bluuebunny/crossref_metadata_2025_split huggingface dataset:
Actinobacterial 16S rRNA genes from freshwater habitats cluster in four distinct lineages
Authors: Warnecke Falk , Amann Rudolf , Pernthaler Jakob 
Published: December 2004

SummaryWe analysed the phylogenetic relatedness of 16S rRNA genes from freshwater bacteria affiliated with the class Actinobacteria. A polymerase chain reaction assay was developed to identify reliably rare Actinobacteria‐related inserts within 16S rRNA gene clone libraries. In 18 libraries constructed from seven freshwater systems, altogether 63 actinobacterial sequence types were collected from a total of > 1800 clones. Sixty of the newly obtained sequences grouped within four distinct phylogenetic lineages. They constitute approximately 75% of the nearly complete sequences within these clusters that are presently available. A comparison with > 300 sequences from various soil habitats revealed that two of these monophyletic actinobacterial clades (acI and acII) almost exclusively harbour 16S rRNA sequence types from freshwaters and estuaries. This may indicate that such bacteria are not inoculated to freshwaters from terrestrial sources, but are autochthonous components of freshwater microbial assemblages. In contrast, sequence types from freshwaters, marine sediments and soils were clearly mixed in another of the actinobacterial lineages (acIV). Sequence divergence within acIV was the highest of all four lineages (88% minimum similarity), which potentially reflects its radiation across several habitat types. Within the freshwater lineages, groups of essentially identical sequence types were retrieved from geographically distant aquatic systems with strikingly different hydrological and limnological characteristics. This points to the necessity to investigate genotypic variability, in situ abundances and activities of these Actinobacteria in freshwater plankton in greater detail by cultivation‐independent techniques.
Characterization of two alkane hydroxylase genes from the marine hydrocarbonoclastic bacterium Alcanivorax borkumensis
Authors: Van Beilen Jan B. , Marín Mercedes M. , Smits Theo H. M. , Röthlisberger Martina , Franchini Alessandro G. , Witholt Bernard , Rojo Fernando 
Published: December 2004

SummaryThe marine γ‐Proteobacterium Alcanivorax borkumensis is highly specialized in the assimilation of aliphatic hydrocarbons, and makes up a large part of the biomass in oil‐polluted marine environments. In addition to the previously identified alkane hydroxylase AlkB1, a second alkane hydroxylase (AlkB2) showing 65% identity to the Pseudomonas aeruginosa AlkB2 alkane hydroxylase was identified. Unlike alkB1, alkB2 is not flanked by genes involved in alkane metabolism. Heterologous expression of the A. borkumensis AP1 alkB1 and alkB2 genes showed that they encode functional alkane hydroxylases with substrate ranges similar to those of their P. putida and P. aeruginosa homologues. The transcription initiation sites and levels of the alkB1, alkB2 and alkS mRNA transcripts were determined. Expression of both alkB1 and alkB2 was induced by alkanes, but transcripts corresponding to alkB1 were much more abundant than those of alkB2. An inverted repeat similar to the binding site for the P. putida GPo1 transcriptional activator AlkS was present upstream of the promoters for alkB1 and alkB2, although that of alkB2 was less well conserved, and only the transcriptional fusion of promoter PalkB1 to the reporter gene lacZ efficiently responded to n‐octane. Contrary to what has been found for the P. putida GPo1 alkane degradation pathway, expression of the A. borkumensis AP1 alkS gene was not induced by alkanes, and an AlkS binding site was not present upstream of the promoter for alkS. This indicates that, in spite of the clear similarities, the A. borkumensis alk‐genes are regulated by a strategy different from that of the P. putida GPo1 alk genes.
Diversity of prokaryotes and methanogenesis in deep subsurface sediments from the Nankai Trough, Ocean Drilling Program Leg 190
Authors: Newberry Carole J. , Webster Gordon , Cragg Barry A. , Parkes R. John , Weightman Andrew J. , Fry John C. 
Published: December 2004

