bluuebunny/crossref_metadata_2025 huggingface dataset:
Discovery of Novel Antifungal (1,3)-β-
d
-Glucan Synthase Inhibitors
Authors: Onishi J. , Meinz M. , Thompson J. , Curotto J. , Dreikorn S. , Rosenbach M. , Douglas C. , Abruzzo G. , Flattery A. , Kong L. , Cabello A. , Vicente F. , Pelaez F. , Diez M. T. , Martin I. , Bills G. , Giacobbe R. , Dombrowski A. , Schwartz R. , Morris S. , Harris G. , Tsipouras A. , Wilson K. , Kurtz M. B.
Published: July 2002

The increasing incidence of life-threatening fungal infections has driven the search for new, broad-spectrum fungicidal agents that can be used for treatment and prophylaxis in immunocompromised patients. Natural-product inhibitors of cell wall (1,3)-β-
d
-glucan synthase such as lipopeptide pneumocandins and echinocandins as well as the glycolipid papulacandins have been evaluated as potential therapeutics for the last two decades. As a result, MK-0991 (caspofungin acetate; Cancidas), a semisynthetic analogue of pneumocandin B
o
, is being developed as a broad-spectrum parenteral agent for the treatment of aspergillosis and candidiasis. This and other lipopeptide antifungal agents have limited oral bioavailability. Thus, we have sought new chemical structures with the mode of action of lipopeptide antifungal agents but with the potential for oral absorption. Results of natural-product screening by a series of newly developed methods has led to the identification of four acidic terpenoid (1,3)-β-
d
-glucan synthase inhibitors. Of the four compounds, the in vitro antifungal activity of one, enfumafungin, is comparable to that of L-733560, a close analogue of MK-0991. Like the lipopeptides, enfumafungin specifically inhibits glucan synthesis in whole cells and in (1,3)-β-
d
-glucan synthase assays, alters the morphologies of yeasts and molds, and produces a unique response in
Saccharomyces cerevisiae
strains with point mutations in
FKS1
, the gene which encodes the large subunit of glucan synthase.
Efficacy of FK463, a New Lipopeptide Antifungal Agent, in Mouse Models of Pulmonary Aspergillosis
Authors: Matsumoto Satoru , Wakai Yoshimi , Nakai Toru , Hatano Kazuo , Ushitani Tomoe , Ikeda Fumiaki , Tawara Shuichi , Goto Toshio , Matsumoto Fumio , Kuwahara Shogo
Published: July 2002

The efficacy of FK463, a novel water-soluble lipopeptide, was evaluated in mouse models of pulmonary aspergillosis and was compared with that of amphotericin B (AMPH-B). In the pulmonary aspergillosis models induced by intranasal inoculation, FK463 exhibited good efficacy, with 50% effective doses in the range of 0.26 to 0.51 mg/kg of body weight; these values were comparable to those of AMPH-B. In an
Aspergillus
target organ assay with immunosuppressed mice, under conditions of constant plasma levels of FK463, using a subcutaneously implanted osmotic pressure pump, a significant reduction in viable fungal cells was observed at plasma FK463 levels of 0.55 to 0.80 μg/ml or higher. We conclude that FK463 is highly effective in the treatment of pulmonary aspergillosis in this animal model. These results indicate that FK463 may be a potent parenterally administered antifungal agent for pulmonary aspergillosis.
Potent and Selective Activity of a Combination of Thymidine and 1843U89, a Folate-Based Thymidylate Synthase Inhibitor, against
Plasmodium falciparum
Authors: Jiang Lei , Lee Pei-Chieh , White John , Rathod Pradipsinh K.
Published: July 2002

Unlike mammalian cells, malarial parasites are completely dependent on the de novo pyrimidine pathway and lack the enzymes to salvage preformed pyrimidines. In the present study, first, it is shown that 1843U89, even without polyglutamylation, is a potent folate-based inhibitor of purified malarial parasite thymidylate synthase. The binding was noncompetitive with respect to methylenetetrahydrofolate, and 1843U89 had a

K
i

of 1 nM. The compound also had potent antimalarial activity in vitro.
Plasmodium falciparum
cells in culture were inhibited by 1843U89, with a 50% inhibitory concentration of about 70 nM. The compound was effective against drug-sensitive as well as drug-resistant clones of
P. falciparum
. As predicted by the biochemistry of the parasite, the potent inhibition of parasite proliferation by 1843U89 could not be reversed with 10 μM thymidine. In contrast, in the presence of 10 μM thymidine, mammalian cells were unaffected by 1843U89 even at concentrations as high as 0.1 mM, thus offering a selectivity window of more than 1,000-fold. On this basis, folate-based thymidylate synthase inhibitors may represent a powerful additional tool that can be used to combat drug-resistant malaria.
Concentrations of Chloroquine and Malaria Parasites in Blood in Nigerian Children
Authors: Mockenhaupt Frank P. , May Jürgen , Bergqvist Yngve , Ademowo Olusegun G. , Olumese Peter E. , Falusi Adeyinka G. , Großterlinden Lars , Meyer Christian G. , Bienzle Ulrich
Published: July 2002

