bluuebunny/crossref_metadata_2025_split huggingface dataset:
Microbial Diversity of Biofilms in Dental Unit Water Systems
Authors: Singh Ruby , Stine O. Colin , Smith David L. , Spitznagel John K. , Labib Mohamed E. , Williams Henry N. 
Published: June 2003

ABSTRACT

We investigated the microbial diversity of biofilms found in dental unit water systems (DUWS) by three methods. The first was microscopic examination by scanning electron microscopy (SEM), acridine orange staining, and fluorescent in situ hybridization (FISH). Most bacteria present in the biofilm were viable. FISH detected the β and γ, but not the α, subclasses of
Proteobacteria.
In the second method, 55 cultivated biofilm isolates were identified with the Biolog system, fatty acid analysis, and 16S ribosomal DNA (rDNA) sequencing. Only 16S identified all 55 isolates, which represented 13 genera. The most common organisms, as shown by analyses of 16S rDNA, belonged to the genera
Afipia
(28%) and
Sphingomonas
(16%). The third method was a culture-independent direct amplification and sequencing of 165 subclones from community biofilm 16S rDNA. This method revealed 40 genera: the most common ones included
Leptospira
(20%),
Sphingomonas
(14%),
Bacillus
(7%),
Escherichia
(6%),
Geobacter
(5%), and
Pseudomonas
(5%). Some of these organisms may be opportunistic pathogens. Our results have demonstrated that a biofilm in a health care setting may harbor a vast diversity of organisms. The results also reflect the limitations of culture-based techniques to detect and identify bacteria. Although this is the greatest diversity reported in DUWS biofilms, other genera may have been missed. Using a technique based on jackknife subsampling, we projected that a 25-fold increase in the number of subclones sequenced would approximately double the number of genera observed, reflecting the richness and high diversity of microbial communities in these biofilms.
Metabolic Engineering of
Escherichia coli
for Production of Enantiomerically Pure (
R
)-(−)-Hydroxycarboxylic Acids
Authors: Lee Sang Yup , Lee Young 
Published: June 2003

ABSTRACT

A heterologous metabolism of polyhydroxyalkanoate (PHA) biosynthesis and degradation was established in
Escherichia coli
by introducing the
Ralstonia eutropha
PHA biosynthesis operon along with the
R. eutropha
intracellular PHA depolymerase gene. By with this metabolically engineered
E. coli
, enantiomerically pure (
R
)-3-hydroxybutyric acid (R3HB) could be efficiently produced from glucose. By employing a two-plasmid system, developed as the PHA biosynthesis operon on a medium-copy-number plasmid and the PHA depolymerase gene on a high-copy-number plasmid, R3HB could be produced with a yield of 49.5% (85.6% of the maximum theoretical yield) from glucose. By integration of the PHA biosynthesis genes into the chromosome of
E. coli
and by introducing a plasmid containing the PHA depolymerase gene, R3HB could be produced without plasmid instability in the absence of antibiotics. This strategy can be used for the production of various enantiomerically pure (
R
)-hydroxycarboxylic acids from renewable resources.
1,8-Dihydroxynaphthalene (DHN)-Melanin Biosynthesis Inhibitors Increase Erythritol Production in
Torula corallina
, and DHN-Melanin Inhibits Erythrose Reductase
Authors: Lee Jung-Kul , Jung Hyung-Moo , Kim Sang-Yong 
Published: June 2003

ABSTRACT

The yeast
Torula corallina
is a strong erythritol producer that is used in the industrial production of erythritol. However, melanin accumulation during culture represents a serious problem for the purification of erythritol from the fermentation broth. Melanin biosynthesis inhibitors such as 3,4-dihydroxyphenylalanine and 1,8-dihydroxynaphthalene (DHN)-melanin inhibitors were added to the
T. corallina
cultures. Only the DHN-melanin inhibitors showed an effect on melanin production, which suggests that the melanin formed during the culturing of
T. corallina
is derived from DHN. This finding was confirmed by the detection of a shunt product of the pentaketide pathway, flaviolin, and elemental analysis. Among the DHN-melanin inhibitors, tricyclazole was the most effective. Supplementation with tricyclazole enhanced the production of erythritol while significantly inhibiting the production of DHN-melanin and DHN-melanin biosynthetic enzymes, such as trihydroxynaphthalene reductase. The erythrose reductase from
T. corallina
was purified to homogeneity by ion-exchange and affinity chromatography. Purified erythrose reductase was significantly inhibited in vitro in a noncompetitive manner by elevated levels of DHN-melanin. In contrast, the level of erythrose reductase activity was unaffected by increasing concentrations of tricyclazole. These results suggest that supplemental tricyclazole reduces the production of DHN-melanin, which may lead to a reduction in the inhibition of erythrose reductase and a higher yield of erythritol. This is the first report to demonstrate that melanin biosynthesis inhibitors increase the production of a sugar alcohol in
T. corallina
.
Use of PCR for Direct Detection of
Campylobacter
Species in Bovine Feces
Authors: Inglis G. Douglas , Kalischuk Lisa D. 
Published: June 2003

