bluuebunny/crossref_metadata_2025 huggingface dataset:
Treatment of Visceral Leishmaniasis with Sterically Stabilized Liposomes Containing Camptothecin
Authors: Proulx Marie-Eve , Désormeaux André , Marquis Jean-François , Olivier Martin , Bergeron Michel G.
Published: July 2002

The efficacy of 20(
S
)-camptothecin (CPT), free and incorporated into sterically stabilized liposomes, has been investigated in vitro against
Leishmania donovani
promastigotes and in vivo in a murine model of visceral leishmaniasis. Incubation of
L. donovani
promastigotes with free or liposomal CPT inhibited the growth of parasites in a dose-dependent manner. Tissue distribution studies revealed that the intraperitoneal administration of liposomal CPT was efficient for the delivery of high drug levels to the liver and spleen. Treatment of infected mice with intraperitoneal injections of free and liposomal CPT significantly reduced the parasite loads in the livers by 43 and 55%, respectively, compared with the loads for untreated controls. However, both treatments caused normochromic anemia and neutropenia.
Stability and Compatibility of Ceftazidime Administered by Continuous Infusion to Intensive Care Patients
Authors: Servais Hélène , Tulkens Paul M.
Published: July 2002

The stability and compatibility of ceftazidime have been examined in the context of its potential use in concentrated solutions for continuous infusion in patients suffering from severe nosocomial pneumonia and receiving other intravenous medications by the same route. Ceftazidime stability in 4 to 12% solutions was found satisfactory (<10% degradation) for 24 h if kept at a temperature of 25°C (77°F) maximum. Studies mimicking the simultaneous administration of ceftazidime and other drugs as done in clinics showed physical incompatibilities with vancomycin, nicardipine, midazolam, and propofol and a chemical incompatibility with
N
-acetylcystein. Concentrated solutions (50 mg/ml) of erythromycin or clarithromycin caused the appearance of a precipitate, whereas gentamicin, tobramycin, amikacin, isepamicin, fluconazole, ketamine, sufentanil, valproic acid, furosemide, uradipil, and a standard amino acid solution were physically and chemically compatible.
Interaction of Common Azole Antifungals with P Glycoprotein
Authors: Wang Er-jia , Lew Karen , Casciano Christopher N. , Clement Robert P. , Johnson William W.
Published: July 2002

Both eucaryotic and procaryotic cells are resistant to a large number of antibiotics because of the activities of export transporters. The most studied transporter in the mammalian ATP-binding cassette transporter superfamily, P glycoprotein (P-gp), ejects many structurally unrelated amphiphilic and lipophilic xenobiotics. Observed clinical interactions and some in vitro studies suggest that azole antifungals may interact with P-gp. Such an interaction could both affect the disposition and exposure to azole antifungal therapeutics and partially explain the clinical drug interactions observed with some antifungals. Using a whole-cell assay in which the retention of a marker substrate is evaluated and quantified, we studied the abilities of the most widely prescribed orally administered azole antifungals to inhibit the function of this transporter. In a cell line presenting an overexpressed amount of the human P-gp transporter, itraconazole and ketoconazole inhibited P-gp function with 50% inhibitory concentrations (IC
50
s) of ∼2 and ∼6 μM, respectively. Cyclosporin A was inhibitory with an IC
50
of 1.4 μM in this system. Uniquely, fluconazole had no effect in this assay, a result consistent with known clinical interactions. The effects of these azole antifungals on ATP consumption by P-gp (representing transport activity) were also assessed, and the
K

m

values were congruent with the IC
50
s. Therefore, exposure of tissue to the azole antifungals may be modulated by human P-gp, and the clinical interactions of azole antifungals with other drugs may be due, in part, to inhibition of P-gp transport.
Very Low Cefotaxime Concentrations Select for Hypermutable
Streptococcus pneumoniae
Populations
Authors: Negri María-Cristina , Morosini María-Isabel , Baquero María-Rosario , Campo Rosa del , Blázquez Jesús , Baquero Fernando
Published: July 2002

A mixed culture of an hypermutable
hexA Streptococcus pneumoniae
mutant strain and its
hexA
+
isogenic ancestor was challenged with low cefotaxime concentrations. Despite identical original cefotaxime MICs, the
hexA
mutant population was significantly selected at very low concentrations, and all of the tested selected variants harbored the Thr550→Ala mutation in
pbp2x
. Since cefotaxime selects hypermutators, the risk of secondary acquisition of antibiotic resistance is increased; as expected, the cefotaxime-resistant mutants had a mutation frequency 10 times higher in response to to ciprofloxacin. The present study presents a model (not necessarily reflecting the clinical setting) illustrating the risk of selection of mutators in the evolution of multiple resistance.
In Vitro and In Vivo Activities of Gatifloxacin against
Mycobacterium tuberculosis
Authors: Alvirez-Freites Enrique J. , Carter Janna L. , Cynamon Michael H.
Published: July 2002

Gatifloxacin (GAT) and moxifloxacin (MXF) were evaluated in vitro to determine their activities against
Mycobacterium tuberculosis
. GAT was subsequently compared in a dose range study to isoniazid (INH) in a murine tuberculosis model. GAT was somewhat less active than INH. GAT and MXF were evaluated in mice infected with
M. tuberculosis
and were found to have similar activities. GAT was studied alone and in combination with ethambutol, ethionamide (ETA), and pyrazinamide (PZA) and compared to INH and rifampin (RIF). GAT appears to have sufficient activity alone and in combination with ETA with or without PZA to merit evaluation for treatment of tuberculosis.
Malaria Parasites Giving Rise to Recrudescence In Vitro
Authors: Nakazawa Shusuke , Maoka Takashi , Uemura Haruki , Ito Yoshihiro , Kanbara Hiroji
Published: July 2002

