bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of Three Methods of Determining MICs for Filamentous Fungi Using Different End Point Criteria and Incubation Periods
Authors: Llop C. , Pujol I. , Aguilar C. , Sala J. , Riba D. , Guarro J.
Published: July 2002

Three different methods were used to determine the in vitro activities of amphotericin B, ketoconazole, itraconazole, and flucytosine against 30 isolates of different genera of filamentous fungi. MICs were determined visually, with or without agitation, and spectrophotometrically by using a broth microdilution method. For amphotericin B, there was one end point reading criterion (the minimum concentration of antifungal that inhibited 100% of growth), but for azoles and flucytosine there were two (the minimum concentrations that inhibited 50 and 75% of fungal growth, respectively) after 48 and 72 h of incubation. All tests were performed in triplicate. An intraclass correlation coefficient (ICC) was used to evaluate the reproducibility of each of the methods and the correlation among them. The reproducibility of the three methods was very high (ICC of 0.808 to 0.992), particularly in the case of azoles and flucytosine. In general, the degree of reproducibility was highest for azoles and amphotericin B after 72 h of incubation and for flucytosine after 48 h of incubation. The degree of correlation among the three methods was very high (ICC of >0.98) with all of the antifungals under all the conditions tested. The end point reading criteria and the time of incubation affected neither the reproducibility of the methods nor their correlation, and their effect on MICs was statistically significant.
Genes for Production of the Enediyne Antitumor Antibiotic C-1027 in
Streptomyces globisporus
Are Clustered with the
cagA
Gene That Encodes the C-1027 Apoprotein
Authors: Liu Wen , Shen Ben
Published: July 2002

C-1027, the most potent member of the enediyne antitumor antibiotic family, is produced by
Streptomyces globisporus
C-1027 and consists of an apoprotein (encoded by the
cagA
gene) and a nonpeptidic chromophore. The C-1027 chromophore could be viewed as being derived biosynthetically from a benzoxazolinate, a deoxyamino hexose, a β-amino acid, and an enediyne core. By adopting a strategy for cloning of the C-1027 biosynthesis gene cluster by mapping a putative dNDP-glucose 4,6-dehydratase (NGDH) gene to
cagA
, we have localized 75 kb of contiguous DNA from
S. globisporus
. DNA sequence analysis of two regions of the cloned gene cluster revealed two genes,
sgcA
and
sgcB
, that encode an NGDH enzyme and a transmembrane efflux protein, respectively, and confirmed that the
cagA
gene resides approximately 14 kb upstream of the
sgcAB
locus. The involvement of the cloned gene cluster in C-1027 biosynthesis was demonstrated by disrupting the
sgcA
gene to generate C-1027-nonproducing mutants and by complementing the
sgcA
mutants in vivo to restore C-1027 production. These results represent the first cloning of a gene cluster for enediyne antitumor antibiotic biosynthesis and provide a starting point for future genetic and biochemical investigations of C-1027 biosynthesis.
A Canadian National Surveillance Study of Urinary Tract Isolates from Outpatients: Comparison of the Activities of Trimethoprim-Sulfamethoxazole, Ampicillin, Mecillinam, Nitrofurantoin, and Ciprofloxacin
Authors: Zhanel George G. , Karlowsky James A. , Harding Godfrey K. M. , Carrie Anita , Mazzulli Tony , Low Donald E. ,   The Canadian Urinary Isolate Study Group, Hoban Daryl J.
Published: July 2002

Ampicillin, trimethoprim-sulfamethoxazole, mecillinam, nitrofurantoin, and ciprofloxacin mean resistance rates for 2,000 urinary tract isolates collected from outpatients across Canada in 1998 were 41.1, 19.2, 14.7, 5.0, and 1.8%, respectively. For
Escherichia coli
isolates alone (
n
= 1,681) comparable rates were 41.0, 18.9, 7.4, 0.1, and 1.2%, respectively. The majority of
E. coli
isolates resistant to ampicillin, trimethoprim-sulfamethoxazole, or ciprofloxacin were susceptible (MIC, <16 μg/ml) to mecillinam.
Population Pharmacokinetics and Pharmacodynamic Modeling of Abacavir (1592U89) from a Dose-Ranging, Double-Blind, Randomized Monotherapy Trial with Human Immunodeficiency Virus-Infected Subjects
Authors: Weller Stephen , Radomski Kristine M. , Lou Yu , Stein Daniel S.
Published: July 2002

Abacavir (formerly 1592U89) is a carbocyclic nucleoside analog with potent anti-human immunodeficiency virus (anti-HIV) activity when administered alone or in combination with other antiretroviral agents. The population pharmacokinetics and pharmacodynamics of abacavir were investigated in 41 HIV type 1 (HIV-1)-infected, antiretroviral naive adults with baseline CD4
+
cell counts of ≥100/mm
3
and plasma HIV-1 RNA levels of >30,000 copies/ml. Data for analysis were obtained from patients who received randomized, blinded monotherapy with abacavir at 100, 300, or 600 mg twice-daily (BID) for up to 12 weeks. Plasma abacavir concentrations from sparse sampling were analyzed by standard population pharmacokinetic methods, and the effects of dose, combination therapy, gender, weight, and age on parameter estimates were investigated. Bayesian pharmacokinetic parameter estimates were calculated to determine the peak concentration of abacavir in plasma (
C
max
) and the area under the concentration-time curve from time zero to infinity (AUC
0–∞
) for individual subjects. The pharmacokinetics of abacavir were dose proportional over the 100- to 600-mg dose range and were unaffected by any covariates. No significant correlations were observed between the incidence of the five most common adverse events (headache, nausea, diarrhea, vomiting, and malaise or fatigue) and AUC
0–∞
. A significant correlation was observed between
C
max
and nausea by categorical analysis (
P
= 0.019), but this was of borderline significance by logistic regression (odds ratio, 1.45; 95% confidence interval, 0.95 to 2.32). The log
10
time-averaged AUC
0–∞
minus baseline (AAUCMB) values for HIV-1 RNA and CD4
+
cell count correlated significantly with
C
max
and AUC
0–∞
, but with better model fits for AUC
0–∞
. The increase in AAUCMB values for CD4
+
cell count plateaued early for drug exposures that were associated with little change in AAUCMB values for plasma HIV-1 RNA. There was less than a 0.4 log
10
difference over 12 weeks in the HIV-1 RNA levels with the doubling of the abacavir AUC
0–∞
from 300 to 600 mg BID dosing. In conclusion, pharmacodynamic modeling supports the selection of abacavir 300 mg twice-daily dosing.
Use of Preclinical Data for Selection of a Phase II/III Dose for Evernimicin and Identification of a Preclinical MIC Breakpoint
Authors: Drusano G. L. , Preston S. L. , Hardalo C. , Hare R. , Banfield C. , Andes D. , Vesga O. , Craig W. A.
Published: July 2002