ABSTRACT

This study reports on the use of PCR to directly detect and distinguish
Campylobacter
species in bovine feces without enrichment. Inhibitors present in feces are a major obstacle to using PCR to detect microorganisms. The QIAamp DNA stool minikit was found to be an efficacious extraction method, as determined by the positive amplification of internal control DNA added to bovine feces before extraction. With nested or seminested multiplex PCR,
Campylobacter coli
,
C. fetus
,
C. hyointestinalis
, and
C. jejuni
were detected in all fecal samples inoculated at ≈10
4
CFU g
−1
, and 50 to 83% of the samples inoculated at ≈10
3
CFU g
−1
were positive. At ≈10
2
CFU g
−1
,
C. fetus
,
C. hyointestinalis
, and
C. jejuni
(17 to 50% of the samples) but not
C. coli
were detected by PCR. From uninoculated bovine feces, a total of 198 arbitrarily selected isolates of
Campylobacter
were recovered on four commonly used isolation media incubated at three temperatures. The most frequently isolated taxa were
C. jejuni
(152 isolates) and
C. lanienae
(42 isolates), but isolates of
C. fetus
subsp.
fetus
,
Arcobacter butzleri
, and
A. skirrowii
also were recovered (≤2 isolates per taxon). Considerable variability was observed in the frequency of isolation of campylobacters among the four media and three incubation temperatures tested. With genus-specific primers,
Campylobacter
DNA was detected in 75% of the fecal samples, representing an 8% increase in sensitivity relative to that obtained with microbiological isolation across the four media and three incubation temperatures tested. With nested primers,
C. jejuni
and
C. lanienae
were detected in 25 and 67% of the samples, respectively. In no instance was DNA from either
C. coli
,
C. fetus
, or
C. hyointestinalis
detected in uninoculated bovine feces. PCR was more sensitive than isolation on microbiological media for detecting
C. lanienae
(17%) but not
C. jejuni
. Campylobacters are a diverse and fastidious group of bacteria, and the development of direct PCR not only will increase the understanding of
Campylobacter
species diversity and their frequency of occurrence in feces but also will enhance the knowledge of their role in the gastrointestinal tract of livestock and of the factors that influence shedding.
Oxygen-Insensitive Nitroreductases NfsA and NfsB ofEscherichia coliFunction under Anaerobic Conditions as Lawsone-Dependent Azo Reductases
Authors: Rau Jörg , Stolz Andreas 
Published: June 2003

ABSTRACTQuinones can function as redox mediators in the unspecific anaerobic reduction of azo compounds by various bacterial species. These quinones are enzymatically reduced by the bacteria and the resulting hydroquinones then reduce in a purely chemical redox reaction the azo compounds outside of the cells. Recently, it has been demonstrated that the addition of lawsone (2-hydroxy-1,4-naphthoquinone) to anaerobically incubated cells ofEscherichia coliresulted in a pronounced increase in the reduction rates of different sulfonated and polymeric azo compounds. In the present study it was attempted to identify the enzyme system(s) responsible for the reduction of lawsone byE. coliand thus for the lawsone-dependent anaerobic azo reductase activity. An NADH-dependent lawsone reductase activity was found in the cytosolic fraction of the cells. The enzyme was purified by column chromatography and the amino-terminal amino acid sequence of the protein was determined. The sequence obtained was identical to the sequence of an oxygen-insensitive nitroreductase (NfsB) described earlier from this organism. Subsequent biochemical tests with the purified lawsone reductase activity confirmed that the lawsone reductase activity detected was identical with NfsB. In addition it was proven that also a second oxygen-insensitive nitroreductase ofE. coli(NfsA) is able to reduce lawsone and thus to function under adequate conditions as quinone-dependent azo reductase.
Evaluation of DNA Extraction Methods for Use in Combination with SYBR Green I Real-Time PCR To Detect
Salmonella enterica
Serotype Enteritidis in Poultry
Authors: De Medici Dario , Croci Luciana , Delibato Elisabetta , Di Pasquale Simona , Filetici Emma , Toti Laura 
Published: June 2003

