bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of a new solvent‐responsive gene locus in Pseudomonas putida F1 and its functionalization as a versatile biosensor
Authors: Phoenix P. , Keane A. , Patel A. , Bergeron H. , Ghoshal S. , Lau P. C. K.
Published: December 2004

A new gene cluster, designated sepABC and a divergently transcribed sepR, was found downstream of the two‐component todST phosphorelay system that regulates toluene degradation (the tod pathway) in Pseudomonas putida F1 (PpF1). The deduced amino acid sequences encoded by sepABC show a high homology to bacterial proteins known to be involved in solvent efflux or multidrug pumps. SepA, SepB and SepC are referred to be periplasmic, inner membrane and outer membrane efflux proteins respectively. Effects on growth of various PpF1 mutants compared to that of the wild type in the presence of toluene indicated a possible protective role of the solvent efflux system in a solvent‐stressed environment. Growth tests with the complemented mutants confirmed the involvement of the Sep proteins in conferring solvent tolerance. The sepR gene encodes a 260‐residue polypeptide that is a member of the E. coli IclR repressor protein family. The repressor role of SepR was established by conducting tests with a sep‐lacZ transcriptional fusion in Escherichia coli and PpF1, expression of SepR as a maltose‐binding fusion protein in a DNA binding assay, and mRNA analysis. Southern hybridization experiments and analysis of the P. putida KT2440 genome sequence indicated that sepR is a relatively rare commodity compared to homologues of the sepABC genes. We developed a whole‐cell bioluminescent biosensor, PpF1G4, which contains a chromosomally based sep‐lux transcriptional fusion. The biosensor showed significant induction of the sepABC genes by a wide variety of aromatic molecules, including benzene, toluene, ethylbenzene, and all three isomers of xylene (BTEX), naphthalene, and complex mixtures of aliphatic and aromatic hydrocarbons. PpF1G4 represents a second‐generation biosensor that is not based on a catabolic promoter but is nonetheless inducible by aromatic pollutants and moreover functional under nutrient‐rich conditions.
Heavy metal tolerance and metal homeostasis in Pseudomonas putida as revealed by complete genome analysis
Authors: Cánovas David , Cases Ildefonso , De Lorenzo Víctor
Published: December 2004

The genome of Pseudomonas putida KT2440 encodes an unexpected capacity to tolerate heavy metals and metalloids. The availability of the complete chromosomal sequence allowed the categorization of 61 open reading frames likely to be involved in metal tolerance or homeostasis, plus seven more possibly involved in metal resistance mechanisms. Some systems appeared to be duplicated. These might perform redundant functions or be involved in tolerance to different metals. In total, P. putida was found to bear two systems for arsenic (arsRBCH), one for chromate (chrA), four to six systems for divalent cations (two cadA and two to four czc chemiosmotic antiporters), two systems for monovalent cations: pacS, cusCBA (plus one cryptic silP gene containing a frameshift mutation), two operons for Cu chelation (copAB), one metallothionein for metal(loid) binding, one system for Te/Se methylation (tpmT) and four ABC transporters for the uptake of essential Zn, Mn, Mo and Ni (one nikABCDE, two znuACB and one mobABC). Some of the metal‐related clusters are located in gene islands with atypical genome signatures. The predicted capacity of P. putida to endure exposure to heavy metals is discussed from an evolutionary perspective.
Proteome reference map of Pseudomonas putida strain KT2440 for genome expression profiling: distinct responses of KT2440 and Pseudomonas aeruginosa strain PAO1 to iron deprivation and a new form of superoxide dismutase
Authors: Heim S. , Ferrer M. , Heuer H. , Regenhardt D. , Nimtz M. , Timmis K. N.
Published: December 2004