One of the most challenging issues in the design of phase II/III clinical trials of antimicrobial agents is dose selection. The choice is often based on preclinical data from pharmacokinetic (PK) studies with animals and healthy volunteers but is rarely linked directly to the target organisms except by the MIC, an in vitro measure of antimicrobial activity with many limitations. It is the thesis of this paper that rational dose-selection decisions can be made on the basis of the pharmacodynamics (PDs) of the test agent predicted by a mathematical model which uses four data sets: (i) the distribution of MICs for clinical isolates, (ii) the distribution of the values of the PK parameters for the test drug in the population, (iii) the PD target(s) developed from animal models of infection, and (iv) the protein binding characteristics of the test drug. In performing this study with the new anti-infective agent evernimicin, we collected a large number (
n
= 4,543) of recent clinical isolates of gram-positive pathogens (
Streptococcus pneumoniae
,
Enterococcus faecalis
and
Enterococcus faecium
, and
Staphylococcus aureus
) and determined the MICs using E-test methods (AB Biodisk, Stockholm, Sweden) for susceptibility to evernimicin. Population PK data were collected from healthy volunteers (
n
= 40) and patients with hypoalbuminemia (
n
= 12), and the data were analyzed by using NPEM III. PD targets were developed with a neutropenic murine thigh infection model with three target pathogens:
S. pneumoniae
(
n
= 5),
E. faecalis
(
n
= 2), and
S. aureus
(
n
= 4). Drug exposure or the ratio of the area under the concentration-time curve/MIC (AUC/MIC) was found to be the best predictor of microbiological efficacy. There were three possible microbiological results: stasis of the initial inoculum at 24 h (10
7
CFU), log killing (pathogen dependent, ranging from 1 to 3 log
10
), or 90% maximal killing effect (90%
E
max
). The levels of protein binding in humans and mice were similar. The PK and PD of 6 and 9 mg of evernimicin per kg of body weight were compared; the population values for the model parameters and population covariance matrix were used to generate five Monte Carlo simulations with 200 subjects each. The fractional probability of attaining the three PD targets was calculated for each dose and for each of the three pathogens. All differences in the fractional probability of attaining the target AUC/MIC in this PD model were significant. For
S. pneumoniae
, the probability of attaining all three PD targets was high for both doses. For
S. aureus
and enterococci, there were increasing differences between the 6- and 9-mg/kg evernimicin doses for reaching the 2 log killing (
S. aureus
), 1 log killing (enterococci), or 90%
E
max
AUC/MIC targets. This same approach may also be used to set preliminary in vitro MIC breakpoints.
Prevalence of Molecular Mechanisms of Resistance to Azole Antifungal Agents in
Candida albicans
Strains Displaying High-Level Fluconazole Resistance Isolated from Human Immunodeficiency Virus-Infected Patients
Authors: Perea Sofia , López-Ribot José L. , Kirkpatrick William R. , McAtee Robert K. , Santillán Rebecca A. , Martı́nez Marcos , Calabrese David , Sanglard Dominique , Patterson Thomas F.
Published: July 2002

Molecular mechanisms of azole resistance in
Candida albicans
, including alterations in the target enzyme and increased efflux of drug, have been described, but the epidemiology of the resistance mechanisms has not been established. We have investigated the molecular mechanisms of resistance to azoles in
C. albicans
strains displaying high-level fluconazole resistance (MICs, ≥64 μg/ml) isolated from human immunodeficiency virus (HIV)-infected patients with oropharyngeal candidiasis. The levels of expression of genes encoding lanosterol 14α-demethylase (
ERG11
) and efflux transporters (
MDR1
and CDR) implicated in azole resistance were monitored in matched sets of susceptible and resistant isolates. In addition,
ERG11
genes were amplified by PCR, and their nucleotide sequences were determined in order to detect point mutations with a possible effect in the affinity for azoles. The analysis confirmed the multifactorial nature of azole resistance and the prevalence of these mechanisms of resistance in
C. albicans
clinical isolates exhibiting frank fluconazole resistance, with a predominance of overexpression of genes encoding efflux pumps, detected in 85% of all resistant isolates, being found. Alterations in the target enzyme, including functional amino acid substitutions and overexpression of the gene that encodes the enzyme, were detected in 65 and 35% of the isolates, respectively. Overall, multiple mechanisms of resistance were combined in 75% of the isolates displaying high-level fluconazole resistance. These results may help in the development of new strategies to overcome the problem of resistance as well as new treatments for this condition.
Inhibition of Trypsin-Like Cysteine Proteinases (Gingipains) fromPorphyromonas gingivalisby Tetracycline and Its Analogues
Authors: Imamura Takahisa , Matsushita Kenji , Travis James , Potempa Jan
Published: July 2002