ABSTRACT

The objective of this study was to develop a rapid, reproducible, and robust method for detecting
Salmonella enterica
serotype Enteritidis in poultry samples. First, for the extraction and purification of DNA from the preenrichment culture, four methods (boiling, alkaline lysis, Nucleospin, and Dynabeads DNA Direct System I) were compared. The most effective method was then combined with a real-time PCR method based on the double-stranded DNA binding dye SYBR Green I used with the ABI Prism 7700 system. The specificity of the reaction was determined by the melting temperature (

T
m

) of the amplicon obtained. The experiments were conducted both on samples of chicken experimentally contaminated with serotype Enteritidis and on commercially available poultry samples, which were also used for comparisons with the standard cultural method (i.e., ISO 6579/2001). The results of comparisons among the four DNA extraction methods showed significant differences except for the results from the boiling and Nucleospin methods (the two methods that produced the lowest threshold cycles). Boiling was selected as the preferred extraction method because it is the simplest and most rapid. This method was then combined with SYBR Green I real-time PCR, using primers SEFA-1 and SEFA-2. The specificity of the reaction was confirmed by the

T
m

, which was consistently specific for the amplicon obtained; the mean peak

T
m

obtained with curves specific for serotype Enteritidis was 82.56 ± 0.22°C. The standard curve constructed using the mean threshold cycle and various concentrations of serotype Enteritidis (ranging from 10
3
to 10
8
CFU/ml) showed good linearity (
R
2
= 0.9767) and a sensitivity limit of less than 10
3
CFU/ml. The results of this study demonstrate that the SYBR Green I real-time PCR constitutes an effective and easy-to-perform method for detecting serotype Enteritidis in poultry samples.
Secondary Metabolites of
Flustra foliace
a and Their Influence on Bacteria
Authors: Peters Lars , König Gabriele M. , Wright Anthony D. , Pukall Rüdiger , Stackebrandt Erko , Eberl Leo , Riedel Kathrin 
Published: June 2003

ABSTRACT

The North Sea bryozoan
Flustra foliacea
was investigated to determine its secondary metabolite content. Gas chromatography-mass spectrometry analysis of a dichloromethane extract of the bryozoan enabled 11 compounds to be identified. Preparative high-performance liquid chromatography of the extract resulted in the isolation of 10 brominated alkaloids (compounds 1 to 10) and one diterpene (compound 11). All of these compounds were tested to determine their activities in agar diffusion assays against bacteria derived from marine and terrestrial environments. Compounds 1, 3 to 7, 10, and 11 exhibited significant activities against one or more marine bacterial strains originally isolated from
F. foliacea
but only weak activities against all of the terrestrial bacteria. By using the biosensors
Pseudomonas putida
(pKR-C12),
P. putida
(pAS-C8), and
Escherichia coli
(pSB403) the antagonistic effect on
N
-acyl-homoserine lactone-dependent quorum-sensing systems was investigated. Compounds 8 and 10 caused reductions in the signal intensities in these bioassays ranging from 50 to 20% at a concentration of 20 μg/ml.
Nitric Oxide Reductase (
norB
) Genes from Pure Cultures and Environmental Samples
Authors: Braker Gesche , Tiedje James M. 
Published: June 2003

ABSTRACT

A PCR-based approach was developed to recover nitric oxide (NO) reductase (
norB
) genes as a functional marker gene for denitrifying bacteria.
norB
database sequences grouped in two very distinct branches. One encodes the quinol-oxidizing single-subunit class (qNorB), while the other class is a cytochrome
bc
-type complex (cNorB). The latter oxidizes cytochrome
c
, and the gene is localized adjacent to
norC
. While both
norB
types occur in denitrifying strains, the
qnorB
type was also found in a variety of nondenitrifying strains, suggesting a function in detoxifying NO. Branch-specific degenerate primer sets detected the two
norB
types in our denitrifier cultures. Specificity was confirmed by sequence analysis of the
norB
amplicons and failure to amplify
norB
from nondenitrifying strains. These primer sets also specifically amplified
norB
from freshwater and marine sediments. Pairwise comparison of amplified
norB
sequences indicated minimum levels of amino acid identity of 43.9% for
qnorB
and 38% for
cnorB
. Phylogenetic analysis confirmed the existence of two classes of
norB
genes, which clustered according to the respective primer set. Within the
qnorB
cluster, the majority of genes from isolates and a few environmental clones formed a separate subcluster. Most environmental
qnorB
clones originating from both habitats clustered into two distinct subclusters of novel sequences from presumably as yet uncultivated organisms.
cnorB
clones were located on separate branches within subclusters of genes from known organisms, suggesting an origin from similar organisms.
Nutritional Similarity between Leaf-Associated Nonpathogenic Bacteria and the Pathogen Is Not Predictive of Efficacy in Biological Control of Bacterial Spot of Tomato
Authors: Dianese Alexei C. , Ji Pingsheng , Wilson Mark 
Published: June 2003