Consumption of chloroquine (CQ) and subtherapeutic drug levels in blood are considered to be widespread in areas where malaria is endemic. A cross-sectional study was performed with 405 Nigerian children to assess factors associated with the presence of CQ in blood and to examine correlations of drug levels with malaria parasite species and densities. Infections with
Plasmodium
species and parasite densities were determined by microscopy and PCR assays. Whole-blood CQ concentrations were measured by high-performance liquid chromatography.
Plasmodium falciparum
,
P. malariae
, and
P. ovale
were observed in 80, 16, and 9% of the children, respectively, and CQ was detected in 52% of the children. CQ concentrations were >17 and <100 nmol/liter in 25% of the children, 100 to 499 nmol/liter in 14% of the children, and ≥500 nmol/liter in 13% of the children. Young age, attendance at health posts, and absence of parasitemia were factors independently associated with CQ in blood. With increasing concentrations of CQ, the prevalence of
P. falciparum
infection and parasite densities decreased. However, at concentrations corresponding to those usually attained during regular prophylaxis (≥500 nmol/liter), 62% of children were still harboring
P. falciparum
parasites. In contrast, no infection with
P. malariae
and only one infection with
P. ovale
were observed in children with CQ concentrations of ≥100 nmol/liter. These data show the high prevalence of subcurative CQ concentrations in Nigerian children and confirm the considerable degree of CQ resistance in that country. Subtherapeutic drug levels are likely to further promote CQ resistance and may impair the development and maintenance of premunition in areas where malaria is endemic.
Enhanced Extracellular Production of Aspartyl Proteinase, a Virulence Factor, by
Candida albicans
Isolates following Growth in Subinhibitory Concentrations of Fluconazole
Authors: Wu Tao , Wright Katherine , Hurst Steven F. , Morrison Christine J.
Published: July 2002

We examined the production of secreted aspartyl proteinase (Sap), a putative virulence factor of
Candida albicans
, by a series of 17 isolates representing a single strain obtained from the oral cavity of an AIDS patient before and after the development of clinical and in vitro resistance to fluconazole. Isolates were grown in Sap-inducing yeast carbon base-bovine serum albumin medium containing 0, 0.25, 0.5, or 1 MIC of fluconazole, and cultures were sampled daily for 14 days to determine extracellular Sap activity by enzymatic degradation of bovine serum albumin. Extracellular Sap activity was significantly decreased in a dose-dependent manner for the most fluconazole-susceptible isolate (MIC, 1.0 μg/ml) and significantly increased in a dose-dependent manner for the most fluconazole-resistant isolate (MIC, >64 μg/ml). Enhanced extracellular Sap production could not be attributed to cell death or nonspecific release of Sap, because there was no reduction in the number of CFU and no significant release of enolase, a constitutive enzyme of the glycolytic pathway. Conversely, intracellular Sap concentrations were significantly increased in a dose-dependent manner in the most fluconazole-susceptible isolate and decreased in the most fluconazole-resistant isolate. Enhanced Sap production correlated with the overexpression of a gene encoding a multidrug resistance (
MDR1
) efflux pump occurring in these isolates. These data indicate that exposure to subinhibitory concentrations of fluconazole can result in enhanced extracellular production of Sap by isolates with the capacity to overexpress
MDR1
and imply that patients infected with these isolates and subsequently treated with suboptimal doses of fluconazole may experience enhanced
C. albicans
virulence in vivo.
Serotype Distribution and Antimicrobial Resistance of
Streptococcus pneumoniae
Isolates from Pediatric Patients in Singapore
Authors: Soh Shirlena Wee-Ling , Poh Chit Laa , Lin Raymond V. Tzer Pin
Published: July 2002

One hundred eighty
Streptococcus pneumoniae
strains isolated from children at a pediatric hospital in Singapore from 1997 to 1999 were serotyped and their antimicrobial susceptibility patterns were determined. Sixty-three percent of the isolates were resistant to penicillin. Significantly large numbers of the strains investigated were resistant to trimethoprim-sulfamethoxazole (87.8%), tetracycline (71.7%), erythromycin (67.8%), and chloramphenicol (40%). Penicillin and multidrug resistance was mostly associated with the frequently isolated
S. pneumoniae
isolates of serotypes (serotypes 19F, 23F, 6B, and 14). Isolates of serotype 19F, the serotype most commonly encountered in Singapore (41.1%), had the highest prevalence of penicillin (78.4%) and multidrug resistance (94.6%). Most of the invasive
S. pneumoniae
isolates (8 of 17; 47.1%) were of serotype 14.
Bacteriostatic and Bactericidal Activities of Moxifloxacin against
Coxiella burnetii
Authors: Rolain Jean-Marc , Maurin Max , Raoult Didier
Published: July 2002

The in vitro activity of moxifloxacin against
Coxiella burnetii
was compared to those of pefloxacin, ofloxacin, and doxycycline. MICs of moxifloxacin ranged from 0.5 to 1 μg/ml for the Nine Mile, Priscilla, and Q212 strains. Moxifloxacin was not bactericidal against
C. burnetii
at 4 μg/ml.
Activity of Gatifloxacin Alone or in Combination with Pyrimethamine or Gamma Interferon against
Toxoplasma gondii
Authors: Khan Anis A. , Slifer Teri R. , Araujo Fausto G. , Remington Jack S.
Published: July 2002

The activity of gatifloxacin against
Toxoplasma gondii
, either alone or in combination with pyrimethamine or gamma interferon (IFN-γ), was examined in vitro and in vivo. In vitro, gatifloxacin significantly inhibited intracellular replication of tachyzoites of the RH strain with a 50% inhibitory concentration of 0.21 μg/ml at 48 h after addition of the drug to the cultures. Toxicity for host cells was not observed at this concentration. A synergistic effect (combination indices < 0.5) was demonstrated in vitro following 48 h of treatment with the combination of gatifloxacin and pyrimethamine (1:1 ratio). Doses of gatifloxacin of 100 and 200 mg/kg of body weight/day administered orally to mice for 10 days resulted in significant (
P
values of 0.056 and <0.0001, respectively) prolongation in time to death following infection with a lethal inoculum of tachyzoites. A dose of 400 mg/kg resulted in 20% survival (
P
= 0.0001). Mortality was 100% in untreated control mice and in mice treated with 25 or 50 mg/kg/day. Treatment of infected mice with a combination of gatifloxacin at 200 mg/kg/day and pyrimethamine at 12.5 mg/kg/day resulted in 85% survival, whereas 100 and 80% of mice treated with gatifloxacin alone or pyrimethamine alone, respectively, died (
P
< 0.0001). Moreover, a gatifloxacin dose of 200 mg/kg/day administered orally for 10 days plus 2 μg of recombinant murine IFN-γ/day administered intraperitoneally for 10 days resulted in significant survival compared with IFN-γ alone (
P
< 0.0001) or gatifloxacin alone (
P
< 0.007).
In Vitro and In Vivo Effects of 14α-Demethylase (
ERG11
) Depletion in
Candida glabrata
Authors: Nakayama Hironobu , Nakayama Noboru , Arisawa Mikio , Aoki Yuko
Published: July 2002

