bluuebunny/crossref_metadata_2025_split huggingface dataset:
Molecular Cloning, Sequence Analysis, and Heterologous Expression of the Phosphinothricin Tripeptide Biosynthetic Gene Cluster from
Streptomyces viridochromogenes
DSM 40736
Authors: Blodgett Joshua A. V. , Zhang Jun Kai , Metcalf William W. 
Published: December 2004

ABSTRACT

A fosmid library from genomic DNA of
Streptomyces viridochromogenes
DSM 40736 was constructed and screened for the presence of genes known to be involved in the biosynthesis of phosphinothricin tripeptide (PTT). Eight positives were identified, one of which was able to confer PTT biosynthetic capability upon
Streptomyces lividans
after integration of the fosmid into the chromosome of this heterologous host. Sequence analysis of the 40,241-bp fosmid insert revealed 29 complete open reading frames (ORFs). Deletion analysis demonstrated that a minimum set of 24 ORFs were required for PTT production in the heterologous host. Sequence analysis revealed that most of these PTT genes have been previously identified in either
S. viridochromogenes
or
S. hygroscopicus
(or both), although only 11 out of 24 of these ORFs have experimentally defined functions. Three previously unknown genes within the cluster were identified and are likely to have roles in the stepwise production of phosphonoformate from phosphonoacetaldehyde. This is the first report detailing the entire PTT gene cluster from any producing streptomycete.
bla
SHV
Genes in
Klebsiella pneumoniae
: Different Allele Distributions Are Associated with Different Promoters within Individual Isolates
Authors: Hammond David S. , Schooneveldt Jacqueline M. , Nimmo Graeme R. , Huygens Flavia , Giffard Philip M. 
Published: December 2004

ABSTRACT

Extended-spectrum β-lactamases (ESBLs) emerge by point mutation from non-extended-spectrum precursors. The aims of this study were to reveal the basis for variations in resistance levels found in a collection of 21
Klebsiella pneumoniae
clinical isolates from Brisbane, Australia. Previous studies have shown that 20 of these isolates possess
bla
SHV-11
,
bla
SHV-2a
, and/or
bla
SHV-12
, and there is an association between the copy numbers of the ESBL-encoding genes and resistance levels. In this study, a real-time PCR method for interrogating the polymorphic sites at codons 238 and 240 was developed, and this confirmed the relationship between mutant gene copy numbers and resistance levels. The
bla
SHV
promoter region was cloned from one of the ESBL-expressing isolates, and this showed that
bla
SHV
genes exist downstream of two different promoters within this single isolate. These promoters have both been reported previously, and they differ by virtue of the presence or absence of an IS
26
insertion. The
bla
SHV
copy numbers in
cis
with the different promoters were measured, and the copy number of the IS
26
promoter was correlated with resistance levels. Cloning and analysis of PCR products showed that different
bla
SHV
variants existed in
cis
with individual promoters in individual isolates but that mutant genes were more abundant downstream of the IS
26
promoter. There were no ESBL-positive isolates without this promoter. It was concluded that
bla
SHV
in
cis
with the IS
26
promoter is located on an amplifiable replicon, and the presence of the IS
26
insertion may facilitate the acquisition of an ESBL-positive phenotype.
Virulence Genotype and Phylogenetic Origin in Relation to Antibiotic Resistance Profile among
Escherichia coli
Urine Sample Isolates from Israeli Women with Acute Uncomplicated Cystitis
Authors: Johnson James R. , Kuskowski Michael A. , O'Bryan Timothy T. , Colodner Raul , Raz Raul 
Published: December 2004

ABSTRACT

To clarify the virulence and phylogenetic implications of antimicrobial agent resistance in
Escherichia coli
, 100
E. coli
isolates from urine samples of Israeli women with acute uncomplicated cystitis were analyzed by molecular phylotyping and virulence genotyping for comparison with resistance phenotypes. The differences between the isolates that were resistant and susceptible to trimethoprim-sulfamethoxazole and ampicillin were minimal. In contrast, ciprofloxacin resistance was associated with greatly reduced inferred virulence and categorical shifts away from the highly virulent phylogenetic group B2, which explained much of the virulence effect. The results of amplification fingerprinting suggested that most ciprofloxacin-resistant isolates represented unique clonal groups and were not derived from clonal groups with more highly virulent susceptible isolates. These findings suggest that virulence and antimicrobial resistance are not mutually exclusive in
E. coli
clinical isolates. Instead, the relationship between virulence and antimicrobial resistance varies according to the particular resistance phenotype; for ciprofloxacin resistance, the relationship is strongly influenced by phylogenetic background. The basis for the concentration of ciprofloxacin resistance in non-B2 phylogenetic groups remains unknown.
De Novo Generation of Cationic Antimicrobial Peptides: Influence of Length and Tryptophan Substitution on Antimicrobial Activity
Authors: Deslouches Berthony , Phadke Shruti M. , Lazarevic Vanja , Cascio Michael , Islam Kazi , Montelaro Ronald C. , Mietzner Timothy A. 
Published: December 2004