ABSTRACTIt has been demonstrated that for a nonpathogenic, leaf-associated bacterium, effectiveness in the control of bacterial speck of tomato is correlated with the similarity in the nutritional needs of the nonpathogenic bacterium and the pathogenPseudomonas syringaepv. tomato. This relationship was investigated further in this study by using the pathogenXanthomonas campestrispv. vesicatoria, the causal agent of bacterial spot of tomato, and a collection of nonpathogenic bacteria isolated from tomato foliage. The effects of inoculation of tomato plants with one of 34 nonpathogenic bacteria prior to inoculation with the pathogenX. campestrispv. vesicatoria were quantified by determining (i) the reduction in disease severity (number of lesions per square centimeter) in greenhouse assays and (ii) the reduction in leaf surface pathogen population size (log10of the number of CFU per leaflet) in growth chamber assays. Nutritional similarity between the nonpathogenic bacteria andX. campestrispv. vesicatoria was quantified by using either niche overlap indices (NOI) or relatedness in cluster analyses based upon in vitro utilization of carbon or nitrogen sources reported to be present in tomato tissues or in Biolog GN plates. In contrast to studies withP. syringaepv. tomato, nutritional similarity between the nonpathogenic bacteria and the pathogenX. campestrispv. vesicatoria was not correlated with reductions in disease severity. Nutritional similarity was also not correlated with reductions in pathogen population size. Further, the percentage of reduction in leaf surface pathogen population size was not correlated with the percentage of reduction in disease severity, suggesting that the epiphytic population size ofX. campestrispv. vesicatoria is not related to disease severity and thatX. campestrispv. vesicatoria exhibits behavior in the phyllosphere prior to lesion formation that is different from that ofP. syringaepv. tomato.
Use of Growth Indices from Radiometric Culture for Quantification of Sheep Strains ofMycobacterium aviumsubsp.paratuberculosis
Authors: Reddacliff Leslie A. , Nicholls Paul J. , Vadali Aparna , Whittington Richard J. 
Published: June 2003

ABSTRACTA simple method for using growth indices from radiometric BACTEC cultures was evaluated for the enumeration of Australian sheep strains ofMycobacterium aviumsubsp.paratuberculosis. The numbers of viable organisms in inocula were determined by end-point titration in BACTEC cultures. Growth indices were measured by using a BACTEC 460 machine. There was a linear relationship between the number of days taken for the cumulative growth index to reach 1,000 (dCGI1000) and log10inoculum size. The use of dCGI1000 was shown to be as effective as the use of growth index data from the entire growth cycle for the estimation of inoculum size. For particular isolates characterized by end-point titration, the dCGI1000 of a single BACTEC vial provided estimates of viable numbers within narrow prediction limits. Predictive relationships were also established for the enumeration ofM. aviumsubsp.paratuberculosisfrom field samples by using the dCGI1000 of a single BACTEC vial, with prediction limits of ±1 to 2 log units. Organisms from feces or contaminated soil grew more slowly than those from cultures or tissues, and separate equations were developed for enumeration from these sources.
Ecology of
Vibrio vulnificus
in Estuarine Waters of Eastern North Carolina
Authors: Pfeffer Courtney S. , Hite M. Frances , Oliver James D. 
Published: June 2003

