bluuebunny/crossref_metadata_2025_split huggingface dataset:
Biofilm Formation and Effect of Caspofungin on Biofilm Structure of
Candida
Species Bloodstream Isolates
Authors: Ferreira J. A. G. , Carr J. H. , Starling C. E. F. , de Resende M. A. , Donlan R. M. 
Published: June 2009

ABSTRACT

Candida
biofilms are microbial communities, embedded in a polymeric matrix, growing attached to a surface, and are highly recalcitrant to antimicrobial therapy. These biofilms exhibit enhanced resistance against most antifungal agents except echinocandins and lipid formulations of amphotericin B. In this study, biofilm formation by different
Candida
species, particularly
Candida albicans
,
C. tropicalis
, and
C. parapsilosis
, was evaluated, and the effect of caspofungin (CAS) was assessed using a clinically relevant in vitro model system. CAS displayed in vitro activity against
C. albicans
and
C. tropicalis
cells within biofilms. Biofilm formation was evaluated after 48 h of antifungal drug exposure, and the effects of CAS on preformed
Candida
species biofilms were visualized using scanning electron microscopy (SEM). Several species-specific differences in the cellular morphologies associated with biofilms were observed. Our results confirmed the presence of paradoxical growth (PG) in
C. albicans
and
C. tropicalis
biofilms in the presence of high CAS concentrations. These findings were also confirmed by SEM analysis and were associated with the metabolic activity obtained by biofilm susceptibility testing. Importantly, these results suggest that the presence of atypical, enlarged, conical cells could be associated with PG and with tolerant cells in
Candida
species biofilm populations. The clinical implications of these findings are still unknown.
In Vitro and In Vivo Properties of Dihydrophthalazine Antifolates, a Novel Family of Antibacterial Drugs
Authors: Caspers Patrick , Bury Luc , Gaucher Bérangère , Heim Jutta , Shapiro Stuart , Siegrist Sibylle , Schmitt-Hoffmann Anne , Thenoz Laure , Urwyler Heinrich 
Published: June 2009

ABSTRACT

Racemic 2,4-diaminopyrimidine dihydrophthalazine derivatives BAL0030543, BAL0030544, and BAL0030545 exhibited low in vitro MICs toward small, selected panels of
Enterococcus faecalis
,
Enterococcus faecium
,
Streptococcus pneumoniae
,
Moraxella catarrhalis
, and
Mycobacterium avium
, though the compounds were less active against
Haemophilus influenzae
. The constellation of dihydrofolate reductases (DHFRs) present in 20 enterococci and 40 staphylococci was analyzed and correlated with the antibacterial activities of the dihydrophthalazines and trimethoprim. DHFRs encoded by
dfrB
,
dfrA
(S1 isozyme),
dfrE
, and
folA
were susceptible to the dihydrophthalazines, whereas DHFRs encoded by
dfrG
(S3 isozyme) and
dfrF
were not. Studies with the separated enantiomers of BAL0030543, BAL0030544, and BAL0030545 revealed preferential inhibition of susceptible DHFRs by the (
R
)-enantiomers. BAL0030543, BAL0030544, and BAL0030545 were well tolerated by mice during 5- and 10-day oral toxicity studies at doses of up to 400 mg/kg of body weight. Using a nonoptimized formulation, the dihydrophthalazines displayed acceptable oral bioavailabilities in mice, and efficacy studies with a septicemia model of mice infected with trimethoprim-resistant, methicillin-resistant
Staphylococcus aureus
gave 50% effective dose values in the range of 1.6 to 6.25 mg/kg.
Effect of
Candida glabrata FKS1
and
FKS2
Mutations on Echinocandin Sensitivity and Kinetics of 1,3-β-
d
-Glucan Synthase: Implication for the Existing Susceptibility Breakpoint
Authors: Garcia-Effron Guillermo , Lee Samuel , Park Steven , Cleary John D. , Perlin David S. 
Published: June 2009