Sterol 14α-demethylase (
ERG11
) is the target enzyme of azole antifungals that are widely used for the treatment of fungal infections.
Candida glabrata
is known to be less susceptible to fluconazole than most
Candida albicans
strains, and the incidence of
C. glabrata
infection has been increasing mostly in conjunction with the use of azole antifungals. Recently, it has been reported that
C. glabrata
can rescue the defect of ergosterol biosynthesis by incorporating cholesterol from serum. To explore the effect of inactivating Erg11p in
C. glabrata
, we generated mutant strains in which the
ERG11
gene was placed under the control of tetracycline-regulatable promoters. In these mutants, expression of the
ERG11
gene can be repressed by doxycycline (DOX). All mutants showed a growth defect in the presence of DOX. The numbers of CFU of the mutants were lowered by only 1/10 with DOX treatment. In these mutants, accumulation of 4,14-dimethylzymosterol, which differs from an accumulated abnormal sterol detected in
C. albicans
and
Saccharomyces cerevisiae
treated with fluconazole, was observed by DOX treatment. Although such phenotypes were also observed in serum-containing media by DOX treatment, they were alleviated. Furthermore, the mutant could grow in DOX-treated mice without a severe reduction in the number of cells. Thus, depleting the expression of the
ERG11
gene lowered the number of CFU by only 1/10 due to the accumulation of 4,14-demethylzymosterol in vitro, and it did not result in the defective growth of fungal cells in mice. These results suggested that Erg11p is not an ideal target molecule of antifungals for
C. glabrata
.
Reversal of Chloroquine Resistance in
Plasmodium falciparum
Using Combinations of Chemosensitizers
Authors: van Schalkwyk Donelly A. , Walden Jason C. , Smith Peter J.
Published: July 2002

Research into chloroquine resistance reversal in
Plasmodium falciparum
has revealed a widespread range of functionally and structurally diverse chemosensitizers. However, nearly all of these chemosensitizers reverse resistance optimally only at concentrations that are toxic to humans. Verapamil, desipramine, and trifluoperazine were shown to potentiate chloroquine accumulation in a chloroquine-resistant (CQ
r
) strain of
P. falciparum
, while progesterone, ivermectin, and cyclosporin A were not shown to potentiate chloroquine accumulation. The simultaneous use of two or even three of these chemosensitizers at concentrations within their therapeutic ranges in humans displayed an additive effect in potentiating chloroquine accumulation in the CQ
r
strain. The levels of resistance reversal achieved with these multiple combinations were comparable to those achieved with high concentrations of the single agents used to enhance the activity of chloroquine. No chemosensitizer, whether used singly or in combination, potentiated any change in chloroquine accumulation or a shift in the 50% inhibitory concentration for the chloroquine-sensitive strain. The use of combinations of chemosensitizers at concentrations not toxic to humans could effectively reverse chloroquine resistance without the marked toxicity from the use of a single agent at high concentrations. This cocktail of chemosensitizers may serve as a viable treatment to restore the efficacy of chloroquine in patients with malaria.
Pharmacokinetics of the Protease Inhibitor Indinavir in Human Immunodeficiency Virus Type 1-Infected Children
Authors: Burger David M. , van Rossum Annemarie M. C. , Hugen Patricia W. H. , Suur Marja H. , Hartwig Nico G. , Geelen Sibyl P. M. , Scherpbier Henriette J. , Hoetelmans Richard M. W. , Vulto Arnold G. , de Groot Ronald
Published: July 2002

The objective of this study was to evaluate the pharmacokinetics of indinavir in human immunodeficiency virus-infected children as part of a prospective, open, uncontrolled, multicenter study in The Netherlands. Human immunodeficiency virus type 1-infected children were monitored over 6 months of treatment with zidovudine (120 mg/m2every 8 h [q8h]), lamivudine (4 mg/kg of body weight q12h), and indinavir (33mg/kg of metabolic weight [MW] q8h). Four weeks after the start of treatment, the steady-state pharmacokinetics of indinavir were determined by high-pressure liquid chromatography. If patients had an indinavir area under the concentration-time curve (AUC) of below 10 or above 30 mg/liter · h, a dose increase or a dose reduction was made and pharmacokinetic measurements were repeated 4 weeks later. Nineteen patients started with the dose of 33 mg/kg of MW q8h. The median AUC (range) was 10.5 (2.8 to 51.0) mg/liter · h. The median AUC (range) in 17 children treated with 50 mg/kg of MW q8h was 20.6 (4.1 to 38.7) mg/liter · h. Finally, five patients had a dose increase to 67 mg/kg of MW q8h, resulting in a median AUC (range) of 36.6 (27.2 to 80.0) mg/liter · h. After 6 months of treatment, there were 11 children with an AUC of below 20 mg/liter · h, of whom 5 (45%) had a detectable viral load, while this was the case in none of the 11 children with an AUC of higher than 20 mg/liter · h. We conclude that the optimal dose of indinavir in children to obtain drug exposure similar to that observed in adult patients is 50 mg/kg of MW q8h, which approximates 600 mg/m2q8h. It would even be better to adjust the indinavir dose based on an AUC of greater than 20 mg/liter · h.
Pharmacokinetic Evaluation of Oral Levofloxacin in Human Immunodeficiency Virus-Infected Subjects Receiving Concomitant Antiretroviral Therapy
Authors: Villani P. , Viale P. , Signorini L. , Cadeo B. , Marchetti F. , Villani A. , Fiocchi C. , Regazzi M. B. , Carosi G.
Published: July 2002