ABSTRACT

While several studies on the ecology of
Vibrio vulnificus
in Gulf Coast environments have been reported, there is little information on the distribution of this pathogen in East Coast waters. Thus, we conducted a multiyear study on the ecology of
V. vulnificus
in estuarine waters of the eastern United States, employing extensive multiple regression analyses to reveal the major environmental factors controlling the presence of this pathogen, and of
Vibrio
spp., in these environments. Monthly field samplings were conducted between July 2000 and April 2002 at six different estuarine sites along the eastern coast of North Carolina. At each site, water samples were taken and nine physicochemical parameters were measured.
V. vulnificus
isolates, along with estuarine bacteria,
Vibrio
spp.,
Escherichia coli
organisms, and total coliforms, were enumerated in samples from each site by using selective media. During the last 6 months of the study, sediment samples were also analyzed for the presence of vibrios, including
V. vulnificus
. Isolates were confirmed as
V. vulnificus
by using hemolysin gene PCR or colony hybridization.
V. vulnificus
was isolated only when water temperatures were between 15 and 27°C, and its presence correlated with water temperature and dissolved oxygen and vibrio levels. Levels of
V. vulnificus
in sediments were low, and no evidence for an overwintering in this environment was found. Multiple regression analysis indicated that vibrio levels were controlled primarily by temperature, turbidity, and levels of dissolved oxygen, estuarine bacteria, and coliforms. Water temperature accounted for most of the variability in the concentrations of both
V. vulnificus
(47%) and
Vibrio
spp. (48%).
Use of Phospholipid Fatty Acids To Detect Previous Self-Heating Events in Stored Peat
Authors: Ranneklev Sissel Brit , Bååth Erland 
Published: June 2003

ABSTRACT

The use of the phospholipid fatty acid (PLFA) composition of microorganisms to detect previous self-heating events was studied in naturally self-heated peat and in peat incubated under temperature-controlled conditions. An increased content of total PLFAs was found in self-heated peat compared to that in unheated peat. Two PLFAs, denoted T1 and T2, were detected only in the self-heated peat. Incubation of peat samples at 25 to 55°C for 4 days indicated that T1 and T2 were produced from microorganisms with different optimum temperatures. This was confirmed by isolation of bacteria at 55°C, which produced T2 but not T1. These bacteria produced another PLFA (denoted T3) which coeluted with 18:1ω7. T2 and T3 were identified as ω-cyclohexyltridecanoic acid and ω-cyclohexylundecanoic acid, respectively, indicating that the bacteria belonged to the genus
Alicyclobacillus
. T1 was tentatively identified as ω-cycloheptylundecanoic acid. T2 was detected 8 h after the peat incubation temperature was increased to 55°C, and maximum levels were found within 5 days of incubation. The PLFA 18:1ω7-T3 increased in proportion to T2. T1 was detected after 96 h at 55°C, and its level increased throughout the incubation period, so that it eventually became one of the dominant PLFAs after 80 days. In peat samples incubated at 55°C and then at 25°C, T1 and T2 disappeared slowly. After 3 months, detectable levels were still found. Incubation at 25°C after heating for 3 days at 55°C decreased the amounts of T2 and 18:1ω7-T3 faster than did incubation at 5°C. Thus, not only the duration and temperature during the heating event but also the storage temperature following heating are important for the detection of PLFAs indicating previous self-heating.
Bacterial Community Structure and Location in Stilton Cheese
Authors: Ercolini Danilo , Hill Philip J. , Dodd Christine E. R. 
Published: June 2003

ABSTRACT

The microbial diversity occurring in Stilton cheese was evaluated by 16S ribosomal DNA analysis with PCR-denaturing gradient gel electrophoresis. DNA templates for PCR experiments were directly extracted from the cheese as well as bulk cells harvested from a variety of viable-count media. The variable V3 and V4-V5 regions of the 16S genes were analyzed. Closest relatives of
Lactococcus lactis
,
Enterococcus faecalis
,
Lactobacillus plantarum
,
Lactobacillus curvatus
,
Leuconostoc mesenteroides
,
Staphylococcus equorum
, and
Staphylococcus
sp. were identified by sequencing of the DGGE fragments. Fluorescently labeled oligonucleotide probes were developed to detect
Lactococcus lactis
,
Lactobacillus plantarum
, and
Leuconostoc mesenteroides
in fluorescence in situ hybridization (FISH) experiments, and their specificity for the species occurring in the community of Stilton cheese was checked in FISH experiments carried out with reference cultures. The combined use of these probes and the bacterial probe Eub338 in FISH experiments on Stilton cheese sections allowed the assessment of the spatial distribution of the different microbial species in the dairy matrix. Microbial colonies of bacteria showed a differential location in the different parts of the cheese examined: the core, the veins, and the crust. Lactococci were found in the internal part of the veins as mixed colonies and as single colonies within the core.
Lactobacillus plantarum
was detected only underneath the surface, while
Leuconostoc
microcolonies were homogeneously distributed in all parts observed. The combined molecular approach is shown to be useful to simultaneously describe the structure and location of the bacterial flora in cheese. The differential distribution of species found suggests specific ecological reasons for the establishment of sites of actual microbial growth in the cheese, with implications of significance in understanding the ecology of food systems and with the aim of achieving optimization of the fermentation technologies as well as preservation of traditional products.