SummaryDiversity of Bacteria and Archaea was studied in deep marine sediments by PCR amplification and sequence analysis of 16S rRNA and methyl co‐enzyme M reductase (mcrA) genes. Samples analysed were from Ocean Drilling Program (ODP) Leg 190 deep subsurface sediments at three sites spanning the Nankai Trough in the Pacific Ocean off Shikoku Island, Japan. DNA was amplified, from three depths at site 1173 (4.15, 98.29 and 193.29 mbsf; metres below the sea floor), and phylogenetic analysis of clone libraries showed a wide variety of uncultured Bacteria and Archaea. Sequences of Bacteria were dominated by an uncultured and deeply branching ‘deep sediment group’ (53% of sequences). Archaeal 16S rRNA gene sequences were mainly within the uncultured clades of the Crenarchaeota. There was good agreement between sequences obtained independently by cloning and by denaturing gradient gel electrophoresis. These sequences were similar to others retrieved from marine sediment and other anoxic habitats, and so probably represent important indigenous bacteria. The mcrA gene analysis suggested limited methanogen diversity with only three gene clusters identified within the Methanosarcinales and Methanobacteriales. The cultivated members of the Methanobacteriales and some of the Methanosarcinales can use CO2 and H2 for methanogenesis. These substrates also gave the highest rates in 14C‐radiotracer estimates of methanogenic activity, with rates comparable to those from other deep marine sediments. Thus, this research demonstrates the importance of the ‘deep sediment group’ of uncultured Bacteria and links limited diversity of methanogens to the dominance of CO2/H2 based methanogenesis in deep sub‐seafloor sediments.
The clonal structure of Bacillus thuringiensis isolates from north‐east Poland does not correlate with their cry gene diversity
Authors: Świȩcicka Izabela , Mahillon Jacques 
Published: December 2004

SummaryThe genetic relationship among 103 natural Bacillus thuringiensis isolates was investigated on the basis of polymerase chain reaction amplification of their specific crystal (cry) protein type genes and chromosomal DNA profiling by pulsed‐field gel electrophoresis (PFGE). The strains were recovered from the intestines of small wild rodents and insectivores from the Biebrza National Park and the Łomża Landscape Park of the Narew River Valley in north‐east Poland. The percentage of B. thuringiensis strains harbouring genes coding for toxins active against Lepidoptera (cry1, cry2, cry9) was very high (64%) compared with that of Diptera‐specific strains (cry4, 14%). No strain with cry genes coding for proteins directed against coleopteran larvae and nematodes was found. After digestion with NotI and AscI, only nine PFGE pulsotypes were observed among all isolates, indicating a clonal structure for the B. thuringiensis population from NE Poland. Interestingly, no correlation was observed between the DNA pulsotype strains and their crystal gene content and diversity. These results therefore emphasize the importance of cry gene horizontal transfer occurring among natural isolates of B. thuringiensis.
Microbial indicator groups in acidic mining lakes
Authors: Wenderoth Dirk F. , Abraham Wolf‐Rainer 
Published: December 2004

SummaryThe salt composition of 14 acidic lakes was analysed, and their microbial communities were compared by their 16S rDNA‐based single‐strand conformation polymorphism (SSCP) fingerprints. The lakes were grouped into three chemically distinct types, and operational taxonomic units (OTUs) were identified in the 16S rDNA SSCP fingerprints. Discriminant analysis between these groups using a multivariate approach showed that not a single organism, but rather a set of seven OTUs (Ferromicrobium acidophilum, Bacillus pumilus, Acidophilium organovorum, Paenibacillus chibensis, Acidocella sp. and two different alpha‐Proteobacteria), was found to be indicative for a given lake type. We conclude that, for these lakes, not single indicator strains but indicator groups exist that are controlled by the ionic composition of the lakes.
Interaction of the Plasmid-Encoded Quinolone Resistance Protein Qnr with
Escherichia coli
DNA Gyrase
Authors: Tran John H. , Jacoby George A. , Hooper David C. 
Published: December 2004

ABSTRACT

Quinolone resistance normally arises by mutations in the chromosomal genes for type II topoisomerases and by changes in the expression of proteins that control the accumulation of quinolones inside bacteria. A novel mechanism of plasmid-mediated quinolone resistance was recently reported that involves DNA gyrase protection by a pentapeptide repeat family member called Qnr. This family includes two other members, McbG and MfpA, that are also involved in resistance to gyrase inhibitors. Purified Qnr-His
6
was shown to protect
Escherichia coli
DNA gyrase directly from inhibition by ciprofloxacin. Here we have provided a biochemical basis for the mechanism of quinolone resistance. We have shown that Qnr can bind to the gyrase holoenzyme and its respective subunits, GyrA and GyrB. The binding of Qnr to gyrase does not require the presence of the complex of enzyme, DNA, and quinolone, since binding occurred in the absence of relaxed DNA, ciprofloxacin, or ATP. We hypothesize that the formation of Qnr-gyrase complex occurs before the formation of the cleavage complex. Furthermore, there was a decrease in DNA binding by gyrase when the enzyme interacted with Qnr. Therefore, it is possible that the reaction intermediate recognized by Qnr is one early in the gyrase catalytic cycle, in which gyrase has just begun to interact with DNA. Quinolones bind later in the catalytic cycle and stabilize a ternary complex consisting of the drug, gyrase, and DNA. By lowering gyrase binding to DNA, Qnr may reduce the amount of holoenzyme-DNA targets for quinolone inhibition.
Pharmacodynamic Profile of Telithromycin against Macrolide- and Fluoroquinolone-Resistant
Streptococcus pneumoniae
in a Neutropenic Mouse Thigh Model
Authors: Tessier Pamela R. , Mattoes Holly M. , Dandekar Prachi K. , Nightingale Charles H. , Nicolau David P. 
Published: December 2004

