bluuebunny/crossref_metadata_2025_split huggingface dataset:
Gamma-Proteobacteria
Aquicella lusitana
gen. nov., sp. nov., and
Aquicella siphonis
sp. nov. Infect Protozoa and Require Activated Charcoal for Growth in Laboratory Media
Authors: Santos Paula , Pinhal Isabel , Rainey Fred A. , Empadinhas Nuno , Costa Joana , Fields Barry , Benson Robert , Veríssimo António , da Costa Milton S. 
Published: November 2003

ABSTRACT

Several isolates, belonging to two new species of the same novel genus of gamma-proteobacteria, were recovered from drilled well (borehole) and spa water at São Gemil in central Portugal. These organisms are phylogenetically most closely related to the strictly intracellular uncultured species of the genus
Rickettsiella
, which cause disease in arthropods, and to the facultatively intracellular species of the genus
Legionella
, some of which cause Legionnaires' disease and Pontiac fever. The São Gemil strains grew only on media containing charcoal, as is also true of the species of the genus
Legionella
. Unlike the vast majority of
Legionella
isolates, the new isolates did not require
l
-cysteine or ferric pyrophosphate for growth but like the legionellae had an absolute requirement for α-ketoglutarate. Strains SGT-39
T
and SGT-56 grew consistently between 30 and 43°C, while strains SGT-108
T
and SGT-109 grew between 30 and 40°C. The pH ranges for growth of these organisms were surprisingly narrow: strains SGT-39
T
and SGT-56 grew between pH 6.3 and 7.3, while strains SGT-108
T
and SGT-109 grew between pH 6.3 and 7.0. Both organisms proliferated in the amoeba
Hartmannella vermiformis
but did not grow in U937 human cells. Based on 16S rRNA gene sequence analysis and physiological, biochemical, and chemical analysis we describe two new species of one novel genus; one species is represented by strain SGT-39
T
, for which we propose the name
Aquicella lusitana
, while strain SGT-108
T
represents a second species of the same genus, for which we propose the name
Aquicella siphonis
.
Diversity and Abundance of Uncultured
Cytophaga
-Like Bacteria in the Delaware Estuary
Authors: Kirchman David L. , Yu Liying , Cottrell Matthew T. 
Published: November 2003

ABSTRACT

The
Cytophaga-Flavobacterium
group is known to be abundant in aquatic ecosystems and to have a potentially unique role in the utilization of organic material. However, relatively little is known about the diversity and abundance of uncultured members of this bacterial group, in part because they are underrepresented in clone libraries of 16S rRNA genes. To circumvent a suspected bias in PCR, a primer set was designed to amplify 16S rRNA genes from the
Cytophaga-Flavobacterium
group and was used to construct a library of these genes from the Delaware Estuary. This library had several novel
Cytophaga
-like 16S rRNA genes, of which about 40% could be grouped together into two clusters (DE clusters 1 and 2) defined by sequences initially observed only in the Delaware library; the other 16S rRNA genes were classified into an additional four clades containing sequences from other environments. An oligonucleotide probe was designed for the cluster with the most clones (DE cluster 2) and was used in fluorescence in situ hybridization assays. Bacteria in DE cluster 2 accounted for about 10% of the total prokaryotic abundance in the Delaware Estuary and in a depth profile of the Chukchi Sea (Arctic Ocean). The presence of DE cluster 2 in the Arctic Ocean was confirmed by results from 16S rRNA clone libraries. The contribution of this cluster to the total bacterial biomass is probably larger than is indicated by the abundance of its members, because the average cell volume of bacteria in DE cluster 2 was larger than those of other bacteria and prokaryotes in the Delaware Estuary and Chukchi Sea. DE cluster 2 may be one of the more abundant bacterial groups in the Delaware Estuary and possibly other marine environments.
Power Analysis for Real-Time PCR Quantification of Genes in Activated Sludge and Analysis of the Variability Introduced by DNA Extraction
Authors: Dionisi Hebe M. , Harms Gerda , Layton Alice C. , Gregory Igrid R. , Parker Jack , Hawkins Shawn A. , Robinson Kevin G. , Sayler Gary S. 
Published: November 2003