The genome sequence of Pseudomonas putida strain KT2440, a nutritionally versatile, saprophytic and plant root‐colonizing Gram‐negative soil bacterium, was recently determined by K. E. Nelson et al. (2002, Environ Microbiol 4: 799–808). Here, we present a two‐dimensional gel protein reference map of KT2440 cells grown in mineral salts medium with glucose as carbon source. Proteins were identified by matrix‐assisted laser desorption ionization time‐of‐flight (MALDI‐TOF) analysis, in conjunction with an in‐house database developed from the genome sequence of KT2440, and ≈  200 two‐dimensional gel spots were assigned. The map was used to assess the genomic response of KT2440 to iron limitation stress and to compare this response with that of the closely related facultative human pathogen Pseudomonas aeruginosa strain PAO1. The synthesis of about 25 proteins was affected in both strains, including four prominent upregulated ferric uptake regulator (Fur) protein‐dependent proteins, but there were also striking differences in their proteome responses, for example in the expression of superoxide dismutases (Sod), which may indicate important roles of iron‐responsive functions in the adaptation of these two bacteria to different lifestyles. The Sod enzyme of KT2440 was shown to be a novel heterodimer of the SodA and SodB polypeptides.
Microcolonies, quorum sensing and cytotoxicity determine the survival of Pseudomonas aeruginosa biofilms exposed to protozoan grazing
Authors: Matz Carsten , Bergfeld Tanja , Rice Scott A. , Kjelleberg Staffan
Published: December 2004

This study was based on the hypothesis that biofilms of the opportunistic pathogen Pseudomonas aeruginosa are successfully adapted to situations of protozoan grazing. We tested P. aeruginosa wild type and strains that were genetically altered, in structural and regulatory features of biofilm development, in response to the common surface‐feeding flagellate Rhynchomonas nasuta. Early biofilms of the wild type showed the formation of grazing resistant microcolonies in the presence of the flagellate, whereas biofilms without the predator were undifferentiated. Grazing on biofilms of quorum sensing mutants (lasR and rhlR/lasR) also resulted in the formation of microcolonies, however, in lower numbers and size compared to the wild type. Considerably fewer microcolonies than the wild type were formed by mutant cells lacking type IV pili, whereas no microcolonies were formed by flagella‐deficient cells. The alginate‐overproducing strain PDO300 developed larger microcolonies in response to grazing. These observations suggest a role of quorum sensing in early biofilms and involvement of flagella, type IV pili, and alginate in microcolony formation in the presence of grazing. More mature biofilms of the wild type exhibited acute toxicity to the flagellate R. nasuta. Rapid growth of the flagellate on rhlR/lasR mutant biofilms indicated a key role of quorum sensing in the upregulation of lethal factors and in grazing protection of late biofilms. Both the formation of microcolonies and the production of toxins are effective mechanisms that may allow P. aeruginosa biofilms to resist protozoan grazing and to persist in the environment.
Actinobacterial 16S rRNA genes from freshwater habitats cluster in four distinct lineages
Authors: Warnecke Falk , Amann Rudolf , Pernthaler Jakob
Published: December 2004

We 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

The 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

Diversity 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

The 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

The 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.
A fuzzy approach to active usage parameter control in IEEE 802.11b wireless networks
Authors: Soud David , Kazemian Hassan B.
Published: December 2004

Abstract: Usage parameter control (UPC) provides support for quality of service across heterogeneous networks. For the network operator UPC assists in limiting network usage through traffic shaping, to prevent unacceptable delay. Traditional methods to apply UPC involve the generic cell rate algorithm or ‘leaky bucket’ algorithm, now commonly implemented in asynchronous transmission mode networks. This paper proposes a novel form of UPC for 802.11b wireless networks. The method proposed measures the rate of individual network flows to actively manage link utilization using a fuzzy logic controller (FLC). The FLC monitors the flow rate and adjusts the sending transmissions to stabilize flows as close to the optimum desired rate as possible. Imposing UPC and using the FLC within a packet switched TCP network enforces cooperation between competing streams of traffic. After carrying out experiments within a wireless network, the results obtained significantly improve upon a ‘best effort’ service.
Contraception in the chimpanzee: 12‐year experience at the CIRMF Primate Centre, Gabon
Authors: Bourry Olivier , Peignot Patricia , Rouquet Pierre
Published: December 2004