The purpose of this study was to evaluate the pharmacokinetics (PK) profile of oral levofloxacin in human immunodeficiency virus-positive patients in steady-state treatment with nelfinavir (NFV) or with efavirenz (EFV) and to determine the effects of levofloxacin on the PK parameters of these two antiretroviral agents. For levofloxacin, plasma samples were obtained at steady state during a 24-h dosing interval. Plasma NFV and EFV concentrations were evaluated before and after 4 days of levofloxacin treatment. Levofloxacin PK do not seem affected by NFV and EFV. There was no significant difference between NFV and EFV plasma levels obtained with and without levofloxacin.
Pfmdr1
Alleles and Response to Ultralow-Dose Mefloquine Treatment in Gabonese Patients
Authors: Mawili-Mboumba Denise P. , Kun Jürgen F. J. , Lell Bertrand , Kremsner Peter G. , Ntoumi Francine
Published: July 2002

The identification of parasite molecular markers involved in resistance to antimalarial compounds is of great interest for monitoring the development and spread of resistance in the field. Polymorphisms in
Plasmodium falciparum
multidrug resistance gene 1 (
pfmdr1
) have been associated with chloroquine resistance and mefloquine susceptibility. In the present study, carried out in Lambaréné, Gabon, we investigated the relationship between the presence of mutations at codons 86, 184, 1034, 1042, and 1246 in the
pfmdr1
gene and the success of ultralow-dose mefloquine treatment (1.1 mg/kg of body weight). Sixty-nine patients were included in the study, and depending on the level of in vivo resistance to mefloquine, they were classified as sensitive responders (S), patients with low-grade resistance (RI), and nonresponders (NR). We found that the prevalences of the Tyr-86 mutation among isolates from patients in groups S, RI, and NR were 100, 96, and 90%, respectively, and that the prevalence of the Phe-184 mutation among the isolates was 80% in each group. A prevalence of about 10% point mutations at codons 1042 and 1246 was detected only in isolates from patients in groups RI and NR. There was no statistically significant association between the presence of the Tyr-86 mutation and the in vivo response (
P
= 0.79). Among the parasite isolates from patients with drug-resistant infections, 83% had the wild-type
pfmdr1
genotype (S
1034
-N
1042
-D
1246
). No link between the presence of this genotype and parasite resistance was detected (
P
= 0.42). Among the isolates analyzed, 85 had double mutations (Y
86
-F
184
or Y
86
-Y
1246
) and 11 had triple mutations (Y
86
-D
1042
-Y
1246
, Y
86
-F
184
-Y
1246
, or Y
86
-F
184
-D
1042
). These findings are not consistent with those of previous in vitro studies and suggest that further evaluation of
pfmdr1
gene polymorphism and in vivo mefloquine sensitivity are needed.
Susceptibilities of Herpes Simplex Viruses to Penciclovir and Acyclovir in Eight Cell Lines
Authors: Leary Jeffry J. , Wittrock Robert , Sarisky Robert T. , Weinberg Adriana , Levin Myron J.
Published: July 2002

The commonly used antiviral drugs acyclovir (ACV) and penciclovir (PCV) possess similarly potent antiviral activities in vivo against herpes simplex virus (HSV). Assay methods for sensitivity to ACV are not necessarily transferable to PCV, even though the two drugs have similar in vivo potencies and mechanisms of action. We determined by plaque reduction assay the relative activities of ACV and PCV against five laboratory-adapted strains of HSV types 1 and 2 (including sensitive and resistant strains) in seven human cell lines and one nonhuman primate cell line. Seven characteristics were used to evaluate the cell lines. All cell lines were similar in their plating efficiencies and abilities to discriminate between sensitive and resistant HSV isolates. Vero and MRC-5 cells yielded the most discordant 50% inhibitory concentrations (IC
50
s) for the two HSV types, while Vero and WI-38 VA-13 cells yielded large differences in the IC
50
s of ACV and PCV. The limited life spans and poor plaque morphologies of the fibroblast lines were undesirable characteristics. Among the transformed cell lines producing well-defined plaques, A549 cells provided the best concordance between IC
50
s for the two HSV types and two antiherpes drugs. Comparison experiments with a yield reduction format indicated that the use of assays of this type might allow some of the cell-specific properties observed in plaque reduction assays to be avoided.
Acyclovir, Ganciclovir, and Zidovudine Transfer into Rat Milk
Authors: Alcorn Jane , McNamara Patrick J.
Published: July 2002

Treatment with antiviral agents that accumulate in breast milk may offer a novel approach to reduce the rates of vertical transmission of important viruses and the risk of clinical illness in suckling neonates. The present study evaluated the extent and mechanism of transfer of three antiviral nucleoside analogues into milk in a lactating rat model system. Acyclovir (0.26 mg/h), ganciclovir (0.13 mg/h), and zidovudine (0.5 mg/h) were each infused to steady-state concentrations in six rats 15 to 16 days postpartum. The observed ratios of the concentrations in milk to the concentrations in serum (observed milk-to-serum ratio), calculated from the ratio of the steady-state concentration in serum to the steady-state concentration in milk, determined the extent of drug transfer into milk. To identify the mechanism of transfer into milk, the observed milk-to-serum ratio was compared to a predicted milk-to-serum ratio estimated from an in vitro passive diffusion model of transfer of each drug into milk. High-pressure liquid chromatography methods determined milk and serum drug concentrations. Mean ± standard deviation observed milk-to-serum ratios for acyclovir, ganciclovir, and zidovudine were 5.1 ± 1.4, 1.6 ± 0.33, and 1.0 ± 0.29, respectively, compared with their corresponding predicted ratios of 1.1, 0.85, and 0.71. These results suggest that acyclovir accumulates in milk due to active transport mechanisms, while passive diffusion processes govern the transfer of both ganciclovir and zidovudine into milk. The presence of all three antiviral drugs in milk and the potential for active drug transfer into milk warrants further investigation of the accumulation of other antiviral drugs in milk and their therapeutic benefits in reducing the vertical transmission of viruses and clinical sequelae in the breast-feeding infant.
Bacterial Functional Redundancy along a Soil Reclamation Gradient
Authors: Yin Bei , Crowley David , Sparovek Gerd , De Melo Wanderley Jose , Borneman James
Published: July 2002

