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PubMed1999

Characterization of mercury resistance mechanisms in marine sediment microbial communities.

While estuarine sediments are often severely polluted with mercury, few studies have focused on the mechanisms of adaptation to mercury contamination in marine sediment microbial communities. In this study, we report a high frequency of Gram-negative bacterial isolates that are resistant to the heavy metal mercury obtained from the aerobic culturable marine microbial community. We detected a low frequency of genes homologous to mer(Tn21) in isolates from three out of four different estuarine environments. Other mercury resistant culturable bacterial isolates lacking homology to the known mer genes were able to reduce Hg(II) to its volatile Hg(0) form, indicating the presence of divergent mer genes. In addition, a number of mercury resistant isolates, obtained from three of the four marine sites investigated, exhibited decreased resistance to mercury in the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone. Representative mercury resistant bacterial isolates were identified by phylogenetic analysis as belonging to the alpha and gamma subclasses of the class Proteobacteria.

Journal Article↗

PubMed2006

Subgingival microbiota of periodontally untreated Mexican subjects with generalized aggressive periodontitis.

BACKGROUND AND AIM: Specific microbial profiles that may distinguish between generalized aggressive-periodontitis (GAgP) and generalized chronic-periodontitis (GCP) have, to date, not been described. The purpose of the present study was to describe the subgingival microbial composition of Mexican subjects with GAgP and compare it with that of individuals with GCP and periodontal health (PH). MATERIAL AND METHODS: Seventy-seven subjects with GAgP (n=19), GCP (n=39) and PH (n=19) were selected. Clinical measurements included plaque accumulation, gingival erythema, bleeding on probing, suppuration, pocket depth and attachment level. Up to 28 subgingival plaque samples were obtained from each subject and analysed using the checkerboard DNA-DNA hybridization technique. RESULTS: GAgP and GCP subjects harboured significantly higher levels and/or proportion of Porphyromonas gingivalis, Tannerella forsythia (levels: p<0.001, proportion: p<0.01), Prevotella nigrescens (p<0.05 levels) and "red" complex species (p<0.001 proportion) than PH subjects. All GAgP subjects were carriers of P. gingivalis and P. nigrescens. No significant differences in any of the 40 microbial species tested were detected between GAgP and GCP subjects. CONCLUSIONS: Our results revealed that the microbial differences between GAgP and GCP subjects were only discrete and none of the bacterial species tested seemed to specifically differentiate the subgingival microbial profile of either periodontitis group.

Actinomyces↗

PubMed1995

Genetic diversity of Desulfovibrio spp. in environmental samples analyzed by denaturing gradient gel electrophoresis of [NiFe] hydrogenase gene fragments.

The genetic diversity of Desulfovibrio species in environmental samples was determined by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified [NiFe] hydrogenase gene fragments. Five different PCR primers were designed after comparative analysis of [NiFe] hydrogenase gene sequences from three Desulfovibrio species. These primers were tested in different combinations on the genomic DNAs of a variety of hydrogenase-containing and hydrogenase-lacking bacteria. One primer pair was found to be specific for Desulfovibrio species only, while the others gave positive results with other bacteria also. By using this specific primer pair, we were able to amplify the [NiFe] hydrogenase genes of DNAs isolated from environmental samples and to detect the presence of Desulfovibrio species in these samples. However, only after DGGE analysis of these PCR products could the number of different Desulfovibrio species within the samples be determined. DGGE analysis of PCR products from different bioreactors demonstrated up to two bands, while at least five distinguishable bands were detected in a microbial mat sample. Because these bands most likely represent as many Desulfovibrio species present in these samples, we conclude that the genetic diversity of Desulfovibrio species in the natural microbial mat is far greater than that in the experimental bioreactors.

Base Sequence↗

PubMed2000

Development of catechol 2,3-dioxygenase-specific primers for monitoring bioremediation by competitive quantitative PCR.