Abstract:  The Primate Centre of the International Center for Medical Research in Franceville, Gabon (CIRMF) has had a chimpanzee (Pan troglodytes) colony for more than 20 years. A contraceptive policy was started in 1990, following a rapid increase in the number of individuals in the 1980s. Intrauterine devices were first used in 24 females; 10 failures occurred over a period of about 4 years. Depo‐provera® was then used in 28 chimpanzees for between 4 months and 4 years; 10 failures occurred, the animals gained weight, and cyclic swelling of the external genitalia was markedly modified. Starting in late 2000, 25 females received progestin implants; only one failure has so far occurred, and the main side effect is a complete abrogation of cyclic sexual swelling.
Genetic differences in leverpress escape/avoidance conditioning in seven mouse strains
Authors: Brennan F. X.
Published: December 2004

Studies of inbred mouse strains can provide us with important information about the genetic basis of learning and memory. The present experiment studies the acquisition of a leverpress escape/avoidance task in six commonly studied inbred mouse strains (C57BL/6NCrlBR, DBA/2NCrlBR, C3H/HeJ, FVB/NJ, BALB/cByJ and 129S6/SvEvTac), and one outbred strain, the CD1. Results indicated that the strains formed three discrete performance clusters. The C57BL/6NCrlBR, C3HeB/FeJ, BALB/cByJ, and CD1 strains acquired the avoidance response comparably to Sprague‐Dawley rats, avoiding approximately 40% of shocks by the fourth and final training session. The 129S6/SvEvTac and FVB/NJ were extremely poor at the avoidance task throughout training. The FVB/NJ strain remained in an escape mode, while the 129S6/SvEvTac animals performed few responses of any type. Finally, the DBA/2NCrlBR strain performed exceptionally well, avoiding over 90% of the shocks by the final session. Results are discussed in terms of genetic differences in learning and how the nigrostriatal dopamine system may mediate the observed differences.
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

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

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

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

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

Phosphorodiamidate 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

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

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

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

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

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.
Effect of Antacids and Ranitidine on the Single-Dose Pharmacokinetics of Fosamprenavir
Authors: Ford Susan L. , Wire Mary B. , Lou Yu , Baker Katherine L. , Stein Daniel S.
Published: December 2004

Single doses of MAALOX TC and ranitidine were administered separately with 1,400 mg of fosamprenavir (FPV). MAALOX TC decreased the area under the concentration-time curve from 0 to 24 h (AUC
0-24
) for plasma amprenavir (APV) by 18% and the maximum concentration of drug in serum (
C
max
) by 35%; the plasma APV concentration at 12 h (
C
12
) increased by 14%. Ranitidine at 300 mg decreased the AUC
0-24
for plasma APV by 30% and
C
max
by 51%;
C
12
was unchanged. FPV may be coadministered with antacids without concern and without separation in dosing; however, caution is recommended when FPV is coadministered with histamine
2
- receptor antagonists or proton pump inhibitors.
Recommendation of an Appropriate Medium for In Vitro Drug Susceptibility Testing of the Fish Pathogen
Tenacibaculum maritimum
Authors: Avendaño-Herrera Ruben , Irgang Rute , Núñez Soledad , Romalde Jesús L. , Toranzo Alicia E.
Published: December 2004