ABSTRACT

The new ketolide telithromycin has potent in vitro activity against
Streptococcus pneumoniae
, including strains resistant to penicillin, macrolides, and fluoroquinolones. The aim of the present study was to define the pharmacodynamic profile of telithromycin against
S. pneumoniae
strains with various resistance profiles in an in vivo system. Ten
S. pneumoniae
strains were studied; seven exhibited penicillin resistance, six demonstrated macrolide resistance, and two exhibited gatifloxacin resistance. The telithromycin MICs for all isolates were ≤0.5 μg/ml. Using the murine thigh infection model, CD-1/ICR mice were rendered neutropenic and were then inoculated with 10
5
to 10
6
CFU of
S. pneumoniae
per thigh. Telithromycin was administered orally at doses ranging from 25 to 800 mg/kg of body weight/day, with the doses administered one, two, three, or four times a day. The activity of telithromycin was assessed by determination of the change in the bacterial density in thigh tissue after 24 h of treatment for each treatment group and the untreated controls. Pharmacokinetic studies of telithromycin were conducted in infected, neutropenic animals. The levels of protein binding by telithromycin in mice ranged from 70 to 95% over the observed range of pharmacokinetic concentrations. By using either the total or the free concentrations of telithromycin, the area under the concentration-time curve (AUC)/MIC ratio was a strong determinant of the response against
S. pneumoniae
, regardless of the phenotypic resistance profile. The maximal efficacy (the 95% effective dose) against this cohort of
S. pneumoniae
strains and bacterial inhibition (stasis) of telithromycin were predicted by ratios of the AUC for the free drug concentration/MIC of approximately 1,000 and 200, respectively.
Pharmacodynamic Modeling of Ciprofloxacin Resistance in
Staphylococcus aureus
Authors: Campion Jeffrey J. , McNamara Patrick J. , Evans Martin E. 
Published: December 2004

ABSTRACT

Three pharmacodynamic models of increasing complexity, designed for two subpopulations of bacteria with different susceptibilities, were developed to describe and predict the evolution of resistance to ciprofloxacin in
Staphylococcus aureus
by using pharmacokinetic, viable count, subpopulation, and resistance mechanism data obtained from in vitro system experiments. A two-population model with unique growth and killing rate constants for the ciprofloxacin-susceptible and -resistant subpopulations best described the initial killing and subsequent regrowth patterns observed. The model correctly described the enrichment of subpopulations with low-level resistance in the parent cultures but did not identify a relationship between the time ciprofloxacin concentrations were in the mutant selection window (the interval between the MIC and the mutant prevention concentration) and the enrichment of these subpopulations. The model confirmed the importance of resistant variants to the emergence of resistance by successfully predicting that resistant subpopulations would not emerge when a low-density culture, with a low probability of mutants, was exposed to a clinical dosing regimen or when a high-density culture, with a higher probability of mutants, was exposed to a transient high initial concentration designed to rapidly eradicate low-level resistant
grlA
mutants. The model, however, did not predict or explain the origin of variants with higher levels of resistance that appeared and became the predominant subpopulation during some experiments or the persistence of susceptible bacteria in other experiments where resistance did not emerge. Continued evaluation of the present two-population pharmacodynamic model and development of alternative models is warranted.
Desketoneoenactin-Siderophore Conjugates for
Candida
: Evidence of Iron Transport-Dependent Species Selectivity
Authors: Bernier Geneviève , Girijavallabhan Vinay , Murray Aaron , Niyaz Noormohamed , Ding Pingyu , Miller Marvin J. , Malouin François 
Published: December 2004