Recrudescences were simulated in vitro with drug treatment to examine how drug-sensitive parasites survive the treatment. Various numbers of cultured parasites were treated with lethal doses of pyrimethamine or mefloquine for various lengths of time. Recrudescences were observed in parasite populations with larger initial numbers of parasites when the treatment duration was prolonged. Equal numbers of parasitized erythrocytes were treated with various concentrations of pyrimethamine or mefloquine. There was no clear linear relationship between the incidence of recrudescence and the drug concentration. Parasites that had recrudesced were continuously allowed to recrudesce in the succeeding recrudescence experiments. Both the duration from the cessation of treatment to the time at which the recrudescent parasitemia level reached 1% and the growth rate of recrudescent parasites were equal among these recrudescences. The recrudescent parasites in these experiments were as sensitive to the drugs as the parasites tested before treatment were. These results suggest that a parasite culture may contain parasites in some phases that are not killed by drug for up to 10 days, which explains the recrudescences that occur even after treatment.
Double-Blind, Randomized, Placebo-Controlled Study of Topical 5% Acyclovir-1% Hydrocortisone Cream (ME-609) for Treatment of UV Radiation-Induced Herpes Labialis
Authors: Evans T. G. , Bernstein D. I. , Raborn G. W. , Harmenberg J. , Kowalski J. , Spruance S. L.
Published: July 2002

Immunopathology is recognized as an important component of infectious disease manifestations, and corticosteroids have been used as an adjunct to antimicrobial therapy for a variety of conditions. Antiviral therapy of herpes labialis has been shown to result in only a small reduction in the time to healing and the duration of pain. To determine if topical application of a combination product containing 5% acyclovir and 1% hydrocortisone (ME-609) could provide benefit to herpes labialis patients, 380 immunocompetent adults with a history of herpes labialis were exposed to experimental UV radiation (UVR) to induce a recurrence. On day 2, just before the appearance of the majority of lesions (“delayed” lesions), subjects were randomized to receive active medication or vehicle control six times per day for 5 days. Overall, 120 of 380 patients developed delayed classical lesions, of whom 50 of 190 (26%) had been treated with ME-609 and 70 of 190 (37%) had received placebo (a reduction of 29% [
P
= 0.02]). Healing time, measured as the time to normal skin, was reduced by treatment with ME-609 (9.0 days for treated patients versus 10.1 days for the controls [
P
= 0.04]). There was a trend toward a reduction in the maximum lesion size in the ME-609 recipients compared to that in the controls (43 versus 60 mm
2
, respectively [
P
= 0.07]). The treatment had no effect on lesion pain, but ME-609 treatment reduced the number of patients with moderate or severe tenderness. Compared to treatment with a placebo, treatment with the combination antiviral-immunomodulatory cream provided benefit to patients with experimental UVR-induced herpes labialis, reducing classical lesion incidence, healing time, lesion size, and lesion tenderness. ME-609 is a novel product that merits further evaluation as a treatment for cold sores.
Cross-Resistance of Pink Bollworm (
Pectinophora gossypiella
) to
Bacillus thuringiensis
Toxins
Authors: Tabashnik Bruce E. , Liu Yong-Biao , de Maagd Ruud A. , Dennehy Timothy J.
Published: July 2002

Two strains of pink bollworm (
Pectinophora gossypiella
) selected in the laboratory for resistance to
Bacillus thuringiensis
toxin Cry1Ac had substantial cross-resistance to Cry1Aa and Cry1Ab but not to Cry1Bb, Cry1Ca, Cry1Da, Cry1Ea, Cry1Ja, Cry2Aa, Cry9Ca, H04, or H205. The narrow spectrum of resistance and the cross-resistance to activated toxin Cry1Ab suggest that reduced binding of toxin to midgut target sites could be an important mechanism of resistance.
Characterization of pRGO1, a Plasmid from
Propionibacterium acidipropionici
, and Its Use for Development of a Host-Vector System in Propionibacteria
Authors: Kiatpapan Pornpimon , Hashimoto Yoshiteru , Nakamura Hisako , Piao Yong-Zhe , Ono Hisayo , Yamashita Mitsuo , Murooka Yoshikatsu
Published: July 2002

The complete nucleotide sequence of pRGO1, a cryptic plasmid from
Propionibacterium acidipropionici
E214, was determined. pRGO1 is 6,868 bp long, and its G+C content is 65.0%. Frame analysis of the sequence revealed six open reading frames, which were designated Orf1 to Orf6. The deduced amino acid sequences of Orf1 and Orf2 showed extensive similarities to an initiator of plasmid replication, the Rep protein, of various plasmids of gram-positive bacteria. The amino acid sequence of the putative translation product of
orf3
exhibited a high degree of similarity to the amino acid sequences of DNA invertase in several bacteria. For the putative translation products of
orf4
,
orf5
, and
orf6
, on the other hand, no homologous sequences were found. The function of these open reading frames was studied by deletion analysis. A shuttle vector, pPK705, was constructed for shuttling between
Escherichia coli
and a
Propionibacterium
strain containing
orf1
(
repA
),
orf2
(
repB
),
orf5
, and
orf6
from pRGO1, pUC18, and the hygromycin B-resistant gene as a drug marker. Shuttle vector pPK705 successfully transformed
Propionibacterium freudenreichii
subsp.
shermanii
IFO12426 by electroporation at an efficiency of 8 × 10
6
CFU/μg of DNA under optimized conditions. Transformation of various species of propionibacteria with pPK705 was also performed at efficiencies of about 10
4
to 10
7
CFU/μg of DNA. The vector was stably maintained in strains of
P. freudenreichii
subsp.
shermanii
,
P. freudenreichii
,
P. pentosaceum
, and
P. freudenreichii
subsp.
freudenreichii
grown under nonselective conditions. Successful manipulation of a host-vector system in propionibacteria should facilitate genetic studies and lead to creation of genes that are useful industrially.
Denaturing Gradient Gel Electrophoresis Analysis of 16S Ribosomal DNA Amplicons To Monitor Changes in Fecal Bacterial Populations of Weaning Pigs after Introduction of
Lactobacillus reuteri
Strain MM53
Authors: Simpson Joyce M. , McCracken Vance J. , Gaskins H. Rex , Mackie Roderick I.
Published: July 2002