ABSTRACT

Thirteen
Candida glabrata
strains harboring a range of mutations in hot spot regions of
FKS1
and
FKS2
were studied. The mutations were linked to an echinocandin reduced susceptibility phenotype. Sequence alignments showed that 11 out of the 13 mutants harbored a mutation in
FKS1
or
FKS2
not previously implicated in echinocandin reduced susceptibility in
C. glabrata
. A detailed kinetic characterization demonstrated that amino acid substitutions in Fks1p and Fks2p reduced drug sensitivity in mutant 1,3-β-
d
-glucan synthase by 2 to 3 log orders relative to that in wild-type enzyme. These mutations were also found to reduce the catalytic efficiency of the enzyme (
V
max
) and to influence the relative expression of
FKS
genes. In view of the association of
FKS
mutations and reduced susceptibility of 1,3-β-
d
-glucan synthase, an evaluation of the new CLSI echinocandin susceptibility breakpoint was conducted. Only 3 of 13 resistant
fks
mutants (23%) were considered anidulafungin or micafungin nonsusceptible (MIC > 2 μg/ml) by this criterion. In contrast, most
fks
mutants (92%) exceeded a MIC of >2 μg/ml with caspofungin. However, when MIC determinations were performed in the presence of 50% serum, all
C. glabrata fks
mutants showed MICs of ≥2 μg/ml for the three echinocandin drugs. As has been observed with
Candida albicans
, the kinetic inhibition parameter 50% inhibitory concentration may be a better predictor of
FKS
-mediated resistance. Finally, the close association between
FKS1/FKS2
hot spot mutations provides a basis for understanding echinocandin resistance in
C. glabrata
.
Widespread Distribution of Poribacteria in Demospongiae
Authors: Lafi Feras F. , Fuerst John A. , Fieseler Lars , Engels Cecilia , Goh Winnie Wei Ling , Hentschel Ute 
Published: June 2009

ABSTRACT
Poribacteria were found in nine sponge species belonging to six orders of Porifera from three oceans. Phylogenetic analysis revealed four distinct poribacterial clades, which contained organisms obtained from several different geographic regions, indicating that the distribution of poribacteria is cosmopolitan. Members of divergent poribacterial clades were also found in the same sponge species in three different sponge genera.
Rhodococcus rhodochrous
DSM 43269 3-Ketosteroid 9α-Hydroxylase, a Two-Component Iron-Sulfur-Containing Monooxygenase with Subtle Steroid Substrate Specificity
Authors: Petrusma M. , Dijkhuizen L. , van der Geize R. 
Published: June 2009

ABSTRACT

This paper reports the biochemical characterization of a purified and reconstituted two-component 3-ketosteroid 9α-hydroxylase (KSH). KSH of
Rhodococcus rhodochrous
DSM 43269, consisting of a ferredoxin reductase (KshB) and a terminal oxygenase (KshA), was heterologously expressed in
Escherichia coli. E. coli
cell cultures, expressing both KshA and KshB, converted 4-androstene-3,17-dione (AD) into 9α-hydroxy-4-AD (9OHAD) with a >60% molar yield over 48 h of incubation. Coexpression and copurification were critical to successfully obtain pure and active KSH. Biochemical analysis revealed that the flavoprotein KshB is an NADH-dependent reductase using flavin adenine dinucleotide as a cofactor. Reconstitution experiments confirmed that KshA, KshB, and NADH are essential for KSH activity with steroid substrates. KSH hydroxylation activity was inhibited by several divalent metal ions, especially by zinc. The reconstituted KSH displayed subtle steroid substrate specificity; a range of 3-ketosteroids, i.e., 5α-Η, 5β-Η, Δ1, and Δ4 steroids, could act as KSH substrates, provided that they had a short side chain. The formation of 9OHAD from AD by KSH was confirmed by liquid chromatography-mass spectrometry analysis and by the specific enzymatic conversion of 9OHAD into 3-hydroxy-9,10-secoandrost-1,3,5(10)-triene-9,17-dione using 3-ketosteroid Δ1-dehydrogenase. Only a single KSH is encoded in the genome of the human pathogen
Mycobacterium tuberculosis
H37Rv, shown to be important for survival in macrophages. Since no human KSH homolog exists, the
M. tuberculosis
enzyme may provide a novel target for treatment of tuberculosis. Detailed knowledge about the biochemical properties of KSH thus is highly relevant in the research fields of biotechnology and medicine.
Assessment of Bias Associated with Incomplete Extraction of Microbial DNA from Soil
Authors: Feinstein Larry M. , Sul Woo Jun , Blackwood Christopher B. 
Published: June 2009

