L) of B669 demonstrated that riminophenazine-mediated enhancement of PLA2 activity and inhibition of K+ uptake in mycobacteria are rapid and probably related events that precede, by several minutes, any detectable effects on microbial ATP concentrations and uptake of Ca2+. Inclusion of the extracellular and intracellular Ca2+-chelating agents EGTA (0.2-7.2 g/L) and BAPTA/FURA-2 (0.2-9.5 mg/L), individually or in combination, did not prevent the effects of B669 on mycobacterial PLA2 activity or K+ transport, whereas alpha-tocopherol, which neutralizes PLA2 primary hydrolysis products, antagonized the inhibitory effects of the riminophenazines on microbial K+ uptake and growth. These results demonstrate that the antimycobacterial activities of clofazimine and B669 are related to a Ca2+-independent increase in mycobacterial PLA2, leading to interference with microbial K+ transport.

Adenosine Triphosphate↗
PubMed1985

Comparison of the Isolator 1.5 Microbial Tube with a conventional blood culture broth system for detection of bacteremia in children.

A lysis direct plating technique, utilizing the Isolator 1.5 Microbial Tube (Du Pont Company) was compared to a conventional two-bottle culture system during a prospective study conducted in a children's hospital. Specimens were processed so that the amounts of blood allocated to each conventional blood culture bottle and the Isolator tube were as equivalent as possible. Of the 1,823 specimens enrolled, 93 (5.1%) yielded growth of 102 clinically significant isolates. Lysis direct plating and conventional blood culture bottles detected 85 and 89 of the isolates, respectively. No statistically significant differences were observed between the two methods in the overall efficiency of organism recovery or in the detection of any specific organism. Contamination rates during the entire study were 14.1% and 3.6% for lysis direct plating and conventional blood culture bottles, respectively. The mean time to availability of colonies of clinically significant isolates from specimens positive by both methods was 29 hr and 35 hr for lysis direct plating and conventional blood culture bottles, respectively. We conclude that lysis direct plating offers an attractive alternative to conventional blood culture bottles for the processing of blood cultures from pediatric patients.

Bacteria↗
PubMed2001

Evaluation of microbial proteolysis in meat products by capillary electrophoresis.

AIMS: The available methods for evaluating proteolysis in meat products, particularly the contribution of micro-organisms, are expensive, time-consuming and require an unacceptable sample size. To minimize these problems, two capillary electrophoresis-based methods have been developed. METHODS AND RESULTS: Six Gram-positive, catalase-positive cocci, four moulds and three yeasts, isolated from dry-cured ham, were tested on sterile pork slices. Using the Capillary Gel Electrophoresis (CGE) method, changes in sarcoplasmic and myofibrillar proteins due to endogenous and microbial enzymes were detected. The Capillary Zone Electrophoresis (CZE) analysis allowed evaluation of bulk changes by micro-organisms in soluble nitrogen compounds. CONCLUSION: CGE analysis of myofibrillar proteins and CZE determination of soluble nitrogen compounds have proved to be valuable tools for evaluating proteolytic activity of endogenous and microbial origin. SIGNIFICANCE AND IMPACT OF THE STUDY: The CGE and CZE methods developed can be used for a rapid and sensitive analysis of proteolysis in meat products.

Animals↗
PubMedDate not supplied

A screening method for beta-lactams in tissues hydrolyzed with penicillinase I and lactamase II.

Antibiotic residues above tolerance levels are not allowed in foods derived from farm animals. Microbial inhibition assays are used to screen antibiotics in U.S. regulatory laboratories. We developed a screening approach to classify beta-lactams through selective hydrolysis of the beta-lactam ring with Penase or lactamase II, thereby inactivating the beta-lactam activity. Optimum conditions for hydrolysis of beta-lactams with Penase and lactamase II were determined. beta-Lactams were detected by a microbial inhibition assay and with enzyme-linked immunosorbent assays before and after hydrolysis. beta-Lactams (10-100 ppb) were spiked in kidney extracts and hydrolyzed. Results indicate a pattern that tentatively classified the beta-lactams into 3 subgroups. Desfuroyl-ceftiofur-cysteine, a major metabolite of ceftiofur, was clearly detected. Penicillin G, ampicillin, amoxicillin, and cloxacillin were distinguishable from cephapirin, ceftiofur metabolite, and high levels of hetacillin. Liver and kidney tissue samples were analyzed with the combined enzyme hydrolysis and screening assays, which tentatively identified the residues. This approach can speed up screening analysis of beta-lactam residues prior to identification and quantitation by chromatographic analysis, thus enhancing positive identification of residues to provide a safer food supply.