The diversity and stability of the fecal bacterial microbiota in weaning pigs was studied after introduction of an exogenous
Lactobacillus reuteri
strain, MM53, using a combination of cultivation and techniques based on genes encoding 16S rRNA (16S rDNA). Piglets (
n
= 9) were assigned to three treatment groups (control, daily dosed, and 4th-day dosed), and fresh fecal samples were collected daily. Dosed animals received 2.5 × 10
10
CFU of antibiotic-resistant
L. reuteri
MM53 daily or every 4th day. Mean
Lactobacillus
counts for the three groups ranged from 1 × 10
9
to 4 × 10
9
CFU/g of feces. Enumeration of strain
L. reuteri
MM53 on MRS agar (Difco) plates containing streptomycin and rifampin showed that the introduced strain fluctuated between 8 × 10
3
and 5 × 10
6
CFU/g of feces in the two dosed groups. Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA fragments, with primers specific for variable regions 1 and 3 (V1 and V3), was used to profile complexity of fecal bacterial populations. Analysis of DGGE banding profiles indicated that each individual maintained a unique fecal bacterial population that was stable over time, suggesting a strong host influence. In addition, individual DGGE patterns could be separated into distinct time-dependent clusters. Primers designed specifically to restrict DGGE analysis to a select group of lactobacilli allowed examination of interspecies relationships and abundance. Based on relative band migration distance and sequence determination,
L. reuteri
was distinguishable within the V1 region 16S rDNA gene patterns. Daily fluctuations in specific bands within these profiles were observed, which revealed an antagonistic relationship between
L. reuteri
MM53 (band V1-3) and another indigenous
Lactobacillus
assemblage (band V1-6).
Habituation of
Salmonella
spp. at Reduced Water Activity and Its Effect on Heat Tolerance
Authors: Mattick K. L. , Jørgensen F. , Legan J. D. , Lappin-Scott H. M. , Humphrey T. J.
Published: July 2002

The effect of habituation at reduced water activity (a
w
) on heat tolerance of
Salmonella
spp. was investigated. Stationary-phase cells were exposed to a
w
0.95 in broths containing glucose-fructose, sodium chloride, or glycerol at 21°C for up to a week prior to heat challenge at 54°C. In addition, the effects of different a
w
s and heat challenge temperatures were investigated. Habituation at a
w
0.95 resulted in increased heat tolerance at 54°C with all solutes tested. The extent of the increase and the optimal habituation time depended on the solute used. Exposure to broths containing glucose-fructose (a
w
0.95) for 12 h resulted in maximal heat tolerance, with more than a fourfold increase in
D
54
values. Cells held for more than 72 h in these conditions, however, became as heat sensitive as nonhabituated populations. Habituation in the presence of sodium chloride or glycerol gave rise to less pronounced but still significant increases in heat tolerance at 54°C, and a shorter incubation time was required to maximize tolerance. The increase in heat tolerance following habituation in broths containing glucose-fructose (a
w
0.95) was RpoS independent. The presence of chloramphenicol or rifampin during habituation and inactivation did not affect the extent of heat tolerance achieved, suggesting that de novo protein synthesis was probably not necessary. These data highlight the importance of cell prehistory prior to heat inactivation and may have implications for food manufacturers using low-a
w
ingredients.
Biogeography and Degree of Endemicity of Fluorescent
Pseudomonas
Strains in Soil
Authors: Cho Jae-Chang , Tiedje James M.
Published: July 2002

Fluorescent
Pseudomonas
strains were isolated from 38 undisturbed pristine soil samples from 10 sites on four continents. A total of 248 isolates were confirmed as
Pseudomonas
sensu stricto by fluorescent pigment production and group-specific 16S ribosomal DNA (rDNA) primers. These isolates were analyzed by three molecular typing methods with different levels of resolution: 16S rDNA restriction analysis (ARDRA), 16S-23S rDNA intergenic spacer-restriction fragment length polymorphism (ITS-RFLP) analysis, and repetitive extragenic palindromic PCR genomic fingerprinting with a BOX primer set (BOX-PCR). All isolates showed very similar ARDRA patterns, as expected. Some ITS-RFLP types were also found at every geographic scale, although some ITS-RFLP types were unique to the site of origin, indicating weak endemicity at this level of resolution. Using a similarity value of 0.8 or more after cluster analysis of BOX-PCR fingerprinting patterns to define the same genotypes, we identified 85 unique fluorescent
Pseudomonas
genotypes in our collection. There were no overlapping genotypes between sites as well as continental regions, indicating strict site endemism. The genetic distance between isolates as determined by degree of dissimilarity in BOX-PCR patterns was meaningfully correlated to the geographic distance between the isolates' sites of origin. Also, a significant positive spatial autocorrelation of the distribution of the genotypes was observed among distances of <197 km, and significant negative autocorrelation was observed between regions. Hence, strong endemicity of fluorescent
Pseudomonas
genotypes was observed, suggesting that these heterotrophic soil bacteria are not globally mixed.
Development of a Highly Sensitive Nested-PCR Procedure Using a Single Closed Tube for Detection of
Erwinia amylovora
in Asymptomatic Plant Material
Authors: Llop Pablo , Bonaterra Anna , Peñalver Javier , López María M.
Published: July 2002