ABSTRACT

The aims of this study were to determine the power of discrimination of the real-time PCR assay for monitoring fluctuations in microbial populations within activated sludge and to identify sample processing points where methodological changes are needed to minimize the variability in target quantification. DNA was extracted using a commercially available kit from mixed liquor samples taken from the aeration tank of four bench-scale activated-sludge reactors operating at 2-, 5-, 10-, and 20-day solid retention times, with mixed-liquor volatile suspended solid (MLVSS) values ranging from 260 to 2,610 mg/liter. Real-time PCR assays for bacterial and
Nitrospira
16S rRNA genes were chosen because they represent, respectively, a highly abundant and a less-abundant bacterial target subject to clustering within the activated sludge matrix. The mean coefficient of variation in DNA yields (measured as microgram of DNA per milligram of MLVSS) in triplicate extractions of 12 different samples was 12.2%. Based on power analyses, the variability associated with DNA extraction had a small impact on the overall variability of the real-time PCR assay. Instead, a larger variability was associated with the PCR assay. The less-abundant target (
Nitrospira
16S rRNA gene) had more variability than the highly abundant target (bacterial 16S rRNA gene), and samples from the lower-biomass reactors had more variability than samples from the higher-biomass reactors. Power analysis of real-time PCR assays indicated that three to five samples were necessary to detect a twofold increase in bacterial 16S rRNA genes, whereas three to five samples were required to detect a fivefold increase in
Nitrospira
16S rRNA genes.
Molecular Characterization and Substrate Preference of a Polycyclic Aromatic Hydrocarbon Dioxygenase from
Cycloclasticus
sp. Strain A5
Authors: Kasai Yuki , Shindo Kazutoshi , Harayama Shigeaki , Misawa Norihiko 
Published: November 2003

ABSTRACT

Cycloclasticus
sp. strain A5 is able to grow with petroleum polycyclic aromatic hydrocarbons (PAHs), including unsubstituted and substituted naphthalenes, dibenzothiophenes, phenanthrenes, and fluorenes. A set of genes responsible for the degradation of petroleum PAHs was isolated by using the ability of the organism to oxidize indole to indigo. This 10.5-kb DNA fragment was sequenced and found to contain 10 open reading frames (ORFs). Seven ORFs showed homology to previously characterized genes for PAH degradation and were designated
phn
genes, although the sequence and order of these
phn
genes were significantly different from the sequence and order of the known PAH-degrading genes. The
phnA1
,
phnA2
,
phnA3
, and
phnA4
genes, which encode the α and β subunits of an iron-sulfur protein, a ferredoxin, and a ferredoxin reductase, respectively, were identified as the genes coding for PAH dioxygenase. The
phnA4A3
gene cluster was located 3.7 kb downstream of the
phnA2
gene. PhnA1 and PhnA2 exhibited moderate (less than 62%) sequence identity to the α and β subunits of other aromatic ring-hydroxylating dioxygenases, but motifs such as the Fe(II)-binding site and the [2Fe-2S] cluster ligands were conserved.
Escherichia coli
cells possessing the
phnA1A2A3A4
genes were able to convert phenanthrene, naphthalene, and methylnaphthalene in addition to the tricyclic heterocycles dibenzofuran and dibenzothiophene to their hydroxylated forms. Significantly, the
E. coli
cells also transformed biphenyl and diphenylmethane, which are ordinarily the substrates of biphenyl dioxygenases.
Usefulness of
rpoB
Gene Sequencing for Identification of
Afipia
and
Bosea
Species, Including a Strategy for Choosing Discriminative Partial Sequences
Authors: Khamis Atieh , Colson Philippe , Raoult Didier , Scola Bernard La 
Published: November 2003