ABSTRACTDNA extraction bias is a frequently cited but poorly understood limitation of molecular characterizations of environmental microbial communities. To assess the bias of a commonly used soil DNA extraction kit, we varied the cell lysis protocol and conducted multiple extractions on subsamples of clay, sand, and organic soils. DNA, as well as bacterial and fungal ribosomal gene copies as measured by quantitative PCR, continued to be isolated in successive extractions. When terminal restriction fragment length polymorphism was used, a significant shift in community composition due to extraction bias was detected for bacteria but not for fungi. Pyrosequencing indicated that the relative abundances of sequences from rarely cultivated groups such asAcidobacteria,Gemmatimonades, andVerrucomicrobiawere higher in the first extraction than in the sixth but that the reverse was true forProteobacteriaandActinobacteria. This suggests that the well-known phylum-level bacterial cultivation bias may be partially exaggerated by DNA extraction bias. We conclude that bias can be adequately reduced in many situations by pooling three successive extractions, and additional measures should be considered when divergent soil types are compared or when comprehensive community analysis is necessary.
Relative Diversity and Community Structure of Ciliates in Stream Biofilms According to Molecular and Microscopy Methods
Authors: Dopheide Andrew , Lear Gavin , Stott Rebecca , Lewis Gillian 
Published: June 2009

ABSTRACT
Ciliates are an important component of aquatic ecosystems, acting as predators of bacteria and protozoa and providing nutrition for organisms at higher trophic levels. Understanding of the diversity and ecological role of ciliates in stream biofilms is limited, however. Ciliate diversity in biofilm samples from four streams subject to different impacts by human activity was assessed using microscopy and terminal restriction fragment length polymorphism (T-RFLP) analysis of 18S rRNA sequences. Analysis of 3′ and 5′ terminal fragments yielded very similar estimates of ciliate diversity. The diversity detected using microscopy was consistently lower than that suggested by T-RFLP analysis, indicating the existence of genetic diversity not apparent by morphological examination. Microscopy and T-RFLP analyses revealed similar relative trends in diversity between different streams, with the lowest level of biofilm-associated ciliate diversity found in samples from the least-impacted stream and the highest diversity in samples from moderately to highly impacted streams. Multivariate analysis provided evidence of significantly different ciliate communities in biofilm samples from different streams and seasons, particularly between a highly degraded urban stream and less impacted streams. Microscopy and T-RFLP data both suggested the existence of widely distributed, resilient biofilm-associated ciliates as well as ciliate taxa restricted to sites with particular environmental conditions, with cosmopolitan taxa being more abundant than those with restricted distributions. Differences between ciliate assemblages were associated with water quality characteristics typical of urban stream degradation and may be related to factors such as nutrient availability and macroinvertebrate communities. Microscopic and molecular techniques were considered to be useful complementary approaches for investigation of biofilm ciliate communities.
Identification and Characterization of a Novel Intracellular Poly(3-Hydroxybutyrate) Depolymerase from
Bacillus megaterium
Authors: Chen Hui-Ju , Pan Shih-Chuan , Shaw Gwo-Chyuan 
Published: June 2009