Benzene, toluene, xylenes, phenol, naphthalene, and biphenyl are among a group of compounds that have at least one reported pathway for biodegradation involving catechol 2,3-dioxygenase enzymes. Thus, detection of the corresponding catechol 2,3-dioxygenase genes can serve as a basis for identifying and quantifying bacteria that have these catabolic abilities. Primers that can successfully amplify a 238-bp catechol 2,3-dioxygenase gene fragment from eight different bacteria are described. The identities of the amplicons were confirmed by hybridization with a 238-bp catechol 2,3-dioxygenase probe. The detection limit was 10(2) to 10(3) gene copies, which was lowered to 10(0) to 10(1) gene copies by hybridization. Using the dioxygenase-specific primers, an increase in catechol 2, 3-dioxygenase genes was detected in petroleum-amended soils. The dioxygenase genes were enumerated by competitive quantitative PCR with a 163-bp competitor that was amplified using the same primers. Target and competitor sequences had identical amplification kinetics. Potential PCR inhibitors that could coextract with DNA, nonamplifying DNA, soil factors (humics), and soil pollutants (toluene) did not impact enumeration. Therefore, this technique can be used to accurately and reproducibly quantify catechol 2, 3-dioxygenase genes in complex environments such as petroleum-contaminated soil. Direct, non-cultivation-based molecular techniques for detecting and enumerating microbial pollutant-biodegrading genes in environmental samples are powerful tools for monitoring bioremediation and developing field evidence in support of natural attenuation.

Biodegradation, Environmental↗

PubMed2002

Molecular method to assess the diversity of Burkholderia species in environmental samples.

In spite of the importance of many members of the genus Burkholderia in the soil microbial community, no direct method to assess the diversity of this genus has been developed so far. The aim of this work was the development of soil DNA-based PCR-denaturing gradient gel electrophoresis (DGGE), a powerful tool for studying the diversity of microbial communities, for detection and analysis of the Burkholderia diversity in soil samples. Primers specific for the genus Burkholderia were developed based on the 16S rRNA gene sequence and were evaluated in PCRs performed with genomic DNAs from Burkholderia and non-Burkholderia species as the templates. The primer system used exhibited good specificity and sensitivity for the majority of established species of the genus Burkholderia. DGGE analyses of the PCR products obtained showed that there were sufficient differences in migration behavior to distinguish the majority of the 14 Burkholderia species tested. Sequence analysis of amplicons generated with soil DNA exclusively revealed sequences affiliated with sequences of Burkholderia species, demonstrating that the PCR-DGGE method is suitable for studying the diversity of this genus in natural settings. A PCR-DGGE analysis of the Burkholderia communities in two grassland plots revealed differences in diversity mainly between bulk and rhizosphere soil samples; the communities in the latter samples produced more complex patterns.

Burkholderia↗

PubMed2004

Spatial distribution of marine crenarchaeota group I in the vicinity of deep-sea hydrothermal systems.

Distribution profiles of marine crenarchaeota group I in the vicinity of deep-sea hydrothermal systems were mapped with culture-independent molecular techniques. Planktonic samples were obtained from the waters surrounding two geographically and geologically distinct hydrothermal systems, and the abundance of marine crenarchaeota group I was examined by 16S ribosomal DNA clone analysis, quantitative PCR, and whole-cell fluorescence in situ hybridization. A much higher proportion of marine crenarchaeota group I within the microbial community was detected in deep-sea hydrothermal environments than in normal deep and surface seawaters. The highest proportion was always obtained from the ambient seawater adjacent to hydrothermal emissions and chimneys but not from the hydrothermal plumes. These profiles were markedly different from the profiles of epsilon-Proteobacteria, which are abundant in the low temperatures of deep-sea hydrothermal environments.

Base Sequence↗

PubMed2006

Transcriptional profiling of the Bacillus anthracis life cycle in vitro and an implied model for regulation of spore formation.

The life cycle of Bacillus anthracis includes both vegetative and endospore morphologies which alternate based on nutrient availability, and there is considerable evidence indicating that the ability of this organism to cause anthrax depends on its ability to progress through this life cycle in a regulated manner. Here we report the use of a custom B. anthracis GeneChip in defining the gene expression patterns that occur throughout the entire life cycle in vitro. Nearly 5,000 genes were expressed in five distinct waves of transcription as the bacteria progressed from germination through sporulation, and we identified a specific set of functions represented within each wave. We also used these data to define the temporal expression of the spore proteome, and in doing so we have demonstrated that much of the spore's protein content is not synthesized de novo during sporulation but rather is packaged from preexisting stocks. We explored several potential mechanisms by which the cell could control which proteins are packaged into the developing spore, and our analyses were most consistent with a model in which B. anthracis regulates the composition of the spore proteome based on protein stability. This study is by far the most comprehensive survey yet of the B. anthracis life cycle and serves as a useful resource in defining the growth-phase-dependent expression patterns of each gene. Additionally, the data and accompanying bioinformatics analyses suggest a model for sporulation that has broad implications for B. anthracis biology and offer new possibilities for microbial forensics and detection.