ABSTRACTComparison of human immunodeficiency virus lentiviral lytic peptide 1 with other host-derived peptides indicates that antimicrobial properties of membrane-active peptides are markedly influenced by their cationic, hydrophobic, and amphipathic properties. Many common themes, such as Arg composition of the cationic face of an amphipathic helix and the importance of maintaining the hydrophobic face, have been deduced from these observations. These studies suggest that a peptide with these structural properties can be derived de novo by using only a few strategically positioned amino acids. However, the effects of length and helicity on antimicrobial activity and selectivity have not been objectively evaluated in the context of this motif. To address these structure-function issues, multimers of a 12-residue lytic base unit (LBU) peptide composed only of Arg and Val residues aligned to form idealized amphipathic helices were designed. Bacterial killing assays and circular dichroism analyses reveal a strong correlation between antibacterial activity, peptide length, and propensity to form a helix in solvent mimicking the environment of a membrane. Increasing peptide length beyond two LBUs (24-residue peptides) resulted in no appreciable increase in antimicrobial activity. Derivatives (WLBU) of the LBU series were further engineered by substituting Trp residues in the hydrophobic domains. The 24-residue WLBU2 peptide was active at physiologic NaCl concentrations againstStaphylococcus aureusand mucoid and nonmucoid strains ofPseudomonas aeruginosa. Further, WLBU2 displayed the highest antibacterial selectivity of all peptides evaluated in the present study by using a coculture model ofP. aeruginosaand primary human skin fibroblasts. These findings provide fundamental information toward the de novo design of an antimicrobial peptide useful for the management of infectious diseases.
Anti-Human Immunodeficiency Virus Type 1 Microbicide Cellulose Acetate 1,2-Benzenedicarboxylate in a Human In Vitro Model of Vaginal Inflammation
Authors: Fichorova R. N. , Zhou F. , Ratnam V. , Atanassova V. , Jiang S. , Strick N. , Neurath A. R. 
Published: December 2004

ABSTRACT
The sexual transmission of human immunodeficiency virus type 1 (HIV-1) is facilitated by inflammation and related epithelial barrier perturbation. Microbicides for vaginal applications are currently being developed to reduce the risk of HIV-1 transmission. However, little is known about their interference with epithelial immune function. In recent clinical trials, nonoxynol-9 (N-9), a virucide with a long history of intravaginal use as a contraceptive, failed to protect against HIV-1 possibly due to mucosal inflammatory damage. Cellulose acetate 1,2-benzenedicarboxylate, also named CAP (for “controls AIDS pandemic”), is an anti-HIV-1 microbicide selected from pharmaceutical excipients that are regarded as safe for oral administration but have not been assessed for potential effects on inflammatory factors in the vaginal environment. Here we use a sensitive human cell culture system to evaluate proinflammatory profiles of soluble CAP in reference to N-9 and known epithelial activators such as tumor necrosis factor alpha (TNF-α) and bacterial lysates. Within 6 h of exposure, TNF-α and N-9 triggered NF-κB and AP-1/cFos activation and upregulated interleukins and an array of chemokines by vaginal and polarized cervical epithelial cells. The induced proinflammatory status continued after removal of stimuli and was confirmed by enhanced transepithelial neutrophil migration. While sustaining stability and anti-HIV-1 activity in the epithelial environment, CAP did not increase the production of proinflammatory mediators during or after exposure, nor did it modify the epithelial resistance to leukocyte traffic. CAP attenuated some TNF-α-induced responses but did not interfere with epithelial cytokine responsiveness to gonococcal determinants. The described system may be useful for predicting proinflammatory side effects of other microbicide candidates for vaginal application.
Real-Time In Vivo Bioluminescent Imaging for Evaluating the Efficacy of Antibiotics in a Rat
Staphylococcus aureus
Endocarditis Model
Authors: Xiong Yan Q. , Willard Julie , Kadurugamuwa Jagath L. , Yu Jun , Francis Kevin P. , Bayer Arnold S. 
Published: December 2004