Anti-Bacterial Agents↗
PubMed2001

Soft deposits, the key site for microbial growth in drinking water distribution networks.

In this project we studied the microbiological quality of soft pipeline deposits removed from drinking water distribution networks during mechanical cleaning. Drinking water and deposit samples were collected from 16 drinking water distribution networks located at eight towns in different parts of Finland. Soft pipeline deposits were found to be the key site for microbial growth in the distribution networks. The microbial numbers in the soft deposits were significantly higher than numbers in running water. The highest microbial numbers were detected in the main deposit pushed ahead by the first swab. The deposits contained high numbers of heterotrophic bacteria, actinomycetes and fungi. Also coliform bacteria were often isolated from deposit samples. Manganese and copper in the deposits correlated negatively with the numbers of heterotrophic bacteria. After a year, the viable microbial numbers in the new deposits were almost as high as in the old deposits before the first mechanical cleaning. The bacterial biomass production was higher in the new than in the old deposits.

Bacteria↗
PubMed2002

Effects of above-ground plant species composition and diversity on the diversity of soil-borne microorganisms.

A coupling of above-ground plant diversity and below-ground microbial diversity has been implied in studies dedicated to assessing the role of macrophyte diversity on the stability, resilience, and functioning of ecosystems. Indeed, above-ground plant communities have long been assumed to drive below-ground microbial diversity, but to date very little is known as to how plant species composition and diversity influence the community composition of micro-organisms in the soil. We examined this relationship in fields subjected to different above-ground biodiversity treatments and in field experiments designed to examine the influence of plant species on soil-borne microbial communities. Culture-independent strategies were applied to examine the role of wild or native plant species composition on bacterial diversity and community structure in bulk soil and in the rhizosphere. In comparing the influence of Cynoglossum officinale (hound's tongue) and Cirsium vulgare (spear thistle) on soil-borne bacterial communities, detectable differences in microbial community structure were confined to the rhizosphere. The colonisation of the rhizosphere of both plants was highly reproducible, and maintained throughout the growing season. In a separate experiment, effects of plant diversity on bacterial community profiles were also only observed for the rhizosphere. Rhizosphere soil from experimental plots with lower macrophyte diversity showed lower diversity, and bacterial diversity was generally lower in the rhizosphere than in bulk soil. These results demonstrate that the level of coupling between above-ground macrophyte communities and below-ground microbial communities is related to the tightness of the interactions involved. Although plant species composition and community structure appear to have little discernible effect on microbial communities inhabiting bulk soil, clear and reproducible changes in microbial community structure and diversity are observed in the rhizosphere.

Bacteria↗
PubMed2006

Microbial diversity of cold-seep sediments in Sagami Bay, Japan, as determined by 16S rRNA gene and lipid analyses.

Microbial communities in Calyptogena sediment and microbial mats of Sagami Bay, Japan, were characterized using 16S rRNA gene sequencing and lipid biomarker analysis. Characterization of 16S rRNA gene isolated from these samples suggested a predominance of bacterial phylotypes related to Gammaproteobacteria (57-64%) and Deltaproteobacteria (27-29%). The Epsilonproteobacteria commonly found in cold seeps and hydrothermal vents were only detected in the microbial mat sample. Significantly different archaeal phylotypes were found in Calyptogena sediment and microbial mats; the former contained only Crenarchaeota clones (100% of the total archaeal clones) and the latter exclusively Euryarchaeota clones, including the anaerobic oxidation of methane archaeal groups ANME-2a and ANME-2c. Many of these lineages are as yet uncultured and undescribed groups of bacteria and archaea. Phospholipid fatty acid analysis suggested the presence of sulphate-reducing and sulphur-oxidizing bacteria. Results of intact glyceryl dialkyl glyceryl tetraether lipid analysis indicated the presence of nonthermophilic marine planktonic archaea. These results suggest that the microbial community in the Sagami Bay seep site is distinct from previously characterized cold-seep environments.

Bacteroides↗
PubMed2003

The early increase in intestinal permeability and systemic endotoxin exposure in patients with severe acute pancreatitis is not associated with systemic bacterial translocation: molecular investigation of microbial DNA in the blood.