Extracellular cysteine proteinases, referred to as gingipains, are considered important virulence factors forPorphyromonas gingivalis, a bacterium recognized as a major etiologic agent of chronic periodontitis. We investigated the effect of tetracycline and its analogues, doxycycline and minocycline, on the enzymatic activities of gingipains. Tetracyclines at 100 μM totally inhibited the amidolytic activity of arginine-specific gingipains (HRgpA and RgpB). In contrast, inhibition of Kgp was less efficient and required a somewhat higher concentration of the antibiotic to achieve the same effect. Among tetracycline derivatives, the most potent gingipain inhibitor was doxycycline, followed by tetracycline and minocycline. RgpB was inhibited by doxycycline in an uncompetitive and reversible manner with a 50% inhibitory concentration of 3 μM. Significantly, inhibition was unaffected by calcium, excluding the chelating activity of tetracyclines as the mechanism of gingipain inactivation. In contrast, the inhibitory activities of the tetracyclines were reduced by cysteine, a reducing agent, suggesting an interference of the drug at the oxidative region with the catalytic system of the enzyme. Doxycycline, at 10 μM, significantly inhibited the RgpB-mediated production of vascular permeability-enhancing activity from human plasma, thus proving an effective inhibition of gingipain in vivo. These results indicate a new activity of tetracyclines as cysteine proteinase inhibitors and may explain the therapeutic efficiency of these antibiotics in the treatment of periodontitis.
Comparison of the Anti-Influenza Virus Activity of RWJ-270201 with Those of Oseltamivir and Zanamivir
Authors: Bantia S. , Parker C. D. , Ananth S. L. , Horn L. L. , Andries K. , Chand P. , Kotian P. L. , Dehghani A. , El-Kattan Y. , Lin T. , Hutchison T. L. , Montgomery J. A. , Kellog D. L. , Babu Y. S.
Published: July 2002

We have recently reported an influenza virus neuraminidase inhibitor, RWJ-270201 (BCX-1812), a novel cyclopentane derivative discovered through structure-based drug design. In this paper, we compare the potency of three compounds, RWJ-270201, oseltamivir, and zanamivir, against neuraminidase enzymes from various subtypes of influenza. RWJ-270201 effectively inhibited all tested influenza A and influenza B neuraminidases in vitro, with 50% inhibitory concentrations of 0.09 to 1.4 nM for influenza A neuraminidases and 0.6 to 11 nM for influenza B neuraminidases. These values were comparable to or lower than those for oseltamivir carboxylate (GS4071) and zanamivir (GG167). RWJ-270201 demonstrated excellent selectivity (>10,000-fold) for influenza virus neuraminidase over mammalian, bacterial, or other viral neuraminidases. Oral administration of a dosage of 1 mg/kg of body weight/day of RWJ-270201 for 5 days (beginning 4 h preinfection) showed efficacy in the murine model of influenza virus infection as determined by lethality and weight loss protection. RWJ-270201 administered intranasally at 0.01 mg/kg/day in the murine influenza model demonstrated complete protection against lethality, whereas oseltamivir carboxylate and zanamivir at the same dose demonstrated only partial protection. In the delayed-treatment murine influenza model, oral administration of a 10-mg/kg/day dose of RWJ-270201 or oseltamivir (GS4104, a prodrug of GS4071) at 24 h postinfection showed significant protection against lethality (
P
< 0.001 versus control). However, when the treatment was delayed for 48 h, no significant protection was observed in either drug group. No drug-related toxicity was observed in mice receiving 100 mg/kg/day of RWJ-270201 for 5 days. These efficacy and safety profiles justify further consideration of RWJ-270201 for the treatment and prevention of human influenza.
Antiviral Activity of Lovastatin against Respiratory Syncytial Virus In Vivo and In Vitro
Authors: Gower Tara L. , Graham Barney S.
Published: July 2002

Respiratory syncytial virus (RSV) is an important human pathogen that can cause severe and life-threatening respiratory infections in infants and immunocompromised adults. We have recently shown that the RSV F glycoprotein, which mediates viral fusion, binds to RhoA. One of the steps in RhoA activation involves isoprenylation at the carboxy terminus of the protein by geranylgeranyltransferase. This modification allows RhoA to be attached to phosphatidyl serine on the inner leaflet of the plasma membrane. Treatment of mice with lovastatin, a drug that inhibits prenylation pathways in the cell by directly inhibiting hydroxymethylglutaryl coenzyme A reductase, diminishes RSV but not vaccinia virus replication when administered up to 24 h after RSV infection and decreases virus-induced weight loss and illness in mice. The inhibition of replication is not likely due to the inhibition of cholesterol biosynthesis, since gemfibrozil, another cholesterol-lowering agent, did not affect virus replication and serum cholesterol levels were not significantly lowered by lovastatin within the time frame of the experiment. Lovastatin also reduces cell-to-cell fusion in cell culture and eliminates RSV replication in HEp-2 cells. These data indicate that lovastatin, more specific isoprenylation inhibitors, or other pharmacological approaches for preventing RhoA membrane localization should be considered for evaluation as a preventive antiviral therapy for selected groups of patients at high risk for severe RSV disease, such as the institutionalized elderly and bone marrow or lung transplant recipients.
Short-Term Measures of Relative Efficacy Predict Longer-Term Reductions in Human Immunodeficiency Virus Type 1 RNA Levels following Nelfinavir Monotherapy
Authors: Mittler John , Essunger Paulina , Yuen Geoffrey J. , Clendeninn Neil , Markowitz Martin , Perelson Alan S.
Published: July 2002