A strategy to measure bacterial functional redundancy was developed and tested with soils collected along a soil reclamation gradient by determining the richness and diversity of bacterial groups capable of in situ growth on selected carbon substrates. Soil cores were collected from four sites along a transect from the Jamari tin mine site in the Jamari National Forest, Rondonia, RO, Brazil: denuded mine spoil, soil from below the canopy of invading pioneer trees, revegetated soil under new growth on the forest edge, and the forest floor of an adjacent preserved forest. Bacterial population responses were analyzed by amending these soil samples with individual carbon substrates in the presence of bromodeoxyuridine (BrdU). BrdU-labeled DNA was then subjected to a 16S-23S rRNA intergenic analysis to depict the actively growing bacteria from each site. The number and diversity of bacterial groups responding to four carbon substrates (
l
-serine,
l
-threonine, sodium citrate, and α-lactose hydrate) increased along the reclamation-vegetation gradient such that the preserved forest soil samples contained the highest functional redundancy for each substrate. These data suggest that bacterial functional redundancy increases in relation to the regrowth of plant communities and may therefore represent an important aspect of the restoration of soil biological functionality to reclaimed mine spoils. They also suggest that bacterial functional redundancy may be a useful indicator of soil quality and ecosystem functioning.
A PCR Assay To Discriminate Human and Ruminant Feces on the Basis of Host Differences in
Bacteroides-Prevotella
Genes Encoding 16S rRNA
Authors: Bernhard Anne E. , Field Katharine G.
Published: July 2002

Our purpose was to develop a rapid, inexpensive method of diagnosing the source of fecal pollution in water. In previous research, we identified
Bacteroides-Prevotella
ribosomal DNA (rDNA) PCR markers based on analysis. These markers length heterogeneity PCR and terminal restriction fragment length polymorphism distinguish cow from human feces. Here, we recovered 16S rDNA clones from natural waters that were close phylogenetic relatives of the markers. From the sequence data, we designed specific PCR primers that discriminate human and ruminant sources of fecal contamination.
Antibacterial Activity Evaluation of Microcin J25 against Diarrheagenic
Escherichia coli
Authors: Sable Sophie , Pons Anne-Marie , Gendron-Gaillard Sandrine , Cottenceau Gilles
Published: July 2002

The inhibitory activities of known microcins were evaluated against some diarrheagenic
Escherichia coli
strains. Some antibacterial properties of microcin J25, the most active one, were studied. A rapid two-step purification was performed. The MIC and the minimum bactericidal concentration of J25 against
E. coli
O157:H7 were 1 and 100 μg ml
−1
, respectively. A 10
4
-CFU ml
−1
contamination by this strain was destroyed in milk and meat extract by 6.25 μg of J25 ml
−1
and in half-diluted egg yolk by 50 μg of J25 ml
−1
.
Evidence for Contribution of Neutral Trehalase in Barotolerance of
Saccharomyces cerevisiae
Authors: Iwahashi Hitoshi , Nwaka Solomon , Obuchi Kaoru
Published: July 2002

In yeast, trehalose accumulation and its hydrolysis, which is catalyzed by neutral trehalase, are believed to be important for thermotolerance. We have shown that trehalose is one of the important factors for barotolerance (resistance to hydrostatic pressure); however, nothing is known about the role of neutral trehalase in barotolerance. To estimate the contribution of neutral trehalase in resisting high hydrostatic pressure, we measured the barotolerance of neutral trehalase I and/or neutral trehalase II deletion strains. Under 180 MPa of pressure for 2 h, the neutral trehalase I deletion strain showed higher barotolerance in logarithmic-phase cells and lower barotolerance in stationary-phase cells than the wild-type strain. Introduction of the neutral trehalase I gene (
NTH1
) into the deletion mutant restored barotolerance defects in stationary-phase cells. Furthermore, we assessed the contribution of neutral trehalase during pressure and recovery conditions by varying the expression of
NTH1
or neutral trehalase activity with a galactose-inducible
GAL1
promoter with either glucose or galactose. The low barotolerance observed with glucose repression of neutral trehalase from the
GAL1
promoter was restored during recovery with galactose induction. Our results suggest that neutral trehalase contributes to barotolerance, especially during recovery.
Relationship between Nitrite Reduction and Active Phosphate Uptake in the Phosphate-Accumulating Denitrifier
Pseudomonas
sp. Strain JR 12
Authors: Barak Yoram , van Rijn Jaap
Published: July 2002