INTRODUCTION: Sepsis is the main cause of late mortality in patients with severe acute pancreatitis and is largely attributed to secondary infection of pancreatic necrosis with gram-negative enteric organisms. This is commonly preceded by a significant increase in intestinal colonization with such microbes and with early increases in intestinal permeability, thus suggesting a mechanism of bacterial translocation. Whilst cultures of blood specimens from these patients often remain sterile, it is conceivable that bacteria might translocate in small volumes with detrimental effects but elude detection by standard microbial culture techniques. AIMS: To investigate the incidence and frequency with which bacterial DNA may exist in the systemic circulation of patients with acute pancreatitis and to relate that to disease severity, changes in intestinal permeability, and systemic endotoxin exposure. METHODOLOGY: Blood samples were obtained at admission and on days 3 and 7 from 26 patients with acute pancreatitis (seven with severe cases) and from 10 healthy controls for DNA extraction and standard microbial cultures. Polymerase chain reaction techniques were used to amplify a gene region (16S ribosomal RNA) found in all bacteria. Levels of serum endotoxin and antibodies to endotoxin core (EndoCAb) were measured at admission, and intestinal permeability to the macromolecule polyethylene glycol 3350 was determined within 72 hours of the onset of symptoms. RESULTS: Blood cultures yielded and enterococci for one patient with a severe attack and coagulase-negative staphylococci for another patient with a mild attack. No bacterial DNA was found in any of the samples. Endotoxemia was detected in 20 patients (five with severe cases), and levels of serum IgM EndoCAb were depleted in patients with severe attacks but remained relatively unchanged during mild attacks (p = 0.033). Intestinal permeability was significantly increased in patients with severe attacks of acute pancreatitis but remained unchanged during mild attacks (p < 0.05). CONCLUSIONS: Whilst severe attacks of acute pancreatitis are associated with early derangement in gut barrier function and systemic endotoxin translocation, there is no molecular evidence for associated bacterial "translocation."

Acute Disease↗
PubMed2002

Microbial inactivation of paprika by a high-temperature short-X time treatment. Influence on color properties.

High-temperature short-time (HTST) treatments have been used to destroy the bioburden of paprika. With this in mind, we have designed a device to treat samples of paprika with a gas whose temperature, pressure, and composition can be selected. Temperatures and treatment times ranged from 130 to 170 degrees C and 4 to 6 s, respectively. The survival of the most commonly found microorganisms in paprika and any alteration in extractable and superficial color were examined. Data showed that the optimum HTST conditions were 145 degrees C, 1.5 kg/cm2 of overpressure, 6 s operation time, and a thermal fluid of saturated steam. No microbial growth was detected during storage after thermal treatment. To minimize the color losses, treated (HTST) paprika samples should be kept under refrigeration.

Capsicum↗
PubMed2003

Bacterial contamination of ex vivo processed PBPC products under clean room conditions.

BACKGROUND: Patients undergoing high-dose radio- and/or chemotherapy and autologous or allogeneic PBPC transplantation are at high risk for infections owing to profound immunosuppression. In this study, the rate of microbial contamination of ex vivo processed PBPC products was analyzed, comparing preparation under clean room conditions to standard laboratory conditions. STUDY DESIGN AND METHODS: After implementation of good manufacturing practice conditions in the two participating institutions, the microbial contamination rate of 366 PBPC harvests from 198 patients was determined under certified clean room conditions (Group A) from 2000 until 2002. To investigate influence of improved environmental conditions along with other parameters, this set of samples was compared with a historical control set of 1413 PBPC products, which have been processed ex vivo under a clean bench in a regular laboratory room and were harvested from 626 patients (Group B) from 1989 until 2000. RESULTS: In Group B microbial contamination was found in 74 PBPC products (5.2%) from 57 patients. In Group A microbial growth was detected in 3 leukapheresis products (0.8%) from 3 patients. After exclusion of PBPC products, which were probably contaminated before manipulation, statistical analysis showed a significant difference (chi2= 10.339; p < 0.001). CONCLUSION: These data suggest an impact of clean room conditions on the bacterial contamination rate of PBPC products. To identify confounding variables, variables like technique of leukapheresis, culture methodology, and microbial colonization of central venous catheters were taken into account. Further variables might be identified in following studies.

Bacteria↗
PubMed2002

New approaches to the study of Antarctic lithobiontic microorganisms and their inorganic traces, and their application in the detection of life in Martian rocks.