ABSTRACT

A gene that codes for a novel intracellular poly(3-hydroxybutyrate) (PHB) depolymerase, designated PhaZ1, has been identified in the genome of
Bacillus megaterium
. A native PHB (nPHB) granule-binding assay showed that purified soluble PhaZ1 had strong affinity for nPHB granules. Turbidimetric analyses revealed that PhaZ1 could rapidly degrade nPHB granules in vitro without the need for protease pretreatment of the granules to remove surface proteins. Notably, almost all the final hydrolytic products produced from the in vitro degradation of nPHB granules by PhaZ1 were 3-hydroxybutyric acid (3HB) monomers. Unexpectedly, PhaZ1 could also hydrolyze denatured semicrystalline PHB, with the generation of 3HB monomers. The disruption of the
phaZ1
gene significantly affected intracellular PHB mobilization during the PHB-degrading stage in
B. megaterium
, as demonstrated by transmission electron microscopy and the measurement of the PHB content. These results indicate that PhaZ1 is functional in intracellular PHB mobilization in vivo. Some of these features, which are in striking contrast with those of other known nPHB granule-degrading PhaZs, may provide an advantage for
B. megaterium
PhaZ1 in fermentative production of the biotechnologically valuable chiral compound (
R
)-3HB.
RNA Colony Blot Hybridization Method for Enumeration of Culturable
Vibrio cholerae
and
Vibrio mimicus
Bacteria
Authors: Grim Christopher J. , Zo Young-Gun , Hasan Nur A. , Ali Afsar , Chowdhury Wasimul B. , Islam Atiqul , Rashid Mohammed H. , Alam Munirul , Morris J. Glenn , Huq Anwar , Colwell Rita R. 
Published: June 2009

ABSTRACT

A species-specific RNA colony blot hybridization protocol was developed for enumeration of culturable
Vibrio cholerae
and
Vibrio mimicus
bacteria in environmental water samples. Bacterial colonies on selective or nonselective plates were lysed by sodium dodecyl sulfate, and the lysates were immobilized on nylon membranes. A fluorescently labeled oligonucleotide probe targeting a phylogenetic signature sequence of 16S rRNA of
V. cholerae
and
V. mimicus
was hybridized to rRNA molecules immobilized on the nylon colony lift blots. The protocol produced strong positive signals for all colonies of the 15 diverse
V. cholerae
-
V. mimicus
strains tested, indicating 100% sensitivity of the probe for the targeted species. For visible colonies of 10 nontarget species, the specificity of the probe was calculated to be 90% because of a weak positive signal produced by
Grimontia
(
Vibrio
)
hollisae
, a marine bacterium. When both the sensitivity and specificity of the assay were evaluated using lake water samples amended with a bioluminescent
V. cholerae
strain, no false-negative or false-positive results were found, indicating 100% sensitivity and specificity for culturable bacterial populations in freshwater samples when
G. hollisae
was not present. When the protocol was applied to laboratory microcosms containing
V. cholerae
attached to live copepods, copepods were found to carry approximately 10,000 to 50,000 CFU of
V. cholerae
per copepod. The protocol was also used to analyze pond water samples collected in an area of cholera endemicity in Bangladesh over a 9-month period. Water samples collected from six ponds demonstrated a peak in abundance of total culturable
V. cholerae
bacteria 1 to 2 months prior to observed increases in pathogenic
V. cholerae
and in clinical cases recorded by the area health clinic. The method provides a highly specific and sensitive tool for monitoring the dynamics of
V. cholerae
in the environment. The RNA blot hybridization protocol can also be applied to detection of other gram-negative bacteria for taxon-specific enumeration.
Development of a Cell Culture Method To Isolate and Enrich
Salmonella enterica
Serotype Enteritidis from Shell Eggs for Subsequent Detection by Real-Time PCR
Authors: Day J. B. , Basavanna U. , Sharma S. K. 
Published: June 2009