A novel method, which involves a nested PCR in a single closed tube, was developed for the sensitive detection of
Erwinia amylovora
in plant material. The external and internal primer pairs used had different annealing temperatures and directed the amplification of a specific DNA fragment from plasmid pEA29. The procedure involved two consecutive PCRs, the first of which was performed at a higher annealing temperature that allowed amplification only by the external primer pair. Using pure cultures of
E. amylovora
, the sensitivity of the nested PCR in one tube was similar to that of a standard nested PCR in two tubes. The specificity and sensitivity were greater than those of standard PCR procedures that used a single primer pair. The presence of inhibitors in plant material, very common in
E. amylovora
hosts, is overcome with this system in combination with a simple DNA extraction protocol because it eliminates many of the inhibitory compounds. In addition, it needs a very small sample volume (1 μl of DNA extracted). With 83 samples of naturally infected material, this method achieved better results than any other PCR technique: standard PCR detected 55% of positive samples, two-tube nested PCR detected 71% of positive samples, and nested PCR in a single closed tube detected 78% of positive samples. When analyzing asymptomatic plant material, the number of positive samples detected by the developed nested PCR was also the highest, compared with the PCR protocols indicated previously (17, 20, and 25% of 251 samples analyzed, respectively). This method is proposed for the detection of endophytic and epiphytic populations of
E. amylovora
in epidemiological studies and for routine use in quarantine surveys, due to its high sensitivity, specificity, speed, and simplicity.
Quantification of Bacterial Groups within Human Fecal Flora by Oligonucleotide Probe Hybridization
Authors: Sghir Abdelghani , Gramet Genevieve , Suau Antonia , Rochet Violaine , Pochart Philippe , Dore Joel
Published: July 2002

To investigate the population structure of the predominant phylogenetic groups within the human adult fecal microbiota, a new oligonucleotide probe designated S-G-Clept-1240-a-A-18 was designed, validated, and used with a set of five 16S rRNA-targeted oligonucleotide probes. Application of the six probes to fecal samples from 27 human adults showed additivity of 70% of the total 16S rRNA detected by the bacterial domain probe. The
Bacteroides
group-specific probe accounted for 37% ± 16% of the total rRNA, while the enteric group probe accounted for less than 1%.
Clostridium leptum
subgroup and
Clostridium coccoides
group-specific probes accounted for 16% ± 7% and 14% ± 6%, respectively, while
Bifidobacterium
and
Lactobacillus
groups made up less than 2%.
Osmoprotection by Pipecolic Acid in
Sinorhizobium meliloti
: Specific Effects of
d
and
l
Isomers
Authors: Gouffi Kamila , Bernard Théophile , Blanco Carlos
Published: July 2002

dl
-Pipecolic acid (
dl
-PIP) promotes growth restoration of
Sinorhizobium meliloti
cells facing inhibitory hyperosmolarity. Surprisingly,
d
and
l
isomers of this imino acid supplied separately were not effective. The uptake of
l
-PIP was significantly favored in the presence of the
d
isomer and by a hyperosmotic stress. Chromatographic analysis of the intracellular solutes showed that stressed cells did not accumulate radiolabeled
l
-PIP. Rather, it participates in the synthesis of the main endogenous osmolytes (glutamate and the dipeptide
N
-acetylglutaminylglutamine amide) during the lag phase, thus providing a means for the stressed cells to recover the osmotic balance.
13
C nuclear magnetic resonance analysis was used to determine the fate of
d
-PIP taken into the cells. In the absence of
l
-PIP, the imported
d
isomer was readily degraded. Supplied together with its
l
isomer,
d
-PIP was accumulated temporarily and thus might contribute together with the endogenous osmolytes to enhance the internal osmotic strength. Furthermore, it started to disappear from the cytosol when the
l
isomer was no longer available in the culture medium (during the late exponential phase of growth). Together, these results show an uncommon mechanism of protection of osmotically stressed cells of
S. meliloti
. It was proved, for the first time, that the presence of the two isomers of the same molecule is necessary for it to manifest an osmoprotective activity. Indeed,
d
-PIP seems to play a major role in cellular osmoadaptation through both its own accumulation and improvement of the utilization of the
l
isomer as an immediate precursor of endogenous osmolytes.
Analysis of the Fecal Microflora of Human Subjects Consuming a Probiotic Product Containing
Lactobacillus rhamnosus
DR20
Authors: Tannock G. W. , Munro K. , Harmsen H. J. M. , Welling G. W. , Smart J. , Gopal P. K.
Published: July 2002