In the present study, Anacker and Ordal agar, marine agar (MA), and
Flexibacter maritimus
medium (FMM) were compared with the dilute versions of Mueller-Hinton agar (DMHA) medium recommended by the National Committee for Clinical Laboratory Standards (NCCLS) for their use in disk diffusion tests with
Tenacibaculum maritimum
strains and to calculate the MICs of five drugs by the Etest method. Preliminary growth tests performed with 32 strains of this pathogen on each medium revealed that all strains failed to grow on DMHA, while the remaining media supported good growth of all isolates. In the susceptibility tests, which were carried out with the other three media, all strains were resistant to oxolinic acid and were highly susceptible to amoxicillin and trimethoprim-sulfamethoxazole, showing a good correspondence with the Etest values, which ranged from 0.064 to 0.75 and 0.006 to 1.5 μg/ml, respectively. Enrofloxacin and oxytetracycline produced significantly smaller inhibition zones and MICs on MA than on the other media assayed. However, fast, clear, and well-defined zones of inhibition were displayed for all strains at 24 h of incubation only on FMM by both the disk diffusion assay and Etest. In addition, FMM prepared with commercial sea salts instead of seawater was also suitable for bacterial isolation as well as for susceptibility testing. On the basis of these results, the use of FMM to determine the in vitro susceptibility of
T. maritimum
and its inclusion in a future revision of the NCCLS M42 report are recommended.
Effect of Antibiotics on Group A Streptococcus Exoprotein Production Analyzed by Two-Dimensional Gel Electrophoresis
Authors: Tanaka Megumi , Hasegawa Tadao , Okamoto Akira , Torii Keizo , Ohta Michio
Published: December 2004

High-dose clindamycin (CLDM) and benzylpenicillin (PCG) are the recommended chemotherapeutic remedies for toxic shock-like syndrome caused by group A streptococci. One reason for this is that it has been shown that CLDM suppresses the expression of some exoproteins, e.g., SpeB, SpeA, and streptolysin O (Slo). We analyzed the effects of antibiotics on the production of whole exoproteins by two-dimensional gel electrophoresis. Unexpectedly, we found that the levels of several exoproteins, Slo, NAD
+
-glycohydrolase (Nga), M protein, and Sic, were increased by CLDM treatment, although we also confirmed previous findings that the levels of various exoproteins, including SpeB, were decreased. The increases in exoprotein levels were also detected by using other protein synthesis inhibitor antibiotics: erythromycin, kanamycin, tetracycline, chloramphenicol, and linezolid. Peptidoglycan synthesis inhibitors (such as PCG, cefazolin, and imipenem), DNA replication inhibitors (such as gatifloxacin), and an RNA polymerase inhibitor (rifampin) did not have significant effects on exoprotein production. The combination of CLDM and PCG had no advantageous effects with regard to exoprotein production compared to the effect achieved with CLDM alone. We also analyzed the transcriptional levels of
slo
and
nga
by reverse transcription-PCR and found that this change was also detected at the transcriptional level. Furthermore, the phenomenon was seen not only in strains of the M1 serotype but also in strains of the other M serotypes. Our study suggests that the clinical effectiveness of CLDM might be due to the inhibition of the production of a limited number of exoproteins.
Antileishmanial Activity of Parthenolide, a Sesquiterpene Lactone Isolated from
Tanacetum parthenium
Authors: Tiuman Tatiana Shioji , Ueda-Nakamura Tânia , Garcia Cortez Diógenes Aparício , Dias Filho Benedito Prado , Morgado-Díaz José Andrés , de Souza Wanderley , Nakamura Celso Vataru
Published: December 2004