We calculated the relative efficacy of treatment, defined as the rate of decline of virus levels in plasma during treatment relative to the rate of decline during highly potent combination therapy, in human immunodeficiency virus type 1 (HIV-1) patients treated for 56 days with different doses of the protease inhibitor nelfinavir. Relative efficacies based on the rate of decline of HIV-1 RNA levels in plasma over the first 14 to 21 days correlated with drug dose and viral load reduction by day 56. Calculation of relative treatment efficacies over the first 2 to 3 weeks of treatment can allow rapid assessment of new antiretroviral agents and dosing regimens, reducing the need to keep subjects in clinical trials on monotherapy for prolonged periods of time. Relative efficacy may also serve as a measure of treatment efficacy in patients in initiating established therapies.
In Vitro Antifungal Activity of KP-103, a Novel Triazole Derivative, and Its Therapeutic Efficacy against Experimental Plantar Tinea Pedis and Cutaneous Candidiasis in Guinea Pigs
Authors: Tatsumi Yoshiyuki , Yokoo Mamoru , Arika Tadashi , Yamaguchi Hideyo
Published: July 2002

The in vitro activity of KP-103, a novel triazole derivative, against pathogenic fungi that cause dermatomycoses and its therapeutic efficacy against plantar tinea pedis and cutaneous candidiasis in guinea pigs were investigated. MICs were determined by a broth microdilution method with morpholinepropanesulfonic acid-buffered RPMI 1640 medium for
Candida
species and with Sabouraud dextrose broth for dermatophytes and by an agar dilution method with medium C for
Malassezia furfur
. KP-103 was the most active of all the drugs tested against
Candida albicans
(geometric mean [GM] MIC, 0.002 μg/ml), other
Candida
species including
Candida parapsilosis
and
Candida glabrata
(GM MICs, 0.0039 to 0.0442 μg/ml), and
M. furfur
(GM MIC, 0.025 μg/ml). KP-103 (1% solution) was highly effective as a treatment for guinea pigs with cutaneous candidiasis and achieved mycological eradication in 8 of the 10 infected animals, whereas none of the imidazoles tested (1% solutions) was effective in even reducing the levels of the infecting fungi. KP-103 was as active as clotrimazole and neticonazole but was less active than lanoconazole and butenafine against
Trichophyton rubrum
(MIC at which 80% of isolates are inhibited [MIC
80
], 0.125 μg/ml) and
Trichophyton mentagrophytes
(MIC
80
, 0.25 μg/ml). However, KP-103 (1% solution) exerted therapeutic efficacy superior to that of neticonazole and comparable to those of lanoconazole and butenafine, yielding negative cultures for all samples from guinea pigs with plantar tinea pedis tested. This suggests that KP-103 has better pharmacokinetic properties in skin tissue than the reference drugs. Because the in vitro activity of KP-103, unlike those of the reference drugs, against
T. mentagrophytes
was not affected by hair as a keratinic substance, its excellent therapeutic efficacy seems to be attributable to good retention of its antifungal activity in skin tissue, in addition to its potency.
All Detectable High-Molecular-Mass Penicillin-Binding Proteins Are Modified in a High-Level β-Lactam-Resistant Clinical Isolate of
Streptococcus mitis
Authors: Amoroso Ana , Demares Diego , Mollerach Marta , Gutkind Gabriel , Coyette Jacques
Published: July 2002

All detectable high-molecular-mass penicillin-binding proteins (HMM PBPs) are altered in a clinical isolate of
Streptococcus mitis
for which the β-lactam MICs are increased from those previously reported in our region (cefotaxime MIC, 64 μg/ml). These proteins were hardly detected at concentrations that saturate all PBPs in clinical isolates and showed, after densitometric analysis, 50-fold-lower radiotracer binding. Resistance was related to mosaic structure in all HMM PBP-coding genes, where critical region replacement was complemented not only by substitutions already reported for the closely related
Streptococcus pneumoniae
but also by other specific replacements that are presumably close to the active-site serine. Mosaic structure was also presumed in a
pbp1a
-sensitive strain used for comparison, confirming that these structures do not unambiguously imply, by themselves, detectable critical changes in the kinetic properties of these proteins.
Optimization of Xanthones for Antimalarial Activity: the 3,6-Bis-ω-Diethylaminoalkoxyxanthone Series
Authors: Kelly Jane Xu , Winter Rolf , Peyton David H. , Hinrichs David J. , Riscoe Michael
Published: July 2002

Hydroxyxanthones have been identified as novel antimalarial agents. The compounds are believed to exert their activity by complexation to heme and inhibition of hemozoin formation. Modification of the xanthone structure was pursued to improve their antimalarial activity. Attachment of R-groups bearing protonatable nitrogen atoms was conducted to enhance heme affinity through ionic interactions with the propionate side chains of the metalloporphyrin and to facilitate drug accumulation in the parasite food vacuole. A series of 3,6-bis-ω-diethylaminoalkoxyxanthones with side chains ranging from 2 to 8 carbon atoms were prepared and evaluated. Measurement of heme affinity for each of the derivatives revealed a strong correlation (
R
2
= 0.97) between affinity and antimalarial potency. The two most active compounds in the series contained 5- and 6-carbon side chains and exhibited low nanomolar 50% inhibitory concentration (IC
50
) values against strains of chloroquine-susceptible and multidrug-resistant
Plasmodium falciparum
in vitro. Both of these xanthones exhibit stronger heme affinity (8.26 × 10
5
and 9.02 × 10
5
M
−1
, respectively) than either chloroquine or quinine under similar conditions and appear to complex heme in a unique manner.
Pharmacokinetics and Safety of an Antirhinoviral Agent, Ruprintrivir, in Healthy Volunteers
Authors: Hsyu Poe-Hirr , Pithavala Yazdi K. , Gersten Merril , Penning Carol A. , Kerr Bradley M.
Published: July 2002