Bacillus anthracis↗

PubMed2006

Comparative study of surgical instruments from sterile-service departments for presence of residual gram-negative endotoxin and proteinaceous deposits.

The ineffective cleaning of surgical instruments may be a vector for the transmission of hospital-acquired infections. The aim of this research was to investigate whether further decontamination procedures need to be instigated in sterile-service departments (SSDs) to reduce the risk of nosocomial illnesses, such as endotoxemia, sepsis, or iatrogenic Creutzfeldt-Jakob disease (to date, 1,147 cases of confirmed Creutzfeldt-Jakob disease deaths in the United Kingdom since 1990 have been reported). Instrument sets were obtained from nine anonymous United Kingdom National Health Service (NHS) primary care trust SSDs. The investigation implemented an advanced light microscopy technique, episcopic differential interference contrast microscopy with the sensitive fluorescent reagents SYPRO Ruby and 4',6-diamidino-2-phenylindole dihydrochloride (DAPI), to detect proteinaceous and microbial contamination levels. Gram-negative lipopolysaccharide (LPS) endotoxin was monitored using a dansylated polymyxin B fluorochrome agent. None of the 260 instruments examined displayed signs of microbial colonization or LPS endotoxin contamination. However, over 60 percent of the instruments showed a high degree of protein soiling (0.4 to 4.2 mug protein/mm(2)). Some instruments appeared soiled with crystalline deposits that may consist of a potentially hazardous material contributing to inflammation and/or surgical shock. It is clear that the overall standard for cleaning must be raised in order to fulfill the imminent introduction of new European standards and to reduce the risk of cross-patient contamination and iatrogenic transmission.

Decontamination↗

PubMed2001

Evaluation of BACTEC MYCO/F Lytic medium for recovery of mycobacteria, fungi, and bacteria from blood.

MYCO/F Lytic medium (MFL), a liquid medium developed for use with the BACTEC 9240 blood culture system, was compared to the Isolator system (IS) for the recovery of fungi and to the BACTEC 13A medium for the recovery of mycobacteria. Recovery of bacteria was compared to routine BACTEC Plus Aerobic/F (AF) blood cultures. Microbial growth was detected in 203 (17%) of 1,166 blood cultures. Fifty-seven specimens were positive for fungi: 35 were positive with both IS and MFL; six were positive with IS only (three Candida albicans, one Histoplasma capsulatum, one Candida glabrata, and one Fusarium species isolate); three were positive with AF only (two C. albicans and one Candida parapsilosis isolate); and 13 were positive with MFL only (five C. glabrata, three C. albicans, two Candida krusei, two Candida tropicalis, and one C. parapsilosis isolate; P > 0.05 versus IS). Eighteen of 19 blood cultures positive for H. capsulatum grew in both IS and MFL, although the time to detection for MFL was greater. The mean time to detection for all fungi was 8.15 days for IS and 12.07 days for MFL. Seven hundred forty specimens were also cultured for mycobacteria with MFL and 13A. Forty-four grew mycobacteria; 38 were positive with both 13A and MFL; and 16 were positive with MFL only. Mycobacterium avium was recovered from 41 specimens; 36 were positive for both systems and 5 were positive for MFL alone. MFL was also compared to the AF bottle for the same 740 specimens. MFL and AF both detected 34 of the 40 clinically significant bacteria, while IS detected only 15 of 40. In summary, MFL is an excellent medium for the recovery of fungi, mycobacteria, and bacteria; however, the time to detection of H. capsulatum is increased.

Bacteremia↗

PubMed2005

Isolation of novel bacteria and actinomycetes using soil-extract agar medium.

Novel bacteria were discovered using an isolation technique consisting of (i) selection of microorganisms that grew on soil-extract agar medium, but not on conventional media, and (ii) detection of small microbial colonies with a microscope. Three bacterial strains thus isolated were provisionally designated Shinshu-th1, -th2, -th 3, and five actinomycete strains, Shinshu-MS-01, -02, -03, -04, -05, respectively. Sequence analysis of their 16S rDNA showed that th1 had 95--96% homology with three unculturable bacteria, and th2 had 96% similarity to Bradyrhizobium sp., one unculturable and one unidentified bacterial strain. A phylogenetic study indicated that both strains were alpha-Proteobacteria belonging to the order Rhizobiales and the family Bradyrhizobiaceae. Since they had low homology (96%) with their close relatives, it is possible that th1 and th2 belong to a new genus. The actinomycetes Shinshu-MS-02 and -03 had 95--96% homology with four strains of Actinomadura, -04 had 95--96% similarity to Streptosporangium and Microbispora, and -05 had 97--98% homology with three strains of Acrocarpospora, Herbidospora and Planotetraspora. According to the phylogenetic study, both 02 and 03 are possibly new species of Actinomadura, -04 of Streptosporangium, and -05 of Acrocarpospora. Shinshu-th 3 and -MS-01 were identified as Mycobacterium cookii and Frankia sp., respectively, having 99% homology with these species.