The in vitro activity of parthenolide against
Leishmania amazonensis
was investigated. Parthenolide is a sesquiterpene lactone purified from the hydroalcoholic extract of aerial parts of
Tanacetum parthenium
. This isolated compound was identified through spectral analyses by UV, infrared,
1
H and
13
C nuclear magnetic resonance imaging, DEPT (distortionless enhancement by polarization transfer), COSY (correlated spectroscopy), HMQC (heteronuclear multiple-quantum coherence), and electron spray ionization-mass spectrometry. Parthenolide showed significant activity against the promastigote form of
L. amazonensis
, with 50% inhibition of cell growth at a concentration of 0.37 μg/ml. For the intracellular amastigote form, parthenolide reduced by 50% the survival index of parasites in macrophages when it was used at 0.81 μg/ml. The purified compound showed no cytotoxic effects against J774G8 macrophages in culture and did not cause lysis in sheep blood when it was used at higher concentrations that inhibited promastigote forms. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis with gelatin as the substrate showed that the enzymatic activity of the enzyme cysteine protease increased following treatment of the promastigotes with the isolated compound. This finding was correlated with marked morphological changes induced by parthenolide, such as the appearance of structures similar to large lysosomes and intense exocytic activity in the region of the flagellar pocket, as seen by electron microscopy. These results provide new perspectives on the development of novel drugs with leishmanicidal activities obtained from natural products.
The Pathogenesis of Fatal Outcome in Murine Pulmonary Aspergillosis Depends on the Neutrophil Depletion Strategy
Authors: Stephens-Romero Shane D. , Mednick Aron J. , Feldmesser Marta
Published: December 2004

Aspergillus fumigatuscauses invasive disease in severely immunocompromised hosts but is readily cleared when host innate defenses are intact. Animal models for evaluation of therapeutic strategies to combat invasive aspergillosis that closely mimic human disease are desirable. We determined optimal dosing regimens for neutrophil depletion and evaluated the course of infection following aerosol infection in mice by determining survival, organ fungal burden, and histopathology in mice in which neutropenia was induced by three methods, administration of granulocyte-depleting monoclonal antibody RB6-8C5 (MAb RB6), administration of cyclophosphamide, and administration of both agents. Administration of either individual agent resulted in a requirement for relatively high conidial inocula to achieve 100% mortality in both BALB/c and C57BL/6 mice, although the infection appeared to be somewhat more lethal in C57BL/6 mice. Death following induction of neutropenia with MAb RB6 occurred when a relatively low fungal burden was present in the lung and may have been related to the inflammatory response associated with neutrophil recovery. In contrast, administration of both agents reduced the lethal inoculum in each mouse strain by approximately 1 log10, and C57BL/6 mice that received both agents had a higher fungal burden and less inflammation in the lung at the time of death than BALB/c mice or mice of either strain that received MAb RB6 alone. Our data suggest that the relationship among fungal burden, inflammation, and death is complex and can be influenced by the immunosuppression regimen, the mouse strain, and the inoculum.
Thioredoxin Peroxidase Secreted byFasciola hepaticaInduces the Alternative Activation of Macrophages
Authors: Donnelly Sheila , O'Neill Sandra M. , Sekiya Mary , Mulcahy Grace , Dalton John P.
Published: December 2004

Alternatively activated macrophages (AAMφ) are primarily associated with the chronic stages of parasitic infections and the development of a polarized Th2 response. We have shown thatFasciola hepaticainfection of BALB/c mice induces a polarized Th2 response during both the latent and chronic stage of disease. The activation status of macrophages was analyzed in this model of helminth infection by evaluating the expression of genetic markers of alternative activation, namely, Fizz1, Ym1, and Arg1. AAMφ were recruited to the peritoneum of mice within 24 h ofF. hepaticainfection and after intraperitoneal injection of parasite excretory-secretory (ES) products. Administration of a recombinant antioxidant thioredoxin peroxidase (TPx), which is contained within the ES products, also induced the recruitment of AAMφ to the peritoneum. In vitro studies showed that this recombinant TPx directly converts RAW 264.7 macrophages to an alternatively activated phenotype characterized by the production of high levels of interleukin-10 (IL-10), prostaglandin E2, corresponding with low levels of IL-12. Our data suggest that the Th2 responses induced by the helminthF. hepaticaare mediated through the secretion of molecules, one of which is TPx, that induce the recruitment and alternative activation of macrophages.
Critical Proinflammatory and Anti-Inflammatory Functions of Different Subsets of CD1d-Restricted Natural Killer T Cells duringTrypanosoma cruziInfection
Authors: Duthie Malcolm S. , Kahn Maria , White Maria , Kapur Raj P. , Kahn Stuart J.
Published: December 2004