The composition of the fecal microflora of 10 healthy subjects was monitored before (6-month control period), during (6-month test period), and after (3-month posttest period) the administration of a milk product containing
Lactobacillus rhamnosus
DR20 (daily dose, 1.6 × 10
9
lactobacilli). Monthly fecal samples were examined by a variety of methods, including bacteriological culture analysis, fluorescent in situ hybridization with group-specific DNA probes, denaturing gradient gel electrophoresis of the V2-V3 region of 16S rRNA genes amplified by PCR, gas-liquid chromatography, and bacterial enzyme activity analysis. The composition of the
Lactobacillus
population of each subject was analyzed by pulsed-field gel electrophoresis of bacterial DNA digests in order to differentiate between DR20 and other strains present in the samples. Representative isolates of lactobacilli were identified to the species level by sequencing the V2-V3 region of their 16S rRNA genes and comparing the sequences obtained (BLAST search) to sequences in the GenBank database. DR20 was detected in the feces of all of the subjects during the test period, but at different frequencies. The presence of DR20 among the numerically predominant strains was related to the presence or absence of a stable indigenous population of lactobacilli during the control period. Strain DR20 did not persist at levels of >10
2
cells per g in the feces of most of the subjects after consumption of the product ceased; the only exception was one subject in which this strain was detected for 2 months during the posttest period. We concluded that consumption of the DR20-containing milk product transiently altered the
Lactobacillus
and enterococcal contents of the feces of the majority of consumers without markedly affecting biochemical or other bacteriological factors.
Analysis of Ammonia-Oxidizing Bacteria from Hypersaline Mono Lake, California, on the Basis of 16S rRNA Sequences
Authors: Ward B. B. , Martino D. P. , Diaz M. C. , Joye S. B.
Published: July 2002

Ammonia-oxidizing bacteria were detected by PCR amplification of DNA extracted from filtered water samples throughout the water column of Mono Lake, California. Ammonia-oxidizing members of the β subdivision of the division
Proteobacteria
(β-subdivision
Proteobacteria
) were detected using previously characterized PCR primers; target sequences were detected by direct amplification in both surface water and below the chemocline. Denaturing gradient gel electrophoresis analysis indicated the presence of at least four different β-subdivision ammonia oxidizers in some samples. Subsequent sequencing of amplified 16S rDNA fragments verified the presence of sequences very similar to those of cultured
Nitrosomonas
strains. Two separate analyses, carried out under different conditions (different reagents, locations, PCR machines, sequencers, etc.), 2 years apart, detected similar ranges of sequence diversity in these samples. It seems likely that the physiological diversity of nitrifiers exceeds the diversity of their ribosomal sequences and that these sequences represent members of the
Nitrosomonas europaea
group that are acclimated to alkaline, high-salinity environments. Primers specific for
Nitrosococcus oceanus
, a marine ammonia-oxidizing bacterium in the γ subdivision of the
Proteobacteria
, did not amplify target from any samples.
Changes in Bacterial Community Structure in the Colon of Pigs Fed Different Experimental Diets and after Infection with
Brachyspira hyodysenteriae
Authors: Leser Thomas D. , Lindecrona Rikke Hvid , Jensen Tim K. , Jensen Bent B. , Møller Kristian
Published: July 2002

Bacterial communities in the large intestines of pigs were compared using terminal restriction fragment length polymorphism (T-RFLP) analysis targeting the 16S ribosomal DNA. The pigs were fed different experimental diets based on either modified standard feed or cooked rice supplemented with dietary fibers. After feeding of the animals with the experimental diets for 2 weeks, differences in the bacterial community structure in the spiral colon were detected in the form of different profiles of terminal restriction fragments (T-RFs). Some of the T-RFs were universally distributed, i.e., they were found in all samples, while others varied in distribution and were related to specific diets. The reproducibility of the T-RFLP profiles between individual animals within the diet groups was high. In the control group, the profiles remained unchanged throughout the experiment and were similar between two independent but identical experiments. When the animals were experimentally infected with
Brachyspira hyodysenteriae
, causing swine dysentery, many of the T-RFs fluctuated, suggesting a destabilization of the microbial community.
Direct and Fe(II)-Mediated Reduction of Technetium by Fe(III)-Reducing Bacteria
Authors: Lloyd J. R. , Sole V. A. , Van Praagh C. V. G. , Lovley D. R.
Published: July 2002

The dissimilatory Fe(III)-reducing bacterium
Geobacter sulfurreducens
reduced and precipitated Tc(VII) by two mechanisms. Washed cell suspensions coupled the oxidation of hydrogen to enzymatic reduction of Tc(VII) to Tc(IV), leading to the precipitation of TcO
2
at the periphery of the cell. An indirect, Fe(II)-mediated mechanism was also identified. Acetate, although not utilized efficiently as an electron donor for direct cell-mediated reduction of technetium, supported the reduction of Fe(III), and the Fe(II) formed was able to transfer electrons abiotically to Tc(VII). Tc(VII) reduction was comparatively inefficient via this indirect mechanism when soluble Fe(III) citrate was supplied to the cultures but was enhanced in the presence of solid Fe(III) oxide. The rate of Tc(VII) reduction was optimal, however, when Fe(III) oxide reduction was stimulated by the addition of the humic analog and electron shuttle anthaquinone-2,6-disulfonate, leading to the rapid formation of the Fe(II)-bearing mineral magnetite. Under these conditions, Tc(VII) was reduced and precipitated abiotically on the nanocrystals of biogenic magnetite as TcO
2
and was removed from solution to concentrations below the limit of detection by scintillation counting. Cultures of Fe(III)-reducing bacteria enriched from radionuclide-contaminated sediment using Fe(III) oxide as an electron acceptor in the presence of 25 μM Tc(VII) contained a single
Geobacter
sp. detected by 16S ribosomal DNA analysis and were also able to reduce and precipitate the radionuclide via biogenic magnetite. Fe(III) reduction was stimulated in aquifer material, resulting in the formation of Fe(II)-containing minerals that were able to reduce and precipitate Tc(VII). These results suggest that Fe(III)-reducing bacteria may play an important role in immobilizing technetium in sediments via direct and indirect mechanisms.
Phylogeny of Microorganisms Populating a Thick, Subaerial, Predominantly Lithotrophic Biofilm at an Extreme Acid Mine Drainage Site
Authors: Bond Philip L. , Smriga Steven P. , Banfield Jillian F.
Published: July 2002