ABSTRACT

Therapeutic options for invasive
Staphylococcus aureus
infections have become limited due to rising antimicrobial resistance, making relevant animal model testing of new candidate agents more crucial than ever. In the present studies, a rat model of aortic infective endocarditis (IE) caused by a bioluminescently engineered, biofilm-positive
S. aureus
strain was used to evaluate real-time antibiotic efficacy directly. This strain was vancomycin and cefazolin susceptible but gentamicin resistant. Bioluminescence was detected and quantified daily in antibiotic-treated and control animals with IE, using a highly sensitive in vivo imaging system (IVIS). Persistent and increasing cardiac bioluminescent signals (BLS) were observed in untreated animals. Three days of vancomycin therapy caused significant reductions in both cardiac BLS (>10-fold versus control) and
S. aureus
densities in cardiac vegetations (
P
< 0.005 versus control). However, 3 days after discontinuation of vancomycin therapy, a greater than threefold increase in cardiac BLS was observed, indicating relapsing IE (which was confirmed by quantitative culture). Cefazolin resulted in modest decreases in cardiac BLS and bacterial densities. These microbiologic and cardiac BLS differences during therapy correlated with a longer time-above-MIC for vancomycin (>12 h) than for cefazolin (∼4 h). Gentamicin caused neither a reduction in cardiac
S. aureus
densities nor a reduction in BLS. There were significant correlations between cardiac BLS and
S. aureus
densities in vegetations in all treatment groups. These data suggest that bioluminescent imaging provides a substantial advance in the real-time monitoring of the efficacy of therapy of invasive
S. aureus
infections in live animals.
Interaction of Bismuth Subsalicylate with Fruit Juices, Ascorbic Acid, and Thiol-Containing Substrates To Produce Soluble Bismuth Products Active against
Clostridium difficile
Authors: Mahony D. E. , Woods A. , Eelman M. D. , Burford N. , Veldhuyzen van Zanten S. J. O. 
Published: December 2004

ABSTRACT

Bismuth subsalicylate (BSS), the active ingredient of Pepto-Bismol, has been used for many years to treat various disorders of the gastrointestinal tract. Using mass spectrometry and the agar dilution method, we determined that insoluble BSS interacts with certain dietary components and organic substrates to produce water-soluble products with activity against
Clostridium difficile
.
Surface-Active Fungicidal
d
-Peptide Inhibitors of the Plasma Membrane Proton Pump That Block Azole Resistance
Authors: Monk Brian C. , Niimi Kyoko , Lin Susan , Knight Allison , Kardos Thomas B. , Cannon Richard D. , Parshot Rekha , King Amanda , Lun David , Harding David R. K. 
Published: December 2004

ABSTRACT

A 1.8-million-member
d
-octapeptide combinatorial library was constructed in which each member comprised a diversity-containing N-terminal pentapeptide and a C-terminal amidated triarginine motif. The C-terminal motif concentrated the library members at the fungal cell surface. A primary screen for inhibitors of
Saccharomyces cerevisiae
and
Candida albicans
growth, together with an in vitro secondary screen with the
S. cerevisiae
plasma membrane ATPase (Pma1p) as a target, identified the antifungal
d
-octapeptide BM0 (
d
-NH
2
-RFWWFRRR-CONH
2
). Optimization of BM0 led to the construction of BM2 (
d
-NH
2
-RRRFWWFRRR-CONH
2
), which had broad-spectrum fungicidal activity against
S. cerevisiae
,
Candida
species, and
Cryptococcus neoformans
; bound strongly to the surfaces of fungal cells; inhibited the physiological activity of Pma1p; and appeared to target Pma1p, with 50% inhibitory concentrations in the range of 0.5 to 2.5 μM. At sub-MICs (<5 μM), BM2 chemosensitized to fluconazole (FLC)
S. cerevisiae
strains functionally hyperexpressing fungal lanosterol 14α-demethylase and resistance-conferring transporters of azole drugs. BM2 chemosensitized to FLC some FLC-resistant clinical isolates of
C. albicans
and
C. dubliniensis
and chemosensitized to itraconazole clinical isolates of
C. krusei
that are intrinsically resistant to FLC. The growth-inhibitory concentrations of BM2 did not cause fungal cell permeabilization, significant hemolysis of red blood cells, or the death of cultured HEp-2 epithelial cells. BM2 represents a novel class of broad-spectrum, surface-active, Pma1p-targeting fungicides which increases the potencies of azole drugs and circumvents azole resistance.
Comparison of Three PCR Primer Sets for Identification of
vanB
Gene Carriage in Feces and Correlation with Carriage of Vancomycin-Resistant Enterococci: Interference by
vanB
-Containing Anaerobic Bacilli
Authors: Ballard S. A. , Grabsch E. A. , Johnson P. D. R. , Grayson M. L. 
Published: December 2004