A single-dose study and a multiple-dose study of the safety and pharmacokinetics of ruprintrivir, a new selective irreversible inhibitor of human rhinovirus 3C protease, were conducted with healthy adult volunteers. Both studies were double-blind, randomized, placebo-controlled, parallel-group investigations of ruprintrivir administered intranasally at two dose levels. The parent drug and its acid metabolite, AG7185, were measured in plasma samples and nasal washings, and the safety of the treatments was monitored. Intranasal ruprintrivir, administered as single doses of 4 and 8 mg or every 3 h, six times per day, for 7 days was safe and well tolerated. Adverse events were mild, short-lived, and confined to the upper respiratory tract (i.e., nose and throat, taste and smell perceptions). Adverse events were similar after placebo and after single or multiple doses of active drug. Systemic exposure to ruprintrivir was rarely detectable with the highest measured concentration of ≤0.52 ng/ml; the assay had a lower limit of quantification of 0.2 ng/ml. Systemic exposure to the metabolite was also low, with a highest measured concentration of 3.25 ng/ml. Concentrations of AG7185 observed during multiple dosing were higher than those observed after the first dose but were no more than predicted from the single-dose study. Substantial amounts of ruprintrivir were observed intranasally for at least 9 h after multiple doses of ruprintrivir.
Effects of Specific Zidovudine Resistance Mutations and Substrate Structure on Nucleotide-Dependent Primer Unblocking by Human Immunodeficiency Virus Type 1 Reverse Transcriptase
Authors: Meyer Peter R. , Matsuura Suzanne E. , Tolun Adviye A. , Pfeifer Ingrid , So Antero G. , Mellors John W. , Scott Walter A.
Published: July 2002

Nucleotide-dependent unblocking of chain-terminated DNA by human immunodeficiency virus type 1 reverse transcriptase (RT) is enhanced by the presence of mutations associated with 3′-azido-3′-deoxythymidine (AZT) resistance. The increase in unblocking activity was greater for mutant combinations associated with higher levels of in vivo AZT resistance. The difference between mutant and wild-type activity was further enhanced by introduction of a methyl group into the nucleotide substrate and was decreased for a nonaromatic substrate, suggesting that π-π interactions between RT and an aromatic structure may be facilitated by these mutations.
Comparison of Methods for Quantification of Cytochrome
cd
1
-Denitrifying Bacteria in Environmental Marine Samples
Authors: Michotey Valérie , Méjean Vincent , Bonin Patricia
Published: July 2002

Two PCR primer sets were developed for the detection and quantification of cytochrome
cd
1
-denitrifying bacteria in environmental marine samples. The specificity and sensitivity of these primers were tested. Both primer sets were suitable for detection, but only one set, cd3F–cd4R, was suitable for the quantification and enumeration of the functional community using most-probable-number PCR and competitive PCR techniques. Quantification of cytochrome
cd
1
denitrifiers taken from marine sediment and water samples was achieved using two different molecular techniques which target the
nirS
gene, and the results were compared to those obtained by using the classical cultivation method. Enumerations using both molecular techniques yielded similar results in seawater and sediment samples. However, both molecular techniques showed 1,000 or 10 times more cytochrome
cd
1
denitrifiers in the sediment or water samples, respectively, than were found by use of the conventional cultivation method for counting.
Gene Cloning, Sequencing, and Inactivation of the Branched-Chain Aminotransferase of
Lactococcus lactis
LM0230
Authors: Atiles Myrta W. , Dudley Edward G. , Steele James L.
Published: July 2002

A branched-chain aminotransferase gene (
ilvE
) from
Lactococcus lactis
LM0230 was identified on a 9-kb chromosomal insert by complementation in
Escherichia coli
DL39. Sequencing of a 2.0-kbp fragment resulted in the identification of a 1,023-bp open reading frame that could encode a 340-amino-acid protein. Sequence analysis of the deduced amino acid sequence revealed 62% identity to IlvE of
Haemophilus influenzae
and high similarity to IlvEs from a variety of organisms found in GenBank classified as class IV aminotransferases. Under logarithmic growth in complex medium,
ilvE
is transcribed monocistronically as a 1.1-kb transcript. Hydrophobicity plot analysis of the deduced amino acid sequence and the lack of a signal peptide sequence suggest IlvE is a cytosolic protein. A derivative of LM0230 lacking IlvE activity was constructed by gene replacement. Comparison of the IlvE-deficient strain's ability to grow in defined media lacking an amino acid but containing its α-keto acid biosynthetic precursor to that of the wild-type strain indicated that IlvE is the only enzyme capable of synthesis of Ile and Val from their biosynthetic precursors. Comparison of the aminotransferase activity of the IlvE mutant to LM0230 revealed that the mutant retained <2, 4.5, 43, 40, and 76% of its aminotransferase activity with Ile, Val, Leu, Met, and Phe, respectively. No difference in growth or acidification rate between LM0230 and the IlvE-deficient strain was observed in milk.
Effect of Electron Donor and Solution Chemistry on Products of Dissimilatory Reduction of Technetium by
Shewanella putrefaciens
Authors: Wildung R. E. , Gorby Y. A. , Krupka K. M. , Hess N. J. , Li S. W. , Plymale A. E. , McKinley J. P. , Fredrickson J. K.
Published: July 2002