ABSTRACT

We investigated the inhibitory activity of synthetic isocyanurate-based as well as linear mono- and trihydroxamate siderophore-drug conjugates against
Candida
spp. The conjugated drug was 13C-desketoneoenactin (DE). The MICs of siderophore-drug conjugates were determined in the absence and presence of 2,2′-dipyridyl to restrict iron availability. The ability of various siderophore types to promote growth in an iron-restricted medium was also assayed. Addition of a siderophore portion to the drug strongly impaired the inhibitory activity of DE. However, the activity of the drug conjugates was increased by up to 16-fold in iron-depleted medium for species having their growth strongly promoted by most hydroxamate-type siderophores (
C. albicans
,
C. stellatoidea
, and
C. pseudotropicalis
). The uptake of
55
Fe from ferrichrome and from two siderophore-drug conjugates was improved when
C. albicans
cells were grown in a low-iron medium. In the same assay, unlabeled ferrichrome was able to compete with the uptake of
55
Fe from both conjugates, indicating a common mechanism of uptake. A
C. albicans
strain lacking the siderophore transporter CaSit1/CaArn1 was not able to use ferrichrome or the synthetic ornithine-based trihydroxamate siderophore for growth promotion and was much less susceptible to the siderophore-drug conjugates than its isogenic parent strain. In summary, the ability of some
Candida
spp. to use ferrichrome-like siderophores for growth promotion explains the selective activity of hydroxamate-drug conjugates, and this activity seems to be related to the presence, in
C. albicans
, of the siderophore transporter CaSit1/CaArn1. New conjugate designs are necessary to fully restore or improve the initial DE activity.
Antisense Phosphorodiamidate Morpholino Oligomer Length and Target Position Effects on Gene-Specific Inhibition inEscherichia coli
Authors: Deere Jesse , Iversen Pat , Geller Bruce L. 
Published: December 2004

ABSTRACTPhosphorodiamidate morpholino oligomers (PMOs) are synthetic DNA analogs that inhibit gene expression in a sequence-dependent manner. PMOs of various lengths (7 to 20 bases) were tested for inhibition of luciferase expression inEscherichia coli. Shorter PMOs generally inhibited luciferase greater than longer PMOs. Conversely, in bacterial cell-free protein synthesis reactions, longer PMOs inhibited equally or more than shorter PMOs. Overlapping, isometric (10-base) PMOs complementary to the region around the start codon of luciferase inhibited to different extents in bacterial cell-free protein expression reactions. Including the anti-start codon in PMOs was not required for maximal inhibition. PMOs targeted to 5′ nontranslated or 3′ coding regions within luciferase mRNA did not inhibit, except for one PMO targeted to the ribosome-binding site. Inhibition of luciferase expression correlated negatively with the predicted secondary structure of mRNA regions targeted by PMO but did not correlate with C+G content of targeted regions. The effects of PMO length and position were corroborated by using PMOs (6 to 20 bases) targeted toacpP, a gene required for viability. Because inhibition by PMOs of ∼11 bases was unexpected based on previous results in eukaryotes, we tested an 11-base PMO in HeLa cells and reticulocyte cell-free protein synthesis reactions. The 11-base PMO significantly inhibited luciferase expression in HeLa cells, although less than did a 20-base PMO. In reticulocyte cell-free reactions, there was a trend toward more inhibition with longer PMOs. These studies indicate that strategies for designing PMOs are substantially different for prokaryotic than eukaryotic targets.
Analysis of the Genetic Variability of Virulence-Related Loci in Epidemic Clones of Methicillin-Resistant
Staphylococcus aureus
Authors: Gomes A. R. , Vinga S. , Zavolan M. , de Lencastre H. 
Published: December 2004

ABSTRACT

Methicillin-resistant
Staphylococcus aureus
(MRSA) isolates have previously been classified into major epidemic clonal types by pulsed-field gel electrophoresis in combination with multilocus sequence typing (MLST) and staphylococcal cassette chromosome
mec
typing. We aimed to investigate whether genetic variability in potentially polymorphic domains of virulence-related factors could provide another level of differentiation in a diverse collection of epidemic MRSA clones. The target regions of strains representative of epidemic clones and genetically related methicillin-susceptible
S. aureus
isolates from the 1960s that were sequenced included the R domains of
clfA
and
clfB
; the D, W, and M regions of
fnbA
and
fnbB
; and three regions in the
agr
operon. Sequence variation ranged from very conserved regions, such as those for RNAIII and the
agr
interpromoter region, to the highly polymorphic R regions of the
clf
genes. The sequences of the
clf
R domains could be grouped into six major sequence types on the basis of the sequences in their 3′ regions. Six sequence types were also observed for the
fnb
sequences at the amino acid level. From an evolutionary point of view, it was interesting that a small DNA stretch at the 3′
clf
R-domain sequence and the
fnb
sequences agreed with the results of MLST for this set of strains. In particular,
clfB
R-domain sequences, which had a high discriminatory capacity and with which the types distinguished were congruent with those obtained by other molecular typing methods, have potential for use for the typing of
S. aureus
. Clone- and strain-specific sequence motifs in the
clf
and
fnb
genes may represent useful additions to a typing methodology with a DNA array.
Streptococcus pyogenes
Pharyngeal Isolates with Reduced Susceptibility to Ciprofloxacin in Spain: Mechanisms of Resistance and Clonal Diversity
Authors: Albertí Sebastián , Cortés Guadalupe , García-Rey Cesar , Rubio Carmen , Baquero Fernando , García-Rodríguez José Ángel , Bouza Emilio , Aguilar Lorenzo 
Published: December 2004