Actinobacteria↗

PubMed2003

Nitrogen metabolism and recycling in Holstein heifers.

To study the effect of dietary N level on urea kinetics and recycling, four Holstein heifers (267 +/- 3.6 kg) were used in a Youden square design. Isocaloric diets with a N content of 1.44, 1.89, 2.50, 2.97, and 3.40% were fed at approximately 1.8 times maintenance intake. Increasing the N content of the diet increased urinary N excretion (P < 0.001) and N balance (P < 0.01), but did not affect the fecal N excretion (P = 0.21). Increasing the level of dietary N, increased urea production (P < 0.001) and excretion (P < 0.001), but no effect (P = 0.24) could be detected in the amount of N recycled to the gut. Urea recycled with the saliva, however, increased (P < 0.001) both in absolute and relative terms, with increasing dietary N. No difference could be detected on the amount of recycled N that was used for anabolism or returned to the ornithine cycle, but less (P = 0.001) N originating from urea was excreted in feces as dietary N increased. Ruminal ammonia concentration increased (P < 0.001) with increasing N intake, but total tract neutral detergent fiber digestibility was depressed only on the lowest N intake diet. No difference (P = 0.30) was detected in ruminal microbial yield among diets, but more (P < 0.003) N was derived from blood urea at low N intakes, and the efficiency of use of the recycled N decreased (P < 0.001) with increasing levels of dietary N. Adaptive changes to low-N diets were a decrease (P < 0.003) in the renal clearance of urea and an increase (P < 0.001) in the gastrointestinal clearance of urea. Urea transporters were present in the rumen wall of the heifers and differentially expressed depending on dietary N content, but their role in the transfer of urea into the rumen remains uncertain. Different mechanisms of N salvage and recycling were involved when animals were fed low-N diets that ensured a supply of endogenous N to the gastrointestinal tract and, due to the reduced contribution of dietary N, an increased efficiency of the N recycled was observed.

Ammonia↗

PubMed1997

[The nosocomial infection in ICU: characteristics and prevention].

In 84 consecutive patients who were treated in ICU, 35 had microbiological 5 data. 117 strains (bacteria: 77; fungus: 40) were isolated from 24 of the 35 patients for 97 times. The positive rate was 68.6%, or 28.6% of 84 patients. The gram-negative bacilli were still the main source of nosocomial infection in ICU, especially the pseudomonas aeruginosa, clostridium welchii, and E. coli, but gram-positive cocci, especially MRSA infection were also increased. Mycotic infection increased very rapidly. 12 of the 24 patients were found a complicated mycotic infection. Six of the 12 (50%) died. Candida tropicalis and candida guilliermondi replaced candida albicans as the main pathogenic fungus. Sputum is still the most important specimen source of positive culture, followed by wound drainage, blood, urine, central venous catheter as well as feces. It is indicated that the respiratory system is still the main focus of nosocomial infection, and mycotic infection is most dangerous in ICU. Regular monitoring of microbial flora changes, detection correct use of antibiotics by drug sensitivity test, adequate surgical drainage, early and prevention of mycotic infection as well as improvement of ICU design will prevent or minimize the nosocomial infection in ICU.

Adult↗

PubMed1975

Transplantable metastasizing prostate adenocarcinomas in rats.

Three spontaneous prostate adenocarcinomas from aged, randombred, germfree Lobund Wistar rats were transplanted, without change, through several series of conventional Lobund Wistar rats. One tumor type differed histologically from the other two tumor types. Rats with subcutaneously transplanted tumors developed metastatic tumors in the lymph nodes and lungs. No microbial agent was detected in the tumor cells.

Adenocarcinoma↗

PubMedDate not supplied

[Antilysozyme activity and antibiotic resistance of periulcerous zone microflora in patients with gastric and duodenal ulcer].