ABSTRACT

Bacteria belonging to the genera
Afipia
and
Bosea
are amoeba-resisting bacteria that have been recently reported to colonize hospital water supplies and are suspected of being responsible for intensive care unit-acquired pneumonia. Identification of these bacteria is now based on determination of the 16S ribosomal DNA sequence. However, the 16S rRNA gene is not polymorphic enough to ensure discrimination of species defined by DNA-DNA relatedness. The complete
rpoB
sequences of 20 strains were first determined by both PCR and genome walking methods. The percentage of homology between different species ranged from 83 to 97% and was in all cases lower than that observed with the 16S rRNA gene; this was true even for species that differed in only one position. The taxonomy of
Bosea
and
Afipia
is discussed in light of these results. For strain identification that does not require the complete
rpoB
sequence (4,113 to 4,137 bp), we propose a simple computerized method that allows determination of nucleotide positions of high variability in the sequence that are bordered by conserved sequences and that could be useful for design of universal primers. A fragment of 740 to 752 bp that contained the most highly variable area (positions 408 to 420) was amplified and sequenced with these universal primers for 47 strains. The variability of this sequence allowed identification of all strains and correlated well with results of DNA-DNA relatedness. In the future, this method could be also used for the determination of variability “hot spots” in sets of housekeeping genes, not only for identification purposes but also for increasing the discriminatory power of sequence typing techniques such as multilocus sequence typing.
New Method Using Sedimentation and Immunomagnetic Separation for Isolation and Enumeration of
Cryptosporidium parvum
Oocysts and
Giardia lamblia
cysts
Authors: Massanet-Nicolau Jaime 
Published: November 2003

ABSTRACT

A new method for the isolation of
Cryptosporidium parvum
oocysts and
Giardia lamblia
cysts from biosolid samples has been developed that utilizes sedimentation and immunomagnetic separation. The method was used to recover stained cysts and oocysts (spike organisms) from primary settled sewage sludge, anaerobically digested sewage sludge, and bovine manure. Recovery efficiencies associated with this method were approximately 40 to 60% and were significantly greater than those associated with similar methods based on sucrose flotation (
P
< 0.001). The recovery efficiency of the sedimentation-based method showed no significant reduction as a result of sample storage for up to 21 days (
P
> 0.05). Recovery efficiencies were determined by spiking samples with prestained cysts and oocysts, allowing them to be differentiated from those naturally present in the biosolid samples. The prestained cysts and oocysts had been fixed in 5% formalin, and the recovery efficiencies associated with this method may be different from recovery efficiencies for fresh cysts or oocysts.
Web-Based Phylogenetic Assignment Tool for Analysis of Terminal Restriction Fragment Length Polymorphism Profiles of Microbial Communities
Authors: Kent Angela D. , Smith Dan J. , Benson Barbara J. , Triplett Eric W. 
Published: November 2003

ABSTRACT
Culture-independent DNA fingerprints are commonly used to assess the diversity of a microbial community. However, relating species composition to community profiles produced by community fingerprint methods is not straightforward. Terminal restriction fragment length polymorphism (T-RFLP) is a community fingerprint method in which phylogenetic assignments may be inferred from the terminal restriction fragment (T-RF) sizes through the use of web-based resources that predict T-RF sizes for known bacteria. The process quickly becomes computationally intensive due to the need to analyze profiles produced by multiple restriction digests and the complexity of profiles generated by natural microbial communities. A web-based tool is described here that rapidly generates phylogenetic assignments from submitted community T-RFLP profiles based on a database of fragments produced by known 16S rRNA gene sequences. Users have the option of submitting a customized database generated from unpublished sequences or from a gene other than the 16S rRNA gene. This phylogenetic assignment tool allows users to employ T-RFLP to simultaneously analyze microbial community diversity and species composition. An analysis of the variability of bacterial species composition throughout the water column in a humic lake was carried out to demonstrate the functionality of the phylogenetic assignment tool. This method was validated by comparing the results generated by this program with results from a 16S rRNA gene clone library.
Diversity and Succession of the Intestinal Bacterial Community of the Maturing Broiler Chicken
Authors: Lu Jiangrang , Idris Umelaalim , Harmon Barry , Hofacre Charles , Maurer John J. , Lee Margie D. 
Published: November 2003