An unusually thick (∼1 cm) slime developed on a slump of finely disseminated pyrite ore within an extreme acid mine drainage site at Iron Mountain, near Redding, Calif. The slime was studied over the period of 1 year. The subaerial form of the slime distinguished it from more typical submerged streamers. Phylogenetic analysis of 16S rRNA genes revealed a diversity of sequences that were mostly novel. Nearest relatives to the majority of sequences came from iron-oxidizing acidophiles, and it appears that iron oxidation is the predominant metabolic characteristic of the organisms in the slime. The most abundant of the 16S rRNA genes detected were from organisms related to
Leptospirillum
species. The dominant sequence (71% of clones) may represent a new genus. Sequences within the
Archaea
of the
Thermoplasmales
lineage were detected. Most of these were only distantly related to known microorganisms. Also, sequences affiliating with
Acidimicrobium
were detected. Some of these were closely related to “
Ferromicrobium acidophilus
,” and others were affiliated with a lineage only represented by environmental clones. Unexpectedly, sequences that affiliated within the delta subdivision of the
Proteobacteria
were detected. The predominant metabolic feature of bacteria of this subdivision is anaerobic sulfate or metal reduction. Thus, microenvironments of low redox potential possibly exist in the predominantly oxidizing environments of the slime. These results expand our knowledge of the biodiversity of acid mine drainage environments and extend our understanding of the ecology of extremely acidic systems.
Flexible Community Structure Correlates with Stable Community Function in Methanogenic Bioreactor Communities Perturbed by Glucose
Authors: Fernandez Ana S. , Hashsham Syed A. , Dollhopf Sherry L. , Raskin Lutgarde , Glagoleva Olga , Dazzo Frank B. , Hickey Robert F. , Criddle Craig S. , Tiedje James M.
Published: July 2002

Methanogenic bioreactor communities were used as model ecosystems to evaluate the relationship between functional stability and community structure. Replicated methanogenic bioreactor communities with two different community structures were established. The effect of a substrate loading shock on population dynamics in each microbial community was examined by using morphological analysis, small-subunit (SSU) rRNA oligonucleotide probes, amplified ribosomal DNA (rDNA) restriction analysis (ARDRA), and partial sequencing of SSU rDNA clones. One set of replicated communities, designated the high-spirochete (HS) set, was characterized by good replicability, a high proportion of spiral and short thin rod morphotypes, a dominance of spirochete-related SSU rDNA genes, and a high percentage of
Methanosarcina
-related SSU rRNA. The second set of communities, designated the low-spirochete (LS) set, was characterized by incomplete replicability, higher morphotype diversity dominated by cocci, a predominance of
Streptococcus
-related and deeply branching
Spirochaetales
-related SSU rDNA genes, and a high percentage of
Methanosaeta
-related SSU rRNA. In the HS communities, glucose perturbation caused a dramatic shift in the relative abundance of fermentative bacteria, with temporary displacement of spirochete-related ribotypes by
Eubacterium
-related ribotypes, followed by a return to the preperturbation community structure. The LS communities were less perturbed, with
Streptococcus
-related organisms remaining prevalent after the glucose shock, although changes in the relative abundance of minor members were detected by morphotype analysis. A companion paper demonstrates that the more stable LS communities were less functionally stable than the HS communities (S. A. Hashsham, A. S. Fernandez, S. L. Dollhopf, F. B. Dazzo, R. F. Hickey, J. M. Tiedje, and C. S. Criddle, Appl. Environ. Microbiol. 66:4050–4057, 2000).
Rapid, Quantitative PCR Monitoring of Growth of
Clostridium botulinum
Type E in Modified-Atmosphere-Packaged Fish
Authors: Kimura B. , Kawasaki S. , Nakano H. , Fujii T.
Published: July 2002

A rapid, quantitative PCR assay (TaqMan assay) which quantifies
Clostridium botulinum
type E by amplifying a 280-bp sequence from the botulinum neurotoxin type E (BoNT/E) gene is described. With this method, which uses the hydrolysis of an internal fluoregenic probe and monitors in real time the increase in the intensity of fluorescence during PCR by using the ABI Prism 7700 sequence detection system, it was possible to perform accurate and reproducible quantification of the
C. botulinum
type E toxin gene. The sensitivity and specificity of the assay were verified by using 6 strains of
C. botulinum
type E and 18 genera of 42 non-
C. botulinum
type E strains, including strains of
C. botulinum
types A, B, C, D, F, and G. In both pure cultures and modified-atmosphere-packaged fish samples (jack mackerel), the increase in amounts of
C. botulinum
DNA could be monitored (the quantifiable range was 10
2
to 10
8
CFU/ml or g) much earlier than toxin could be detected by mouse assay. The method was applied to a variety of seafood samples with a DNA extraction protocol using guanidine isothiocyanate. Overall, an efficient recovery of
C. botulinum
cells was obtained from all of the samples tested. These results suggested that quantification of BoNT/E DNA by the rapid, quantitative PCR method was a good method for the sensitive assessment of botulinal risk in the seafood samples tested.
Development and Characterization of a Xylose-Dependent System for Expression of Cloned Genes in
Bacillus subtilis
: Conditional Complementation of a Teichoic Acid Mutant
Authors: Bhavsar Amit P. , Zhao Xumei , Brown Eric D.
Published: July 2002