Phosphate uptake by the phosphate-accumulating denitrifier
Pseudomonas
sp. JR12 was examined with different combinations of electron and carbon donors and electron acceptors. Phosphate uptake in acetate-supplemented cells took place with either oxygen or nitrate but did not take place when nitrite served as the final electron acceptor. Furthermore, nitrite reduction rates by this denitrifier were shown to be significantly reduced in the presence of phosphate. Phosphate uptake assays in the presence of the H
+
-ATPase inhibitor
N,N
′-dicyclohexylcarbodiimide (DCCD), in the presence of the uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP), or with osmotic shock-treated cells indicated that phosphate transport over the cytoplasmic membrane of this bacterium was mediated by primary and secondary transport systems. By examining the redox transitions of whole cells at 553 nm we found that phosphate addition caused a significant oxidation of a
c
-type cytochrome. Based on these findings, we propose that this
c
-type cytochrome serves as an intermediate in the electron transfer to both nitrite reductase and the site responsible for active phosphate transport. In previous studies with this bacterium we found that the oxidation state of this
c
-type cytochrome was significantly higher in acetate-supplemented, nitrite-respiring cells (incapable of phosphate uptake) than in phosphate-accumulating cells incubated with different combinations of electron donors and acceptors. Based on the latter finding and results obtained in the present study it is suggested that phosphate uptake in this bacterium is subjected to a redox control of the active phosphate transport site. By means of this mechanism an explanation is provided for the observed absence of phosphate uptake in the presence of nitrite and inhibition of nitrite reduction by phosphate in this organism. The implications of these findings regarding denitrifying, phosphate removal wastewater plants is discussed.
Sequencing Bands of Ribosomal Intergenic Spacer Analysis Fingerprints for Characterization and Microscale Distribution of Soil Bacterium Populations Responding to Mercury Spiking
Authors: Ranjard Lionel , Brothier Elisabeth , Nazaret Sylvie
Published: July 2002

Two major emerging bands (a 350-bp band and a 650-bp band) within the RISA (ribosomal intergenic spacer analysis) profile of a soil bacterial community spiked with Hg(II) were selected for further identification of the populations involved in the response of the community to the added metal. The bands were cut out from polyacrylamide gels, cloned, characterized by restriction analysis, and sequenced for phylogenetic affiliation of dominant clones. The sequences were the intergenic spacer between the
rrs
and
rrl
genes and the first 130 nucleotides of the
rrl
gene. Comparison of sequences derived from the 350-bp band to The GenBank database permitted us to identify the bacteria as being mostly close relatives to low G+C firmicutes (
Clostridium
-like genera), while the 650-bp band permitted us to identify the bacteria as being mostly close relatives to β-proteobacteria (
Ralstonia
-like genera). Oligonucleotide probes specific for the identified dominant bacteria were designed and hybridized with the RISA profiles derived from the control and spiked communities. These studies confirmed the contribution of these populations to the community response to the metal. Hybridization of the RISA profiles from subcommunities (bacterial pools associated with different soil microenvironments) also permitted to characterize the distribution and the dynamics of these populations at a microscale level following mercury spiking.
Rapid Method for Coextraction of DNA and RNA from Natural Environments for Analysis of Ribosomal DNA- and rRNA-Based Microbial Community Composition
Authors: Griffiths Robert I. , Whiteley Andrew S. , O'Donnell Anthony G. , Bailey Mark J.
Published: July 2002

A rapid protocol for the extraction of total nucleic acids from environmental samples is described. The method facilitates concomitant assessment of microbial 16S rRNA diversity by PCR and reverse transcription-PCR amplification from a single extraction. Denaturing gradient gel electrophoresis microbial community analysis differentiated the active component (rRNA derived) from the total bacterial diversity (ribosomal DNA derived) down the horizons of an established grassland soil.
Morphologic, Host Specificity, and Molecular Characterization of a HungarianCryptosporidium meleagridisIsolate
Authors: Sréter Tamás , Kovács Gábor , da Silva Alexandre J. , Pieniazek Norman J. , Széll Zoltán , Dobos-Kovács Mihály , Márialigeti Károly , Varga István
Published: July 2002

This study was undertaken in order to characterizeCryptosporidium meleagridisisolated from a turkey in Hungary and to compare the morphologies, host specificities, organ locations, and small-subunit RNA (SSU rRNA) gene sequences of this organism and otherCryptosporidiumspecies. The phenotypic differences betweenC. meleagridisandCryptosporidium parvumHungarian calf isolate (zoonotic genotype) oocysts were small, although they were statistically significant. Oocysts ofC. meleagridiswere successfully passaged in turkeys and were transmitted from turkeys to immunosuppressed mice and from mice to chickens. The location ofC. meleagridiswas the small intestine, like the location ofC. parvum. A comparison of sequence data for the variable region of the SSU rRNA gene ofC. meleagridisisolated from turkeys with otherCryptosporidiumsequence data in the GenBank database revealed that the HungarianC. meleagridissequence is identical to aC. meleagridissequence recently described for a North Carolina isolate. Thus,C. meleagridisis a distinct species that occurs worldwide and has a broad host range, like theC. parvumzoonotic strain (also called the calf or bovine strain) andCryptosporidium felis. Because birds are susceptible toC. meleagridisand to some zoonotic strains ofC. parvum, these animals may play an active role in contamination of surface waters not only withCryptosporidium baileyibut also withC. parvum-like parasites.
Electricity Generation in Microbial Fuel Cells Using Neutral Red as an Electronophore
Authors: Park Doo Hyun , Zeikus J. Gregory
Published: July 2002