ABSTRACT

The diversity of bacterial floras in the ilea and ceca of chickens that were fed a vegetarian corn-soy broiler diet devoid of feed additives was examined by analysis of 1,230 partial 16S rRNA gene sequences. Nearly 70% of sequences from the ileum were related to those of
Lactobacillus
, with the majority of the rest being related to
Clostridiaceae
(11%),
Streptococcus
(6.5%), and
Enterococcus
(6.5%). In contrast,
Clostridiaceae
-related sequences (65%) were the most abundant group detected in the cecum, with the other most abundant sequences being related to
Fusobacterium
(14%),
Lactobacillus
(8%), and
Bacteroides
(5%). Statistical analysis comparing the compositions of the different 16S rRNA libraries revealed that population succession occurred during some sampling periods. The significant differences among cecal libraries at 3 and 7 days of age, at 14 to 28 days of age, and at 49 days of age indicated that successions occurred from a transient community to one of increasing complexity as the birds aged. Similarly, the ileum had a stable bacterial community structure for birds at 7 to 21 days of age and between 21 to 28 days of age, but there was a very unique community structure at 3 and 49 days of age. It was also revealed that the composition of the ileal and cecal libraries did not significantly differ when the birds were 3 days old, and in fact during the first 14 days of age, the cecal microflora was a subset of the ileal microflora. After this time, the ileum and cecum had significantly different library compositions, suggesting that each region developed its own unique bacterial community as the bird matured.
Long-Term Succession of Structure and Diversity of a Biofilm Formed in a Model Drinking Water Distribution System
Authors: Martiny Adam C. , Jørgensen Thomas M. , Albrechtsen Hans-Jørgen , Arvin Erik , Molin Søren 
Published: November 2003

ABSTRACT

In this study, we examined the long-term development of the overall structural morphology and community composition of a biofilm formed in a model drinking water distribution system with biofilms from 1 day to 3 years old. Visualization and subsequent quantification showed how the biofilm developed from an initial attachment of single cells through the formation of independent microcolonies reaching 30 μm in thickness to a final looser structure with an average thickness of 14.1 μm and covering 76% of the surface. An analysis of the community composition by use of terminal restriction fragment length polymorphisms showed a correlation between the population profile and the age of the sample, separating the samples into young (1 to 94 days) and old (571 to 1,093 days) biofilms, whereas a limited spatial variation in the biofilm was observed. A more detailed analysis with cloning and sequencing of 16S rRNA fragments illustrated how a wide variety of cells recruited from the bulk water initially attached and resulted in a species richness comparable to that in the water phase. This step was followed by the growth of a bacterium which was related to
Nitrospira
, which constituted 78% of the community by day 256, and which resulted in a reduction in the overall richness. After 500 days, the biofilm entered a stable population state, which was characterized by a greater richness of bacteria, including
Nitrospira
,
Planctomyces
,
Acidobacterium
, and
Pseudomonas
. The combination of different techniques illustrated the successional formation of a biofilm during a 3-year period in this model drinking water distribution system.
Analysis, Characterization, and Loci of the
tuf
Genes in
Lactobacillus
and
Bifidobacterium
Species and Their Direct Application for Species Identification
Authors: Ventura Marco , Canchaya Carlos , Meylan Valèrie , Klaenhammer Todd R. , Zink Ralf 
Published: November 2003

ABSTRACT

We analyzed the
tuf
gene, encoding elongation factor Tu, from 33 strains representing 17
Lactobacillus
species and 8
Bifidobacterium
species. The
tuf
sequences were aligned and used to infer phylogenesis among species of lactobacilli and bifidobacteria. We demonstrated that the synonymous substitution affecting this gene renders elongation factor Tu a reliable molecular clock for investigating evolutionary distances of lactobacilli and bifidobacteria. In fact, the phylogeny generated by these
tuf
sequences is consistent with that derived from 16S rRNA analysis. The investigation of a multiple alignment of
tuf
sequences revealed regions conserved among strains belonging to the same species but distinct from those of other species. PCR primers complementary to these regions allowed species-specific identification of closely related species, such as
Lactobacillus casei
group members. These
tuf
gene-based assays developed in this study provide an alternative to present methods for the identification for lactic acid bacterial species. Since a variable number of
tuf
genes have been described for bacteria, the presence of multiple genes was examined. Southern analysis revealed one
tuf
gene in the genomes of lactobacilli and bifidobacteria, but the
tuf
gene was arranged differently in the genomes of these two taxa. Our results revealed that the
tuf
gene in bifidobacteria is flanked by the same gene constellation as the
str
operon, as originally reported for
Escherichia coli
. In contrast, bioinformatic and transcriptional analyses of the DNA region flanking the
tuf
gene in four
Lactobacillus
species indicated the same four-gene unit and suggested a novel
tuf
operon specific for the genus
Lactobacillus
.
Influence of Storage on Monodispersed Cells of
Mycobacterium terrae
Used for Quantitative Carrier Test prEN 14563
Authors: Woelk Eva , Goroncy-Bermes Peter , Sand Wolfgang 
Published: November 2003