Antilysozyme activity (ALA) as well as antibiotic resistance were detected in 133 microbial cultures isolated from bioptic specimens of the mucous membrane of the ulcerous and periulcerous zones, taken from patients with gastric and duodenal ulcer. In 85.7-94.7% of cases Gram positive cocci and in 62.5% of cases Gram-positive bacilli showed no ALA. 50% of Gram negative bacteria cultures lacked ALA, while the remaining 50% exhibited this activity, on the average, 2.36 +/- 1.40 mkg/ml. Lysozyme activity was determined in 33.3% of the isolated staphylococci strains both with and without ALA. Staphylococci isolated from the gastric mucosa of healthy controls had no ALA in 33.3% of cases, and in 66.7% of cases ALA was equal to 2 mkg/ml. Gram positive coccal microflora showed, mainly, high sensitivity to antibiotics. In Gram negative bacteria antibiotic resistance was determined in 44.3 +/- 21.2% of the isolates. In Gram negative microorganisms correlation between ALA and antibiotic resistance was observed. From the periulcerous zone of patients with gastric and duodenal ulcer persistence associated Gram negative microorganisms were mainly isolated.

Bacteria↗

PubMed2003

Recent advances in the development of immunostimulatory oligonucleotides.

Some immune cells recognize distinct molecular structures present in pathogens through specific pattern recognition receptors that are able to distinguish prokaryotic DNA from vertebrate DNA. The detection of invading microbial DNA is based on the recognition of unmethylated deoxycytidyl-deoxyguanosin dinucleotide (CpG) motifs. Synthetic oligonucleotides (ODNs) containing these CpG motifs are able to activate both innate and acquired immune responses through a signaling pathway involving Toll-like receptor 9 (TLR9). Depending on the sequence, length, as well as number and positions of CpG motifs in an ODN, distinct immunostimulatory profiles can be observed. These immunostimulatory profiles can be further modified and fine-tuned by appropriate chemical modifications, leading to preclinical and clinical development of CpG ODNs in cancer, allergy, asthma and infectious diseases.

Adjuvants, Immunologic↗

PubMed2003

Molecular diagnostics of atypical pneumonia.

The emergence of nucleic acid-based molecular techniques has significantly enhanced laboratory diagnosis and monitoring of atypical pneumonia. These techniques have not only provided rapid and sensitive detection of fastidious microbial organisms but have also played critical roles in identifying and characterizing emerging pathogens that cause atypical pneumonia. Other benefits that molecular techniques can bring to the field include organism differentiation, quantitation, typing, and antibiotic resistance profiles. Gradually becoming standardized and widely available, the future will see some promising molecular methods become a mainstay in clinical laboratories for recognition and diagnosis of atypical pneumonia pathogens.

Chlamydia↗

PubMed1992

[Ultrastructure of liposomes (interliposomal bonds)].

Interliposomal bonds (ILBS) analogous to intercellular bonds (ICBs) in microbial cultures were detected by electron microscopy in the liposomal materials obtained after encapsulation of substances of various chemical structure. Possible nonspecific formation of the bonds between biological membrane-limited objects (ILBs and ICBs) was suggested and formation of such bonds in liposome encapsulated drugs was believed to be of importance.

Anti-Bacterial Agents↗

PubMed1992

[Efficacy of sodium imipenem/cilastatin on patients of complicated urinary tract infections--following the failure of prior antimicrobial agents].

Clinical efficacy of sodium imipenem/cilastatin was studied on 45 patients with complicated urinary tract infections, on whom prior antimicrobial agents were ineffective. A 0.5g dose of IPM/CS was administered by drop infusion twice a day for 5 days. The clinical efficacy was evaluated according to the criteria of the Japanese UTI committee. Of a total of 65 strains, 29 strains of gram positive bacteria and 36 strains of gram negative bacteria, were detected as causative microbials. Bacteriologically, 23 strains of gram positive bacteria (79%) and 34 strains of gram negative bacteria (94%) were eradicated following the treatment. S. aureus, E. faecalis and Flavobacterium sp. were less sensitive to IPM/CS. Overall clinical effectiveness rate of IPM/CS in the present study was 87%. Adverse drug side effects were observed in six patients, namely they were eruption, headache and slight elevation of serum transaminase. These findings suggest that IPM/CS is an effective agent for the treatment of complicated UTI even when the prior medicine was ineffective.

Adolescent↗