We have developed a xylose-dependent expression system for tight and modulated expression of cloned genes in
Bacillus subtilis
. The expression system is contained on plasmid pSWEET for integration at the
amyE
locus of
B. subtilis
and incorporates components of the well-characterized, divergently transcribed xylose utilization operon. The system contains the xylose repressor encoded by
xylR
, the promoter and 5′ portion of
xylA
containing an optimized catabolite-responsive element, and intergenic
xyl
operator sequences. We have rigorously compared this expression system to the isopropyl-β-
d
-thiogalactopyranoside-induced
spac
system using a thermostable β-galactosidase reporter (BgaB) and found the
xyl
promoter-operator to have a greater capacity for modulated expression, a higher induction/repression ratio (279-fold for the
xyl
system versus 24-fold with the
spac
promoter), and lower levels of expression in the absence of an inducer. We have used this system to probe an essential function in wall teichoic acid biosynthesis in
B. subtilis
. Expression of the teichoic acid biosynthesis gene
tagD
, encoding glycerol-3-phosphate cytidylyltransferase, from the xylose-based expression system integrated at
amyE
exhibited xylose-dependent complementation of the temperature-sensitive mutant
tag-12
when grown at the nonpermissive temperature. Plasmid pSWEET thus provides a robust new expression system for conditional complementation in
B. subtilis
.
Aspects of Bioavailability of Mercury for Methylation in Pure Cultures of
Desulfobulbus propionicus
(1pr3)
Authors: Benoit J. M. , Gilmour C. C. , Mason R. P.
Published: July 2002

We have previously hypothesized that sulfide inhibits Hg methylation by decreasing its bioavailability to sulfate-reducing bacteria (SRB), the important methylators of Hg in natural sediments. With a view to designing a bioassay to test this hypothesis, we investigated a number of aspects of Hg methylation by the SRB
Desulfobulbus propionicus
, including (i) the relationship between cell density and methylmercury (MeHg) production, (ii) the time course of Hg methylation relative to growth stage, (iii) changes in the bioavailability of an added inorganic Hg (Hg
I
) spike over time, and (iv) the dependence of methylation on the concentration of dissolved Hg
I
present in the culture. We then tested the effect of sulfide on MeHg production by this microorganism. These experiments demonstrated that under conditions of equal bioavailability, per-cell MeHg production was constant through log-phase culture growth. However, the methylation rate of a new Hg spike dramatically decreased after the first 5 h. This result was seen whether methylation rate was expressed as a fraction of the total added Hg or the filtered Hg
I
concentration, which suggests that Hg bioavailability decreased through both changes in Hg complexation and formation of solid phases. At low sulfide concentration, MeHg production was linearly related to the concentration of filtered Hg
I
. The methylation of filtered Hg
I
decreased about fourfold as sulfide concentration was increased from 10
−6
to 10
−3
M. This decline is consistent with a decrease in the bioavailability of Hg
I
, possibly due to a decline in the dissolved neutral complex, HgS
0
.
Changes in Populations of Rhizosphere Bacteria Associated with Take-All Disease of Wheat
Authors: McSpadden Gardener Brian B. , Weller David M.
Published: July 2002

Take-all, caused by
Gaeumannomyces
graminis
var.
tritici
, is one of the most important fungal diseases of wheat worldwide. Knowing that microbe-based suppression of the disease occurs in monoculture wheat fields following severe outbreaks of take-all, we analyzed the changes in rhizosphere bacterial communities following infection by the take-all pathogen. Several bacterial populations were more abundant on diseased plants than on healthy plants, as indicated by higher counts on a
Pseudomonas
-selective medium and a higher fluorescence signal in terminal restriction fragment length polymorphism analyses of amplified 16S ribosomal DNA (rDNA). Amplified rDNA restriction analysis (ARDRA) of the most abundant cultured populations showed a shift in dominance from
Pseudomonas
to
Chryseobacterium
species in the rhizosphere of diseased plants. Fluorescence-tagged ARDRA of uncultured rhizosphere washes revealed an increase in ribotypes corresponding to several bacterial genera, including those subsequently identified by partial 16S sequencing as belonging to species of alpha-, beta-, and gamma-proteobacteria, sphingobacteria, and flavobacteria. The functional significance of some of these populations was investigated in vitro. Of those isolated, only a small subset of the most abundant
Pseudomonas
spp. and a
phlD

+

Pseudomonas
sp. showed any significant ability to inhibit
G.
graminis
var.
tritici
directly. When cultured strains were mixed with the inhibitory
phlD

+

Pseudomonas
strain, the
Chryseobacterium
isolates showed the least capacity to inhibit this antagonist of the pathogen, indicating that increases in
Chryseobacterium
populations may facilitate the suppression of take-all by 2,4-diacetylphloroglucinol-producing
phlD

+

pseudomonads.
Family- and Genus-Level 16S rRNA-Targeted Oligonucleotide Probes for Ecological Studies of Methanotrophic Bacteria
Authors: Gulledge Jay , Ahmad Azeem , Steudler Paul A. , Pomerantz William J. , Cavanaugh Colleen M.
Published: July 2002