To help provide a fundamental basis for use of microbial dissimilatory reduction processes in separating or immobilizing
99
Tc in waste or groundwaters, the effects of electron donor and the presence of the bicarbonate ion on the rate and extent of pertechnetate ion [Tc(VII)O
4
−
] enzymatic reduction by the subsurface metal-reducing bacterium
Shewanella putrefaciens
CN32 were determined, and the forms of aqueous and solid-phase reduction products were evaluated through a combination of high-resolution transmission electron microscopy, X-ray absorption spectroscopy, and thermodynamic calculations. When H
2
served as the electron donor, dissolved Tc(VII) was rapidly reduced to amorphous Tc(IV) hydrous oxide, which was largely associated with the cell in unbuffered 0.85% NaCl and with extracellular particulates (0.2 to 0.001 μm) in bicarbonate buffer. Cell-associated Tc was present principally in the periplasm and outside the outer membrane. The reduction rate was much lower when lactate was the electron donor, with extracellular Tc(IV) hydrous oxide the dominant solid-phase reduction product, but in bicarbonate systems much less Tc(IV) was associated directly with the cell and solid-phase Tc(IV) carbonate may have been present. In the presence of carbonate, soluble (<0.001 μm) electronegative, Tc(IV) carbonate complexes were also formed that exceeded Tc(VII)O
4
−
in electrophoretic mobility. Thermodynamic calculations indicate that the dominant reduced Tc species identified in the experiments would be stable over a range of E
h
and pH conditions typical of natural waters. Thus, carbonate complexes may represent an important pathway for Tc transport in anaerobic subsurface environments, where it has generally been assumed that Tc mobility is controlled by low-solubility Tc(IV) hydrous oxide and adsorptive, aqueous Tc(IV) hydrolysis products.
Rhamnolipid-Induced Removal of Lipopolysaccharide from
Pseudomonas aeruginosa
: Effect on Cell Surface Properties and Interaction with Hydrophobic Substrates
Authors: Al-Tahhan Ragheb A. , Sandrin Todd R. , Bodour Adria A. , Maier Raina M.
Published: July 2002

Little is known about the interaction of biosurfactants with bacterial cells. Recent work in the area of biodegradation suggests that there are two mechanisms by which biosurfactants enhance the biodegradation of slightly soluble organic compounds. First, biosurfactants can solubilize hydrophobic compounds within micelle structures, effectively increasing the apparent aqueous solubility of the organic compound and its availability for uptake by a cell. Second, biosurfactants can cause the cell surface to become more hydrophobic, thereby increasing the association of the cell with the slightly soluble substrate. Since the second mechanism requires very low levels of added biosurfactant, it is the more intriguing of the two mechanisms from the perspective of enhancing the biodegradation process. This is because, in practical terms, addition of low levels of biosurfactants will be more cost-effective for bioremediation. To successfully optimize the use of biosurfactants in the bioremediation process, their effect on cell surfaces must be understood. We report here that rhamnolipid biosurfactant causes the cell surface of
Pseudomonas
spp. to become hydrophobic through release of lipopolysaccharide (LPS). In this study, two
Pseudomonas aeruginosa
strains were grown on glucose and hexadecane to investigate the chemical and structural changes that occur in the presence of a rhamnolipid biosurfactant. Results showed that rhamnolipids caused an overall loss in cellular fatty acid content. Loss of fatty acids was due to release of LPS from the outer membrane, as demonstrated by 2-keto-3-deoxyoctonic acid and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and further confirmed by scanning electron microscopy. The amount of LPS loss was found to be dependent on rhamnolipid concentration, but significant loss occurred even at concentrations less than the critical micelle concentration. We conclude that rhamnolipid-induced LPS release is the probable mechanism of enhanced cell surface hydrophobicity.
An Antifungal Exo-α-1,3-Glucanase (AGN13.1) from the Biocontrol FungusTrichoderma harzianum
Authors: Ait-Lahsen Hassane , Soler Andrés , Rey Manuel , de la Cruz Jesús , Monte Enrique , Llobell Antonio
Published: July 2002

Trichoderma harzianumsecretes α-1,3-glucanases when it is grown on polysaccharides, fungal cell walls, or autoclaved mycelium as a carbon source (simulated antagonistic conditions). We have purified and characterized one of these enzymes, named AGN13.1. The enzyme was monomeric and slightly basic. AGN13.1 was an exo-type α-1,3-glucanase and showed lytic and antifungal activity against fungal plant pathogens. Northern and Western analyses indicated that AGN13.1 is induced by conditions that simulated antagonism. We propose that AGN13.1 contributes to the antagonistic response ofT. harzianum.
Evidence for Extensive Resistance Gene Transfer among
Bacteroides
spp. and among
Bacteroides
and Other Genera in the Human Colon
Authors: Shoemaker N. B. , Vlamakis H. , Hayes K. , Salyers A. A.
Published: July 2002

Transfer of antibiotic resistance genes by conjugation is thought to play an important role in the spread of resistance. Yet virtually no information is available about the extent to which such horizontal transfers occur in natural settings. In this paper, we show that conjugal gene transfer has made a major contribution to increased antibiotic resistance in
Bacteroides
species, a numerically predominant group of human colonic bacteria. Over the past 3 decades, carriage of the tetracycline resistance gene,
tetQ
, has increased from about 30% to more than 80% of strains. Alleles of
tetQ
in different
Bacteroides
species, with one exception, were 96 to 100% identical at the DNA sequence level, as expected if horizontal gene transfer was responsible for their spread. Southern blot analyses showed further that transfer of
tetQ
was mediated by a conjugative transposon (CTn) of the CTnDOT type. Carriage of two erythromycin resistance genes,
ermF
and
ermG
, rose from <2 to 23% and accounted for about 70% of the total erythromycin resistances observed. Carriage of
tetQ
and the
erm
genes was the same in isolates taken from healthy people with no recent history of antibiotic use as in isolates obtained from patients with
Bacteroides
infections. This finding indicates that resistance transfer is occurring in the community and not just in clinical environments. The high percentage of strains that are carrying these resistance genes in people who are not taking antibiotics is consistent with the hypothesis that once acquired, these resistance genes are stably maintained in the absence of antibiotic selection. Six recently isolated strains carried
ermB
genes. Two were identical to
erm
(B)-P from
Clostridium perfringens
, and the other four had only one to three mismatches. The nine strains with
ermG
genes had DNA sequences that were more than 99% identical to the
ermG
of
Bacillus sphaericus
. Evidently, there is a genetic conduit open between gram-positive bacteria, including bacteria that only pass through the human colon, and the gram-negative
Bacteroides
species. Our results support the hypothesis that extensive gene transfer occurs among bacteria in the human colon, both within the genus
Bacteroides
and among
Bacteroides
species and gram-positive bacteria.
Twelve-Hour PCR-Based Method for Detection of
Salmonella
spp. in Food
Authors: Ferretti R. , Mannazzu I. , Cocolin L. , Comi G. , Clementi F.
Published: July 2002