ABSTRACT

The degree of cell clumping increased with time of storage (1% cell clumps immediately after homogenization and 3 and 6.5% after 48 and 96 h of storage, respectively), and the number of living single cells decreased. Quantitative carrier tests were carried out with these cells using ortho-phthaldialdehyde (OPA) and coco fatty aminoxethylate as biocides. In contrast to OPA, with coco fatty aminoxethylate the reductions obtained with freshly homogenized mycobacteria were significantly higher (
P
= 0.02) than those obtained with mycobacteria kept in the refrigerator for 4 days. Therefore, it is advisable to prepare the test suspension freshly for each test.
FUM9
Is Required for C-5 Hydroxylation of Fumonisins and Complements the Meitotically Defined
Fum3
Locus in
Gibberella moniliformis
Authors: Butchko Robert A. E. , Plattner Ronald D. , Proctor Robert H. 
Published: November 2003

ABSTRACT

Deletion of the
Gibberella moniliformis FUM9
gene resulted in mutants that produce only fumonisins that lack a C-5 hydroxyl group. This phenotype is identical to that of previously described mutants with defective alleles at the meiotically defined
Fum3
locus. Transformation with a wild-type
FUM9
gene into a
Fum3
-defective mutant restored wild-type fumonisin production. These results indicate that the
FUM9
protein catalyzes the C-5 hydroxylation of fumonisins and that
FUM9
and the
Fum3
locus are the same gene.
Update on the Major Clonal Types of Methicillin-Resistant
Staphylococcus aureus
in the Czech Republic
Authors: Melter O. , Aires de Sousa M. , Urbášková P. , Jakubů V. , Žemličková H. , de Lencastre H. 
Published: November 2003

ABSTRACT

The purpose of our study was the molecular characterization of methicillin-resistant
Staphylococcus aureus
(MRSA) strains isolated in 21 hospitals in the Czech Republic in the period 2000-2002 and comparison with previous results from 1996-1997. Strains were analyzed by pulsed-field gel electrophoresis (PFGE) of
Sma
I digests and ribotyping of
Hin
dIII digests hybridized with a 16S-23S DNA probe. The prevalence of the most clinically important macrolide (
ermA
,
ermB
,
ermC
, and
msrA
) and aminoglycoside (
aph3
′,
ant4
′, and
aac6
′
-aph2
") resistance genes was evaluated as well. Selected isolates representative of each clonal type were analyzed by multilocus sequence typing and by a multiplex PCR method capable of identifying the structural type of the staphylococcal cassette chromosome
mec
(SCC
mec
) carried by the bacteria. Our results document the displacement of the Brazilian clone (ST239, SCC
mec
type IIIA, PFGE type B, ribotype H1) by a new clone that we named “Czech clone” (ST239, SCC
mec
type IIIA, PFGE type F, ribotype H6) and the maintenance of the Iberian clone (ST247, SCC
mec
type IA, PFGE type A, ribotype H2) exclusively in one hospital in the Czech Republic. In addition, we found a correlation between the distribution of aminoglycoside resistance genes and MRSA clonal types.
Detection and Identification of
Entamoeba
Species in Stool Samples by a Reverse Line Hybridization Assay
Authors: Verweij Jaco J. , Laeijendecker Daphne , Brienen Eric A. T. , van Lieshout Lisette , Polderman Anton M. 
Published: November 2003

ABSTRACT

Classically, detection of
Entamoeba histolytica
is performed by microscopic examination for characteristic cysts and/or trophozoites in fecal preparations. Differentiation of
E. histolytica
cysts and those of nonpathogenic amoebic species is made on the basis of the appearance and the size of the cysts. However, by classical means objective tools for confirmation and quality control do not exist. Therefore, a reverse line blot hybridization assay was developed to detect a variety of
Entamoeba
species and genetic variants known to infect humans. The assay was performed after amplification with general
Entamoeba
-specific primers. The assay could identify four genetic variants of
Entamoeba polecki
-like cysts as well as
E. histolytica
,
Entamoeba dispar
,
Entamoeba hartmanni
,
Entamoeba moshkovskii
and
Entamoeba coli
and even mixed infections in a range of controls and fecal samples. This technique can be used as an additional standard for diagnosis, epidemiology, and quality control for amoebic infections.