ABSTRACT

We assessed the sensitivities and specificities of three previously described PCR primers on enrichment broth cultures of feces for the accurate detection of fecal carriage of vancomycin-resistant enterococci (VRE). In addition, we investigated specimens that were
vanB
PCR positive but VRE culture negative for the presence of other
vanB
-containing pathogens. Feces from 59 patients (12 patients carrying
vanB Enterococcus faecium
strains and 47 patients negative for VRE carriage) were cultured for 36 h in aerobic brain heart infusion (BHI) broth, anaerobic BHI (AnO
2
BHI) broth, or aerobic Enterococcosel (EC) broth. DNA was extracted from the cultures and tested for the presence of
vanB
by using the PCR primers of Dutka-Malen et al. (S. Dutka-Malen, S. Evers, and P. Courvalin, J. Clin. Microbiol. 33:24-27, 1995), Bell et al. (J. M. Bell, J. C. Paton, and J. Turnidge, J. Clin. Microbiol. 36:2187-2190, 1998), and Stinear et al. (T. P. Stinear, D. C. Olden, P. D. R. Johnson, J. K. Davies, and M. L. Grayson, Lancet 357:855-856, 2001). The sensitivity (specificity) of PCR compared with the results of culture on BHI, AnO
2
BHI, and EC broths were 67% (96%), 50% (94%), and 17% (100%), respectively, with the primers of Dutka-Malen et al.; 92% (60%), 92% (45%), and 92% (83%), respectively, with the primers of Bell et al.; and 92% (49%), 92% (43%), and 100% (51%) respectively, with the primers of Stinear et al. The primers of both Bell et al. and Stinear et al. were significantly more sensitive than those of Dutka-Malen et al. in EC broth (
P
= 0.001 and
P
< 0.001, respectively). The poor specificities for all primer pairs were due in part to the isolation and identification of six anaerobic gram-positive bacilli,
Clostridium hathewayi
(
n
= 3), a
Clostridium innocuum
-like organism (
n
= 1),
Clostridium bolteae
(
n
= 1), and
Ruminococcus lactaris
-like (
n
= 1), from five fecal specimens that were
vanB
positive but VRE culture negative. All six organisms were demonstrated to contain a
vanB
gene identical to that of VRE.
VanB-
containing bowel anaerobes may result in false-positive interpretation of PCR-positive fecal enrichment cultures as VRE, regardless of the primers and protocols used.
Role of EspA and Intimin in Expression of Proinflammatory Cytokines from Enterocytes and Lymphocytes by Rabbit Enteropathogenic
Escherichia coli
-Infected Rabbits
Authors: Ramirez Karina , Huerta Rocio , Oswald Eric , Garcia-Tovar Carlos , Hernandez Jose Manuel , Navarro-Garcia Fernando 
Published: December 2004

ABSTRACT

Enteropathogenic
Escherichia coli
(EPEC) produces attaching and effacing (A/E) lesions and watery diarrhea, both of which are intimin and EspA dependent. In this work, we explored the mucosal immune response by detecting cytokine induction in rabbits with diarrhea caused by rabbit EPEC (REPEC). Orally inoculated rabbits exhibited weight loss and mucosal inflammation, developed watery diarrhea, and died (day 7). At day 6 postinoculation, animals were analyzed for the induction of proinflammatory cytokines in enterocytes. The role of lymphocyte-dependent immunity was determined through the expression of proinflammatory cytokines by lymphocytes from Peyer's patches (PP) and the spleen. EspA and intimin mutants were used to explore the role of A/E lesions in the expression of these cytokines. REPEC-infected rabbit enterocytes showed increased interleukin 1β (IL-1β), IL-6, IL-8, and tumor necrosis factor alpha (TNF-α) mRNA expression, but that of anti-inflammatory IL-10 was increased only slightly. In contrast, intimin mutant-infected rabbits were unable to produce this proinflammatory cytokine profile but did produce a remarkable increase in IL-10 expression. Bacteria lacking EspA increased the expression of IL-8 and TNF-α, but that of IL-10 was increased only slightly. PP lymphocytes also produced proinflammatory cytokines, which were dependent on EspA (except for TNF-α) and intimin, while IL-10 was induced by EspA and intimin mutants. In contrast, spleen lymphocytes (systemic compartment) were unable to produce IL-1β and TNF-α. These data show the importance of the proinflammatory cytokines secreted by enterocytes and those expressed locally by PP lymphocytes, which can activate effector mechanisms at the epithelium. Furthermore, this cytokine profile, including IL-6 and IL-1β, which may be involved in the diarrhea produced by EPEC, depends on intimin.
NF-κB Activation duringRickettsia rickettsiiInfection of Endothelial Cells Involves the Activation of Catalytic IκB Kinases IKKα and IKKβ and Phosphorylation-Proteolysis of the Inhibitor Protein IκBα
Authors: Clifton Dawn R. , Rydkina Elena , Freeman Robert S. , Sahni Sanjeev K. 
Published: December 2004