Microbial life in the harsh conditions of Antarctica's cold desert may be considered an analogue of potential life on early Mars. In order to explore the development and survival of this epilithic and endolithic form of microbial life, our most sophisticated, state-of-the-art visualization technologies have to be used to their full potential. The study of any ecosystem requires a knowledge of its components and the processes that take place within it. If we are to understand the structure and function of each component of the microecosystems that inhabit lithic substrates, we need to be able to quantify and identify the microorganisms present in each lithobiontic ecological niche and to accurately characterize the mineralogical features of these hidden microhabitats. Once we have established the techniques that will allow us to observe and identify these microorganisms and mineral substrates in situ, and have confirmed the presence of water, the following questions can be addressed: How are the microorganisms organized in the fissures or cavities? Which microorganisms are present and how many are there? Additional questions that logically follow include: What are the existing water relationships in the microhabitat and what effects do the microorganisms have on the mineral composition? Mechanical and chemical changes in minerals and mineralization of microbial cells can give rise to physical and/or chemical traces (biomarkers) and to microbial fossil formation. In this report, we describe the detection of chains of magnetite within the Martian meteorite ALH84001, as an example of the potential use of SEM-BSE in the search for plausible traces of life on early Mars.

Antarctic Regions↗
PubMed2006

Bacteraemia in patients with hidradenitis suppurativa undergoing carbon dioxide laser surgery: detection and quantification of bacteria by lysis-filtration.

BACKGROUND: Hidradenitis suppurativa (HS) is a cicatrising and persistent disease of apocrine gland-bearing areas in adults. The severity of this condition varies from a few suppurating lesions to widespread, disabling disease. The aetiology is obscure, but suggested contributory factors include a genetic predisposition, comedones occluding the pilosebaceous apparatus, bacterial infections, and hormonal factors. Treatment consists mainly of surgery, while medical therapies serve principally as adjunct therapy. OBJECTIVES: The aim of the study was to determine the number and type of bacteria circulating in the bloodstream in patients with HS undergoing surgical treatment with a carbon dioxide laser stripping-secondary intention technique. METHODS: Twenty-one patients (20 females and 1 male, mean age 36, range 20-55 years) were included in the study. One blood sample (8.3 ml) was taken before surgery, one during the operation and the last one 10 min after surgery. Five healthy persons (all females, mean age 36, range 23-48 years) not undergoing any operation were used as the controls. The blood was cultured by a lysis-filtration technique which had been shown to be very sensitive. Since the filter catches the microorganisms and colonies are formed during culturing, the number of bacteria in the samples is easily determined. RESULTS: In 6 patients, all samples were negative, which indicates that the method of surgery itself caused no spread of bacteria from the lesions. Bacterial growth in the first blood sample was found in 9 patients, from the second sample in 10 and from the third one in 6. In 1 patient, bacteria were detected in three samples. At least 12 bacterial species were identified. The dominating bacteria were coagulase-negative staphylococci of which most were subtyped as Staphylococcus warneri. Among the anaerobic microorganisms, Propionibacterium acnes and P.granulosum were the most frequently isolated bacteria. The bacterial findings in the blood samples accord well with the results from a previous study in which cultures were taken from the deep parts of the HS lesions. In the 5 controls, no microbial growth was detected. CONCLUSION: The carbon dioxide laser stripping technique caused no additional spread of bacteria into the bloodstream. The evaluation of cultures containing microorganisms from normal skin flora is controversial. Since the bacteria encountered in this study are in close agreement with the findings in cultures from the deeper parts of HS lesions they seem to be relevant. The growth of bacteria in the first blood sample taken before surgery may indicate that some of these patients have bacteria continuously circulating in their blood.

Adult↗
PubMed2006

Characterisation of the dominant bacterial population in modified atmosphere packaged farmed halibut (Hippoglossus hippoglossus) based on 16S rDNA-DGGE.

It is not well understood why Atlantic halibut (Hippoglossus hippoglossus) has longer shelf-life than most other white fish species. Our approach was to examine the microbiological diversity of the spoilage microbiota during modified atmosphere (MA) packaging of farmed Atlantic halibut. Portions were packaged with gas mixtures of CO(2):N(2) and CO(2):O(2) (50%:50%) and with air as a reference. The packages were stored at 4 degrees C and samples were taken 6 times during the 23 days of storage. Analyses with molecular techniques (PCR-DGGE) determined profiles of the bacterial populations in the various samples and sequencing detected the bacterial species present. In addition, samples were analysed for microbial, chemical and sensory parameters. The shelf-life was 10-13 days when stored in air and between 13 and 20 days for MA packages, with oxygen-enriched packages suggested as the better gas mixture, based on microbial growth and sensory scores. From sequence analyses of the bacterial population Photobacterium phosphoreum and Pseudomonas spp. were found to dominate in the halibut. Brochothrix thermosphacta was found in most samples at the end of the storage period. Shewanella putrefaciens was found sporadically and in low concentrations based on microbial methods, but not detected by PCR-DGGE.

Animals↗