Neutral red (NR) was utilized as an electron mediator in microbial fuel cells consuming glucose to study both its efficiency during electricity generation and its role in altering anaerobic growth and metabolism of
Escherichia coli
and
Actinobacillus succinogenes
. A study of chemical fuel cells in which NADH, NR, and ferricyanide were the electron donor, the electronophore, and the electron acceptor, respectively, showed that electrical current produced from NADH was proportional to the concentration of NADH. Fourfold more current was produced from NADH in chemical fuel cells when NR was the electron mediator than when thionin was the electron mediator. In microbial fuel cells in which
E. coli
resting cells were used the amount of current produced from glucose when NR was the electron mediator (3.5 mA) was 10-fold more than the amount produced when thionin was the electron mediator (0.4 mA). The amount of electrical energy generated (expressed in joules per mole of substrate) and the amount of current produced from glucose (expressed in milliamperes) in NR-mediated microbial fuel cells containing either
E. coli
or
A. succinogenes
were about 10- and 2-fold greater, respectively, when resting cells were used than when growing cells were used. Cell growth was inhibited substantially when these microbial fuel cells were making current, and more oxidized end products were formed under these conditions. When sewage sludge (i.e., a mixed culture of anaerobic bacteria) was used in the fuel cell, stable (for 120 h) and equivalent levels of current were obtained with glucose, as observed in the pure-culture experiments. These results suggest that NR is better than other electron mediators used in microbial fuel cells and that sludge production can be decreased while electricity is produced in fuel cells. Our results are discussed in relation to factors that may improve the relatively low electrical efficiencies (1.2 kJ/mol) obtained with microbial fuel cells.
Increased Growth of the Microalga
Chlorella vulgaris
when Coimmobilized and Cocultured in Alginate Beads with the Plant-Growth-Promoting Bacterium
Azospirillum brasilense
Authors: Gonzalez Luz E. , Bashan Yoav
Published: July 2002

Coimmobilization of the freshwater microalga
Chlorella vulgaris
and the plant-growth-promoting bacterium
Azospirillum brasilense
in small alginate beads resulted in a significantly increased growth of the microalga. Dry and fresh weight, total number of cells, size of the microalgal clusters (colonies) within the bead, number of microalgal cells per cluster, and the levels of microalgal pigments significantly increased. Light microscopy revealed that both microorganisms colonized the same cavities inside the beads, though the microalgae tended to concentrate in the more aerated periphery while the bacteria colonized the entire bead. The effect of indole-3-acetic acid addition to microalgal culture prior to immobilization of microorganisms in alginate beads partially imitated the effect of
A. brasilense
. We propose that coimmobilization of microalgae and plant-growth-promoting bacteria is an effective means of increasing microalgal populations within confined environments.
Reduction of Fe(III), Cr(VI), U(VI), and Tc(VII) by
Deinococcus radiodurans
R1
Authors: Fredrickson J. K. , Kostandarithes H. M. , Li S. W. , Plymale A. E. , Daly M. J.
Published: July 2002

Deinococcus radiodurans
is an exceptionally radiation-resistant microorganism capable of surviving acute exposures to ionizing radiation doses of 15,000 Gy and previously described as having a strictly aerobic respiratory metabolism. Under strict anaerobic conditions,
D. radiodurans
R1 reduced Fe(III)-nitrilotriacetic acid coupled to the oxidation of lactate to CO
2
and acetate but was unable to link this process to growth.
D. radiodurans
reduced the humic acid analog anthraquinone-2,6-disulfonate (AQDS) to its dihydroquinone form, AH
2
DS, which subsequently transferred electrons to the Fe(III) oxides hydrous ferric oxide and goethite via a previously described electron shuttle mechanism.
D. radiodurans
reduced the solid-phase Fe(III) oxides in the presence of either 0.1 mM AQDS or leonardite humic acids (2 mg ml
−1
) but not in their absence.
D. radiodurans
also reduced U(VI) and Tc(VII) in the presence of AQDS. In contrast, Cr(VI) was directly reduced in anaerobic cultures with lactate although the rate of reduction was higher in the presence of AQDS. The results are the first evidence that
D. radiodurans
can reduce Fe(III) coupled to the oxidation of lactate or other organic compounds. Also,
D. radiodurans
, in combination with humic acids or synthetic electron shuttle agents, can reduce U and Tc and thus has potential applications for remediation of metal- and radionuclide-contaminated sites where ionizing radiation or other DNA-damaging agents may restrict the activity of more sensitive organisms.
Identification and Characterization of Three Differentially Expressed Genes, Encoding
S
-Adenosylhomocysteine Hydrolase, Methionine Aminopeptidase, and a Histone-Like Protein, in the Toxic Dinoflagellate
Alexandrium fundyense
Authors: Taroncher-Oldenburg Gaspar , Anderson Donald M.
Published: July 2002

Genes showing differential expression related to the early G
1
phase of the cell cycle during synchronized circadian growth of the toxic dinoflagellate
Alexandrium fundyense
were identified and characterized by differential display (DD). The determination in our previous work that toxin production in
Alexandrium
is relegated to a narrow time frame in early G
1
led to the hypothesis that transcriptionally up- or downregulated genes during this subphase of the cell cycle might be related to toxin biosynthesis. Three genes, encoding
S
-adenosylhomocysteine hydrolase (
Sahh
), methionine aminopeptidase (
Map
), and a histone-like protein (
HAf
), were isolated.
Sahh
was downregulated, while
Map
and
HAf
were upregulated, during the early G
1
phase of the cell cycle.
Sahh
and
Map
encoded amino acid sequences with about 90 and 70% similarity to those encoded by several eukaryotic and prokaryotic
Sahh
and
Map
genes, respectively. The partial
Map
sequence also contained three cobalt binding motifs characteristic of all
Map
genes.
HAf
encoded an amino acid sequence with 60% similarity to those of two histone-like proteins from the dinoflagellate
Crypthecodinium cohnii
Biecheler. This study documents the potential of applying DD to the identification of genes that are related to physiological processes or cell cycle events in phytoplankton under conditions where small sample volumes represent an experimental constraint. The identification of an additional 21 genes with various cell cycle-related DD patterns also provides evidence for the importance of pretranslational or transcriptional regulation in dinoflagellates, contrary to previous reports suggesting the possibility that translational mechanisms are the primary means of circadian regulation in this group of organisms.
The Macrocyclic Peptide Antibiotic Micrococcin P1Is Secreted by the Food-Borne BacteriumStaphylococcus equorumWS 2733 and InhibitsListeria monocytogeneson Soft Cheese
Authors: Carnio Markus C. , Höltzel Alexandra , Rudolf Melanie , Henle Thomas , Jung Günther , Scherer Siegfried
Published: July 2002