A PCR-based method for the detection of
Salmonella
spp. in food was developed. The method, set up on typical salami from the Italian region of Marche, is sensitive and specific and shows excellent correlation with the conventional method of reference when naturally contaminated foods are analyzed. Moreover, it can be easily performed within a maximum of 12 h from food sampling, thus allowing prompt detection of
Salmonella
spp. in the food stocks analyzed.
Suboxic Deposition of Ferric Iron by Bacteria in Opposing Gradients of Fe(II) and Oxygen at Circumneutral pH
Authors: Sobolev Dmitri , Roden Eric E.
Published: July 2002

The influence of lithotrophic Fe(II)-oxidizing bacteria on patterns of ferric oxide deposition in opposing gradients of Fe(II) and O
2
was examined at submillimeter resolution by use of an O
2
microelectrode and diffusion microprobes for iron. In cultures inoculated with lithotrophic Fe(II)-oxidizing bacteria, the majority of Fe(III) deposition occurred below the depth of O
2
penetration. In contrast, Fe(III) deposition in abiotic control cultures occurred entirely within the aerobic zone. The diffusion microprobes revealed the formation of soluble or colloidal Fe(III) compounds during biological Fe(II) oxidation. The presence of mobile Fe(III) in diffusion probes from live cultures was verified by washing the probes in anoxic water, which removed ca. 70% of the Fe(III) content of probes from live cultures but did not alter the Fe(III) content of probes from abiotic controls. Measurements of the amount of Fe(III) oxide deposited in the medium versus the probes indicated that ca. 90% of the Fe(III) deposited in live cultures was formed biologically. Our findings show that bacterial Fe(II) oxidation is likely to generate reactive Fe(III) compounds that can be immediately available for use as electron acceptors for anaerobic respiration and that biological Fe(II) oxidation may thereby promote rapid microscale Fe redox cycling at aerobic-anaerobic interfaces.
Reduction in Exopolysaccharide Viscosity as an Aid to Bacteriophage Penetration through
Pseudomonas aeruginosa
Biofilms
Authors: Hanlon Geoffrey W. , Denyer Stephen P. , Olliff Cedric J. , Ibrahim Lamia J.
Published: July 2002

To cause an infection, bacteriophages must penetrate the alginate exopolysaccharide of
Pseudomonas aeruginosa
to reach the bacterial surface. Despite a lack of intrinsic motility, phage were shown to diffuse through alginate gels at alginate concentrations up to 8% (wt/vol) and to bring about a 2-log reduction in the cell numbers in 20-day-old biofilms of
P. aeruginosa
. The inability of alginate to act as a more effective diffusional barrier suggests that phage may cause a reduction in the viscosity of the exopolysaccharide. Samples (
n
= 5) of commercial alginate and purified cystic fibrosis (CF) alginate were incubated with 2 × 10
8
purified phage per ml for 24 h at 37°C. After incubation the samples and controls were subjected to rheological analysis with a Carrimed controlled stress rheometer. The viscosities of phage-treated samples were reduced by up to 40% compared to those of controls incubated in the absence of phage. The experiment was repeated by using phage concentrations of 10
10
and 10
12
phage per ml and samples taken for analysis at intervals up to 4 h. The results indicated that there was a time- and concentration-dependent reduction in viscosity of up to 40% compared to the viscosities of the controls. Commercial and purified CF alginate samples, both phage treated and untreated, were subjected to gel filtration chromatography by using Sephacryl High Resolution S-400 medium in order to obtain evidence of degradation. The results demonstrated that alginate treated with phage had a lower molecular weight than untreated alginate. The data suggest that bacteriophage migration through
P. aeruginosa
biofilms may be facilitated by a reduction in alginate viscosity brought about by enzymic degradation and that the source of the enzyme may be the bacterial host itself.
Hyperthermostable Endoglucanase from
Pyrococcus horikoshii
Authors: Ando Susumu , Ishida Hiroyasu , Kosugi Yoshitsugu , Ishikawa Kazuhiko
Published: July 2002

An endoglucanase homolog from the hyperthermophilic archaeon
Pyrococcus horikoshii
was expressed in
Escherichia coli
, and its enzymatic characteristics were examined. The expressed protein was a hyperthermostable endoglucanase which hydrolyzes celluloses, including Avicel and carboxymethyl cellulose, as well as β-glucose oligomers. This enzyme is the first endoglucanase belonging to glycosidase family 5 found from
Pyrococcus
species and is also the first hyperthermostable endoglucanase to which celluloses are the best substrates. This enzyme is expected to be useful for industrial hydrolysis of cellulose at high temperatures, particularly in biopolishing of cotton products.
Isolation and Characterization of Endophytic Colonizing Bacteria from Agronomic Crops and Prairie Plants
Authors: Zinniel Denise K. , Lambrecht Pat , Harris N. Beth , Feng Zhengyu , Kuczmarski Daniel , Higley Phyllis , Ishimaru Carol A. , Arunakumari Alahari , Barletta Raúl G. , Vidaver Anne K.
Published: July 2002