Trypanosoma cruziinfects 15 to 20 million people in Latin America and causes Chagas disease, a chronic inflammatory disease with fatal cardiac and gastrointestinal sequelae. How the immune response causes Chagas disease is not clear, but during the persistent infection both proinflammatory and anti-inflammatory responses are critical. Natural killer T (NKT) cells have been shown to regulate immune responses during infections and autoimmune diseases. We report here that during acuteT. cruziinfection NKT-cell subsets provide distinct functions. CD1d−/−mice, which lack both invariant NKT (iNKT) cells and variant NKT (vNKT) cells, develop a mild phenotype displaying an increase in spleen and liver mononuclear cells, anti-T. cruziantibody response, and muscle inflammation. In contrast, Jα18−/−mice, which lack iNKT cells but have vNKT cells, develop a robust phenotype involving prominent spleen, liver, and skeletal muscle inflammatory infiltrates comprised of NK, dendritic, B and T cells. The inflammatory cells display activation markers; produce more gamma interferon, tumor necrosis factor alpha, and nitric oxide; and show a diminished antibody response. Strikingly, most Jα18−/−mice die. Thus, in response to the same infection, vNKT cells appear to augment a robust proinflammatory response, whereas the iNKT cells dampen this response, possibly by regulating vNKT cells.
Enzymes Involved in Anaerobic Respiration Appear To Play a Role inActinobacillus pleuropneumoniaeVirulence
Authors: Jacobsen Ilse , Hennig-Pauka Isabel , Baltes Nina , Trost Matthias , Gerlach Gerald-F.
Published: December 2004

Actinobacillus pleuropneumoniae, the etiological agent of porcine pleuropneumonia, is able to survive on respiratory epithelia, in tonsils, and in the anaerobic environment of encapsulated sequesters. It was previously demonstrated that a deletion of the anaerobic dimethyl sulfoxide reductase gene (dmsA) results in attenuation in acute disease (N. Baltes, S. Kyaw, I. Hennig-Pauka, and G. F. Gerlach, Infect. Immun.71:6784-6792, 2003). In the present study, using two-dimensional polyacrylamide gel electrophoresis and quadrupole time-of-flight mass spectrometry, we identified an aspartate ammonia-lyase (AspA) which is upregulated upon induction with bronchoalveolar lavage fluid (BALF). This enzyme is involved in the production of fumarate, an alternative electron acceptor under anaerobic conditions. The coding gene (aspA) was cloned and shown to be present in allA. pleuropneumoniaeserotype reference strains. The transcriptional start point was identified downstream of a putative FNR binding motif, and BALF-dependent activation ofaspAwas confirmed by construction of an isogenicA. pleuropneumoniaemutant carrying a chromosomalaspA::luxABtranscriptional fusion. TwoaspAdeletion mutants,A. pleuropneumoniaeΔaspAandA. pleuropneumoniaeΔaspAΔdmsA, were constructed, both showing reduced growth under anaerobic conditions in vitro. Pigs challenged with either of the two mutants in an aerosol infection model showed a lower lung lesion score than that of theA. pleuropneumoniaewild-type (wt) controls. Pigs challenged withA. pleuropneumoniaeΔaspAΔdmsAhad a significantly lower clinical score, and this mutant was rarely reisolated from unaltered lung tissue; in contrast,A. pleuropneumoniaeΔaspAand theA. pleuropneumoniaewt were consistently reisolated in high numbers. These results suggest that enzymes involved in anaerobic respiration are necessary for the pathogen's ability to persist on respiratory tract epithelium and play an important role inA. pleuropneumoniaepathogenesis.