Staphylococcus equorumWS 2733 was found to produce a substance exhibiting a bacteriostatic effect on a variety of gram-positive bacteria. The metabolite was purified to homogeneity by ammonium sulfate precipitation and semipreparative reversed-phase high-performance liquid chromatography. Electrospray mass spectrometry confirmed the high purity of the compound and revealed a molecular mass of 1,143 Da. By two-dimensional nuclear magnetic resonance spectroscopy the substance was identified as micrococcin P1which is a macrocyclic peptide antibiotic that has not yet been reported for the genusStaphylococcus. A total of 95 out of 95Listeriastrains and 130 out of 135 other gram-positive bacteria were inhibited by this substance, while none of 37 gram-negative bacteria were affected. The antilisterial potential of this food-grade strain as a protective starter culture was evaluated by its in situ application in cheese-ripening experiments under laboratory conditions. A remarkable growth reduction ofListeria monocytogenescould be achieved compared to control cheese ripened with a nonbacteriocinogenic type strain ofStaphylococcus equorum. In order to prove that inhibition was due to micrococcin P1, a micrococcin-deficient mutant was constructed which did not inhibitL. monocytogenesin cheese-ripening experiments.
Cloning the Soil Metagenome: a Strategy for Accessing the Genetic and Functional Diversity of Uncultured Microorganisms
Authors: Rondon Michelle R. , August Paul R. , Bettermann Alan D. , Brady Sean F. , Grossman Trudy H. , Liles Mark R. , Loiacono Kara A. , Lynch Berkley A. , MacNeil Ian A. , Minor Charles , Tiong Choi Lai , Gilman Michael , Osburne Marcia S. , Clardy Jon , Handelsman Jo , Goodman Robert M.
Published: July 2002

Recent progress in molecular microbial ecology has revealed that traditional culturing methods fail to represent the scope of microbial diversity in nature, since only a small proportion of viable microorganisms in a sample are recovered by culturing techniques. To develop methods to investigate the full extent of microbial diversity, we used a bacterial artificial chromosome (BAC) vector to construct libraries of genomic DNA isolated directly from soil (termed metagenomic libraries). To date, we have constructed two such libraries, which contain more than 1 Gbp of DNA. Phylogenetic analysis of 16S rRNA gene sequences recovered from one of the libraries indicates that the BAC libraries contain DNA from a wide diversity of microbial phyla, including sequences from diverse taxa such as the low-G+C, gram-positive
Acidobacterium
,
Cytophagales
, and
Proteobacteria
. Initial screening of the libraries in
Escherichia coli
identified several clones that express heterologous genes from the inserts, confirming that the BAC vector can be used to maintain, express, and analyze environmental DNA. The phenotypes expressed by these clones include antibacterial, lipase, amylase, nuclease, and hemolytic activities. Metagenomic libraries are a powerful tool for exploring soil microbial diversity, providing access to the genetic information of uncultured soil microorganisms. Such libraries will be the basis of new initiatives to conduct genomic studies that link phylogenetic and functional information about the microbiota of environments dominated by microorganisms that are refractory to cultivation.
Isolation of Methyl Parathion-Degrading Strain M6 and Cloning of the Methyl Parathion Hydrolase Gene
Authors: Zhongli Cui , Shunpeng Li , Guoping Fu
Published: July 2002

A degradative bacterium, M6, was isolated and presumptively identified as
Plesiomonas
sp. strain M6 was able to hydrolyze methyl parathion to
p
-nitrophenol. A novel organophosphate hydrolase gene designated
mpd
was selected from its genomic library prepared by shotgun cloning. The nucleotide sequence of the
mpd
gene was determined. The gene could be effectively expressed in
Esherichia coli
.
Bacillus thuringiensis
Delta-Endotoxin Cry1 Hybrid Proteins with Increased Activity against the Colorado Potato Beetle
Authors: Naimov Samir , Weemen-Hendriks Mieke , Dukiandjiev Stefan , de Maagd Ruud A.
Published: July 2002

Cry1 delta-endotoxins of
Bacillus thuringiensis
are generally active against lepidopteran insects, but Cry1Ba and Cry1Ia have additional, though low, levels of activity against coleopterans such as the Colorado potato beetle. Here we report the construction of Cry1Ba/Cry1Ia hybrid toxins which have increased activities against this insect species.
Detection of Hepatitis A Virus by the Nucleic Acid Sequence-Based Amplification Technique and Comparison with Reverse Transcription-PCR
Authors: Jean Julie , Blais Burton , Darveau André , Fliss Ismaı̈l
Published: July 2002

A nucleic acid sequence-based amplification (NASBA) technique for the detection of hepatitis A virus (HAV) in foods was developed and compared to the traditional reverse transcription (RT)-PCR technique. Oligonucleotide primers targeting the VP1 and VP2 genes encoding the major HAV capsid proteins were used for the amplification of viral RNA in an isothermal process resulting in the accumulation of RNA amplicons. Amplicons were detected by hybridization with a digoxigenin-labeled oligonucleotide probe in a dot blot assay format. Using the NASBA, as little as 0.4 ng of target RNA/ml was detected per comparison to 4 ng/ml for RT-PCR. When crude HAV viral lysate was used, a detection limit of 2 PFU (4 × 10
2
PFU/ml) was obtained with NASBA, compared to 50 PFU (1 × 10
4
PFU/ml) obtained with RT-PCR. No interference was encountered in the amplification of HAV RNA in the presence of excess nontarget RNA or DNA. The NASBA system successfully detected HAV recovered from experimentally inoculated samples of waste water, lettuce, and blueberries. Compared to RT-PCR and other amplification techniques, the NASBA system offers several advantages in terms of sensitivity, rapidity, and simplicity. This technique should be readily adaptable for detection of other RNA viruses in both foods and clinical samples.