ABSTRACTRocky Mountain spotted fever, a systemic tick-borne illness caused by the obligate intracellular bacteriumRickettsia rickettsii, is associated with widespread infection of the vascular endothelium.R. rickettsiiinfection induces a biphasic pattern of the nuclear factor-κB (NF-κB) activation in cultured human endothelial cells (ECs), characterized by an early transient phase at 3 h and a late sustained phase evident at 18 to 24 h. To elucidate the underlying mechanisms, we investigated the expression of NF-κB subunits, p65 and p50, and IκB proteins, IκBα and IκBβ. The transcript and protein levels of p50, p65, and IκBβ remained relatively unchanged during the course of infection, but Ser-32 phosphorylation of IκBα at 3 h was significantly increased over the basal level in uninfected cells concomitant with a significant increase in the expression of IκBα mRNA. The level of IκBα mRNA gradually returned toward baseline, whereas that of total IκBα protein remained lower than the corresponding controls. The activities of IKKα and IKKβ, the catalytic subunits of IκB kinase (IKK) complex, as measured by in vitro kinase assays with immunoprecipitates from uninfected andR. rickettsii-infected ECs, revealed significant increases at 2 h after infection. The activation of IKK and early phase of NF-κB response were inhibited by heat treatment and completely abolished by formalin fixation of rickettsiae. The IKK inhibitors parthenolide and aspirin blocked the activities of infection-induced IKKα and IKKβ, leading to attenuation of nuclear translocation of NF-κB. Also, increased activity of IKKα was evident later during the infection, coinciding with the late phase of NF-κB activation. Thus, activation of catalytic components of the IKK complex represents an important upstream signaling event in the pathway forR. rickettsii-induced NF-κB activation. Since NF-κB is a critical regulator of inflammatory genes and prevents host cell death during infection via antiapoptotic functions, selective inhibition of IKK may provide a potential target for enhanced clearance of rickettsiae and an effective strategy to reduce inflammatory damage to the host during rickettsial infections.
Development of Allergic Airway Disease in Mice following Antibiotic Therapy and Fungal Microbiota Increase: Role of Host Genetics, Antigen, and Interleukin-13
Authors: Noverr Mairi C. , Falkowski Nicole R. , McDonald Rod A. , McKenzie Andrew N. , Huffnagle Gary B. 
Published: December 2004

ABSTRACTLending support to the hygiene hypothesis, epidemiological studies have demonstrated that allergic disease correlates with widespread use of antibiotics and alterations in fecal microbiota (“microflora”). Antibiotics also lead to overgrowth of the yeastCandida albicans, which can secrete potent prostaglandin-like immune response modulators, from the microbiota. We have recently developed a mouse model of antibiotic-induced gastrointestinal microbiota disruption that is characterized by stable increases in levels of gastrointestinal enteric bacteria andCandida. Using this model, we have previously demonstrated that microbiota disruption can drive the development of a CD4 T-cell-mediated airway allergic response to mold spore challenge in immunocompetent C57BL/6 mice without previous systemic antigen priming. The studies presented here address important questions concerning the universality of the model. To investigate the role of host genetics, we tested BALB/c mice. As with C57BL/6 mice, microbiota disruption promoted the development of an allergic response in the lungs of BALB/c mice upon subsequent challenge with mold spores. In addition, this allergic response required interleukin-13 (IL-13) (the response was absent in IL-13−/−mice). To investigate the role of antigen, we subjected mice with disrupted microbiota to intranasal challenge with ovalbumin (OVA). In the absence of systemic priming, only mice with altered microbiota developed airway allergic responses to OVA. The studies presented here demonstrate that the effects of microbiota disruption are largely independent of host genetics and the nature of the antigen and that IL-13 is required for the airway allergic response that follows microbiota disruption.