ABSTRACT

Salmonella enterica
serotype Enteritidis is a major cause of nontyphoidal salmonellosis from ingestion of contaminated raw or undercooked shell eggs. Current techniques used to identify
Salmonella
serotype Enteritidis in eggs are extremely laborious and time-consuming. In this study, a novel eukaryotic cell culture system was combined with real-time PCR analysis to rapidly identify
Salmonella
serotype Enteritidis in raw shell eggs. The system was compared to the standard microbiological method of the International Organization for Standardization (Anonymous, Microbiology of food and animal feeding stuffs—horizontal method for the detection of
Salmonella
, 2002). The novel technique utilizes a mouse macrophage cell line (RAW 264.7) as the host for the isolation and intracellular replication of
Salmonella
serotype Enteritidis. Exposure of macrophages to
Salmonella
serotype Enteritidis-contaminated eggs results in uptake and intracellular replication of the bacterium, which can subsequently be detected by real-time PCR analysis of the DNA released after disruption of infected macrophages. Macrophage monolayers were exposed to eggs contaminated with various quantities of
Salmonella
serotype Enteritidis. As few as 10 CFU/ml was detected in cell lysates from infected macrophages after 10 h by real-time PCR using primer and probe sets specific for DNA segments located on the
Salmonella
serotype Enteritidis genes
sefA
and
orgC
.
Salmonella
serotype Enteritidis could also be distinguished from other non-serogroup D
Salmonella
serotypes by using the
sefA
- and
orgC
-specific primer and probe sets. Confirmatory identification of
Salmonella
serotype Enteritidis in eggs was also achieved by isolation of intracellular bacteria from lysates of infected macrophages on xylose lysine deoxycholate medium. This method identifies
Salmonella
serotype Enteritidis from eggs in less than 10 h compared to the more than 5 days required for the standard reference microbiological method of the International Organization for Standardization (Microbiology of food and animal feeding stuffs—horizontal method for the detection of
Salmonella
, 2002).
Sulfur Isotope Enrichment during Maintenance Metabolism in the Thermophilic Sulfate-Reducing Bacterium
Desulfotomaculum putei
Authors: Davidson Mark M. , Bisher M. E. , Pratt Lisa M. , Fong Jon , Southam Gordon , Pfiffner Susan M. , Reches Z. , Onstott Tullis C. 
Published: June 2009

ABSTRACT

Values of Δ
34
S (

\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \({=}{\delta}^{34}S_{HS}{-}{\delta}^{34}S_{SO_{4}}\) \end{document}

, where δ
34
S
HS
and

\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \({\delta}^{34}S_{SO_{4}}\) \end{document}

indicate the differences in the isotopic compositions of the HS
−
and SO
4
2−
in the eluent, respectively) for many modern marine sediments are in the range of −55 to −75‰, much greater than the −2 to −46‰ ε
34
S (kinetic isotope enrichment) values commonly observed for microbial sulfate reduction in laboratory batch culture and chemostat experiments. It has been proposed that at extremely low sulfate reduction rates under hypersulfidic conditions with a nonlimited supply of sulfate, isotopic enrichment in laboratory culture experiments should increase to the levels recorded in nature. We examined the effect of extremely low sulfate reduction rates and electron donor limitation on S isotope fractionation by culturing a thermophilic, sulfate-reducing bacterium,
Desulfotomaculum putei
, in a biomass-recycling culture vessel, or “retentostat.” The cell-specific rate of sulfate reduction and the specific growth rate decreased progressively from the exponential phase to the maintenance phase, yielding average maintenance coefficients of 10
−16
to 10
−18
mol of SO
4
cell
−1
h
−1
toward the end of the experiments. Overall S mass and isotopic balance were conserved during the experiment. The differences in the δ
34
S values of the sulfate and sulfide eluting from the retentostat were significantly larger, attaining a maximum Δ
34
S of −20.9‰, than the −9.7‰ observed during the batch culture experiment, but differences did not attain the values observed in marine sediments.
Functional Analysis of
cis
- and
trans
-Acting Elements of the
Candida albicans CDR2
Promoter with a Novel Promoter Reporter System
Authors: Coste Alix T. , Crittin Jérôme , Bauser Christopher , Rohde Bettina , Sanglard Dominique 
Published: June 2009