Endophytic bacteria reside within plant hosts without causing disease symptoms. In this study, 853 endophytic strains were isolated from aerial tissues of four agronomic crop species and 27 prairie plant species. We determined several phenotypic properties and found approximately equal numbers of gram-negative and gram-positive isolates. In a greenhouse study, 28 of 86 prairie plant endophytes were found to colonize their original hosts at 42 days postinoculation at levels of 3.5 to 7.7 log
10
CFU/g (fresh weight). More comprehensive colonization studies were conducted with 373 corn and sorghum endophytes. In growth room studies, none of the isolates displayed pathogenicity, and 69 of the strains were recovered from corn or sorghum seedlings at levels of 8.3 log
10
CFU/plant or higher. Host range greenhouse studies demonstrated that 26 of 29 endophytes were recoverable from at least one host other than corn and sorghum at levels of up to 5.8 log
10
CFU/g (fresh weight). Long-range dent corn greenhouse studies and field trials with 17 wild-type strains and 14 antibiotic-resistant mutants demonstrated bacterial persistence at significant average colonization levels ranging between 3.4 and 6.1 log
10
CFU/g (fresh weight) up to 78 days postinoculation. Three prairie and three agronomic endophytes exhibiting the most promising levels of colonization and an ability to persist were identified as
Cellulomonas
,
Clavibacter
,
Curtobacterium
, and
Microbacterium
isolates by 16S rRNA gene sequence, fatty acid, and carbon source utilization analyses. This study defines for the first time the endophytic nature of
Microbacterium testaceum
. These microorganisms may be useful for biocontrol and other applications.
Experimental Study of Interactions between Purple and Green Sulfur Bacteria in Sandy Sediments Exposed to Illumination Deprived of Near-Infrared Wavelengths
Authors: Massé Astrid , Pringault Olivier , de Wit Rutger
Published: July 2002

Sedimentary biofilms of the green sulfur bacterium
Prosthecochloris aestuarii
strain CE 2404, the purple sulfur bacterium
Thiocapsa roseopersicina
strain 5811, and a mixed culture of both were cultured in fine sand (100- to 300-μm grain size) within counter gradients of oxygen and sulfide. The artificial sediments were exposed to illumination deprived of near-infrared light (NIR) by filtering out the wavelengths longer than 700 nm to simulate the critical light conditions in submerged aquatic sediments. A 16 h of visible light-8 h of dark regimen was used. We studied the effects of these light conditions on the metabolisms of and interactions between both species by comparing the single species biofilms with the mixed biofilm. The photosynthesis rates of
P. aestuarii
were shown to be highly limited by the imposed light conditions, because the sulfide photooxidation rates were strongly stimulated when NIR was added.
T. roseopersicina
performed both aerobic chemosynthesis and photosynthesis, but the photosynthesis rates were low and poorly stimulated by the addition of NIR. This species decreased the penetration depth of oxygen in the sediment by about 1 mm by actively respiring oxygen. This way, the strict anaerobe
P. aestuarii
was able to grow closer to the surface in the mixed culture. As a result,
P. aestuarii
benefited from the presence of
T. roseopersicina
in the mixed culture, which was reflected by an increase in the biomass. In contrast, the density of the latter species was almost completely unaffected by the interaction. Both species coexisted in a layer of the same depth in the mixed culture, and the ecological and evolutionary implications of coexistence are discussed.
Universal Immunoprobe for (Per)Chlorate-Reducing Bacteria
Authors: O'Connor Susan M. , Coates John D.
Published: July 2002

Recent studies in our lab have demonstrated the ubiquity and diversity of microorganisms which couple growth to the reduction of chlorate or perchlorate [(per)chlorate] under anaerobic conditions. We identified two taxonomic groups, the
Dechloromonas
and the
Dechlorosoma
groups, which represent the dominant (per)chlorate-reducing bacteria (ClRB) in the environment. As part of these studies we demonstrated that chlorite dismutation is a central step in the reductive pathway of (per)chlorate that is common to all ClRB and which is mediated by the enzyme chlorite dismutase (CD). Initial studies on CD suggested that this enzyme is highly conserved among the ClRB, regardless of their phylogenetic affiliation. As such, this enzyme makes an ideal target for a probe specific for these organisms. Polyclonal antibodies were commercially raised against the purified CD from the ClRB
Dechloromonas agitata
strain CKB. The obtained antiserum was deproteinated by ammonium sulfate precipitation, and the antigen binding activity was assessed using dot blot analysis of a serial dilution of the antiserum. The titers obtained with purified CD indicated that the antiserum had a high affinity for the CD enzyme, and activity was observed in dilutions as low as 10
−6
of the original antiserum. The antiserum was active against both cell lysates and whole cells of
D. agitata
, but only if the cells were grown anaerobically with (per)chlorate. No response was obtained with aerobically grown cultures. In addition to
D. agitata
, dot blot analysis employed with both whole-cell suspensions and cell lysates of several diverse ClRB representing the alpha, beta, and gamma subclasses of
Proteobacteria
tested positive regardless of phylogenetic affiliation. Interestingly, the dot blot response obtained for each of the ClRB cell lysates was different, suggesting that there may be some differences in the antigenic sites of the CD protein produced in these organisms. In general, no reactions were observed with cells or cell lysates of the organisms closely related to the ClRB which could not grow by (per)chlorate reduction. These studies have resulted in the development of a highly specific and sensitive immunoprobe based on the commonality of the CD enzyme in ClRB which can be used to assess dissimilatory (per)chlorate-reducing populations in environmental samples regardless of their phylogenetic affiliations.