ABSTRACT

A survey of
emm
gene sequences and an analysis of the pulsed-field electrophoretic profiles of 30
Streptococcus pyogenes
isolates with reduced susceptibilities to ciprofloxacin detected the prevalence of isolates with
emm
type 6 and considerable genetic diversity among isolates. The mechanism of ciprofloxacin resistance in these isolates was based on point mutations in topoisomerase IV subunit C encoded by
parC
, mainly replacement of serine-79 by alanine.
Genetic Determinant of Intrinsic Quinolone Resistance in
Fusobacterium canifelinum
Authors: Conrads Georg , Citron Diane M. , Goldstein Ellie J. C. 
Published: December 2004

ABSTRACT

Fourteen fluoroquinolone-resistant fusobacterial strains, originating from cats or dogs, were characterized by sequencing of the 16S-23S and 16S rRNA genes and DNA-DNA hybridization and were described as a new species,
Fusobacterium canifelinum
. All of the strains are intrinsically resistant (MIC, >4 g/ml) to levofloxacin and other fluoroquinolones. Compared to the quinolone resistance-determining region (
gyrA
) of the susceptible relative
F. nucleatum
, we found that Ser79 was replaced with leucine and Gly83 was replaced with arginine.
In Vitro and In Vivo Synergistic Activities of Linezolid Combined with Subinhibitory Concentrations of Imipenem against Methicillin-Resistant
Staphylococcus aureus
Authors: Jacqueline Cédric , Navas Dominique , Batard Eric , Miegeville Anne-Françoise , Le Mabecque Virginie , Kergueris Marie-France , Bugnon Denis , Potel Gilles , Caillon Jocelyne 
Published: December 2004

ABSTRACT

Indifference or moderate antagonism of linezolid combined with other antibiotics in vitro and in vivo have mainly been reported in the literature. We have assessed the in vitro activities of linezolid, alone or in combination with imipenem, against methicillin-resistant
Staphylococcus aureus
(MRSA) strains using the dynamic checkerboard and time-kill curve methods. Linezolid and low concentrations of imipenem had a synergistic effect, leading us to evaluate the in vivo antibacterial activity of the combination using the rabbit endocarditis experimental model. Two MRSA strains were used for in vivo experiments: one was a heterogeneous glycopeptide-intermediate clinical
S. aureus
strain isolated from blood cultures, and the other was the
S. aureus
COL reference strain. Animals infected with one of two MRSA strains were randomly assigned to one of the following treatments: no treatment (controls), linezolid (simulating a dose in humans of 10 mg/kg of body weight every 12 h), a constant intravenous infusion of imipenem (which allowed the steady-state concentration of about 1/32 the MIC of imipenem for each strain to be reached in serum), or the combination of both treatments. Linezolid and imipenem as monotherapies exhibited no bactericidal activity against either strain. The combination of linezolid plus imipenem showed in vivo bactericidal activity that corresponded to a decrease of at least 4.5 log CFU/g of vegetation compared to the counts for the controls. In conclusion, the combination exhibited synergistic and bactericidal activities against two MRSA strains after 5 days of treatment. The combination of linezolid plus imipenem appears to be promising for the treatment of severe MRSA infections and merits further investigations to explore the mechanism underlying the synergy between the two drugs.
Relationship between Copper, Glycopeptide, and Macrolide Resistance among
Enterococcus faecium
Strains Isolated from Pigs in Denmark between 1997 and 2003
Authors: Hasman Henrik , Aarestrup Frank M. 
Published: December 2004

ABSTRACT

A significant relationship between copper resistance (
tcrB
), glycopeptide resistance (Tn
1546
), and macrolide resistance [
erm
(B)] in
Enterococcus faecium
isolated from pigs was found. The
tcrB
gene was located closely upstream of the Tn
1546
element. However, the continued use of copper sulfate has not been able to maintain high levels of macrolide and glycopeptide resistance.