ABSTRACT

Azole resistance in
Candida albicans
can be mediated by the upregulation of the ATP binding cassette transporter genes
CDR1
and
CDR2
. Both genes are regulated by a
cis
-acting element called the drug-responsive element (DRE), with the consensus sequence 5′-CGGAWATCGGATATTTTTTT-3′, and the transcription factor Tac1p. In order to analyze in detail the DRE sequence necessary for the regulation of
CDR1
and
CDR2
and properties of
TAC1
alleles, a one-hybrid system was designed. This system is based on a

P
(CDR2)
-HIS3

reporter system in which complementation of histidine auxotrophy can be monitored by activation of the reporter system by
CDR2
-inducing drugs such as estradiol. Our results show that most of the modifications within the DRE, but especially at the level of CGG triplets, strongly reduce
CDR2
expression. The
CDR2
DRE was replaced by putative DREs deduced from promoters of coregulated genes (
CDR1
,
RTA3
, and
IFU5
). Surprisingly, even if Tac1p was able to bind these putative DREs, as shown by chromatin immunoprecipitation, those from
RTA3
and
IFU5
did not functionally replace the
CDR2
DRE. The one-hybrid system was also used for the identification of gain-of-function (GOF) mutations either in
TAC1
alleles from clinical
C. albicans
isolates or inserted in TAC1 wild-type alleles by random mutagenesis. In all, 17 different GOF mutations were identified at 13 distinct positions. Five of them (G980E, N972D, A736V, T225A, and N977D) have already been described in clinical isolates, and four others (G980W, A736T, N972S, and N972I) occurred at already-described positions, thus suggesting that GOF mutations can occur in a limited number of positions in Tac1p. In conclusion, the one-hybrid system developed here is rapid and powerful and can be used for characterization of
cis
- and
trans
-acting elements in
C. albicans
.
Amino Acid Changes in Elongation Factor Tu of
Mycoplasma pneumoniae
and
Mycoplasma genitalium
Influence Fibronectin Binding
Authors: Balasubramanian Sowmya , Kannan T. R. , Hart P. John , Baseman Joel B. 
Published: June 2009

ABSTRACT

Mycoplasma pneumoniae
and
Mycoplasma genitalium
are closely related organisms that cause distinct clinical manifestations and possess different tissue predilections despite their high degree of genome homology. We reported earlier that surface-localized
M. pneumoniae
elongation factor Tu (EF-Tu
Mp
) mediates binding to the extracellular matrix component fibronectin (Fn) through the carboxyl region of EF-Tu. In this study, we demonstrate that surface-associated
M. genitalium
EF-Tu (EF-Tu
Mg
), in spite of sharing 96% identity with EF-Tu
Mp
, does not bind Fn. We utilized this finding to identify the essential amino acids of EF-Tu
Mp
that mediate Fn interactions by generating modified recombinant EF-Tu proteins with amino acid changes corresponding to those of EF-Tu
Mg
. Amino acid changes in serine 343, proline 345, and threonine 357 were sufficient to significantly reduce the Fn binding of EF-Tu
Mp
. Synthetic peptides corresponding to this region of EF-Tu
Mp
(EF-Tu
Mp
340-358) blocked both recombinant EF-Tu
Mp
and radiolabeled
M. pneumoniae
cell binding to Fn. In contrast, EF-Tu
Mg
340-358 peptides exhibited minimal blocking activity, reinforcing the specificity of EF-Tu-Fn interactions as mediators of microbial colonization and tissue tropism.