Methanotrophic bacteria play a major role in the global carbon cycle, degrade xenobiotic pollutants, and have the potential for a variety of biotechnological applications. To facilitate ecological studies of these important organisms, we developed a suite of oligonucleotide probes for quantitative analysis of methanotroph-specific 16S rRNA from environmental samples. Two probes target methanotrophs in the family
Methylocystaceae
(type II methanotrophs) as a group. No oligonucleotide signatures that distinguish between the two genera in this family,
Methylocystis
and
Methylosinus
, were identified. Two other probes target, as a single group, a majority of the known methanotrophs belonging to the family
Methylococcaceae
(type I/X methanotrophs). The remaining probes target members of individual genera of the
Methylococcaceae
, including
Methylobacter
,
Methylomonas
,
Methylomicrobium
,
Methylococcus
, and
Methylocaldum
. One of the family-level probes also covers all methanotrophic endosymbionts of marine mollusks for which 16S rRNA sequences have been published. The two known species of the newly described genus
Methylosarcina
gen. nov. are covered by a probe that otherwise targets only members of the closely related genus
Methylomicrobium
. None of the probes covers strains of the newly proposed genera
Methylocella
and “
Methylothermus
,” which are polyphyletic with respect to the recognized methanotrophic families. Empirically determined midpoint dissociation temperatures were 49 to 57°C for all probes. In dot blot screening against RNA from positive- and negative-control strains, the probes were specific to their intended targets. The broad coverage and high degree of specificity of this new suite of probes will provide more detailed, quantitative information about the community structure of methanotrophs in environmental samples than was previously available.
Fluorescent Amplified Fragment Length Polymorphism Analysis of Norwegian
Bacillus cereus
and
Bacillus thuringiensis
Soil Isolates
Authors: Ticknor Lawrence O. , Kolstø Anne-Brit , Hill Karen K. , Keim Paul , Laker Miriam T. , Tonks Melinda , Jackson Paul J.
Published: July 2002

We examined 154 Norwegian
B. cereus
and
B. thuringiensis
soil isolates (collected from five different locations), 8
B. cereus
and 2
B. thuringiensis
reference strains, and 2
Bacillus anthracis
strains by using fluorescent amplified fragment length polymorphism (AFLP). We employed a novel fragment identification approach based on a hierarchical agglomerative clustering routine that identifies fragments in an automated fashion. No method is free of error, and we identified the major sources so that experiments can be designed to minimize its effect. Phylogenetic analysis of the fluorescent AFLP results reveals five genetic groups in these group 1 bacilli. The ATCC reference strains were restricted to two of the genetic groups, clearly not representative of the diversity in these bacteria. Both
B. anthracis
strains analyzed were closely related and affiliated with a
B. cereus
milk isolate (ATCC 4342) and a
B. cereus
human pathogenic strain (periodontitis). Across the entire study, pathogenic strains, including
B. anthracis
, were more closely related to one another than to the environmental isolates. Eight strains representing the five distinct phylogenetic clusters were further analyzed by comparison of their 16S rRNA gene sequences to confirm the phylogenetic status of these groups. This analysis was consistent with the AFLP analysis, although of much lower resolution. The innovation of automated genotype analysis by using a replicated and statistical approach to fragment identification will allow very large sample analyses in the future.
Assessing the Diversity of Marine Bacterial β-Glucosidases by Capillary Electrophoresis Zymography
Authors: Arrieta Jesús M. , Herndl Gerhard J.
Published: July 2002

We propose a new method for the fast separation and detection of β-glucosidases in environmental samples. With this approach, β-glucosidases extracted from bacteria are evidenced by substrate-incorporated capillary electrophoresis (CE zymography) and their kinetic parameters can be determined by repeated injections using different substrate concentrations. Preliminary results obtained with natural bacterial communities from the coastal North Sea suggest that the diversity of β-glucosidases in the marine environment might be much higher than previously observed.
Molecular Characterization of a New Abortive Infection System (AbiU) from
Lactococcus lactis
LL51-1
Authors: Dai Gang , Su Ping , Allison Gwen E. , Geller Bruce L. , Zhu Ping , Kim Woojin S. , Dunn Noel W.
Published: July 2002

This study reports on the identification and characterization of a novel abortive infection system, AbiU, from
Lactococcus lactis.
AbiU confers resistance to phages from the three main industrially relevant lactococcal phage species: c2, 936, and P335. The presence of AbiU reduced the efficiency of plaquing against specific phage from each species as follows: 3.7 × 10
−1
, 1.0 × 10
−2
, and 1.0 × 10
−1
, respectively.
abiU
involves two open reading frames,
abiU1
(1,772 bp) and
abiU2
(1,019 bp). Evidence indicates that AbiU1 is responsible for phage resistance and that AbiU2 may downregulate phage resistance against 936 and P335 type phages but not c2 type phage. AbiU appeared to delay transcription of both phage 712 and c2, with the effect being more marked on phage c2.
Engineering Cytochrome P450 BM-3 for Oxidation of Polycyclic Aromatic Hydrocarbons
Authors: Li Qing-Shan , Ogawa Jun , Schmid Rolf D. , Shimizu Sakayu
Published: July 2002

Cytochrome P450 BM-3, a self-sufficient P450 enzyme from
Bacillus megaterium
that catalyzes the subterminal hydroxylation of long-chain fatty acids, has been engineered into a catalyst for the oxidation of polycyclic aromatic hydrocarbons. The activities of a triplet mutant (A74G/F87V/L188Q) towards naphthalene, fluorene, acenaphthene, acenaphthylene, and 9-methylanthracene were 160, 53, 109, 287, and 22/min, respectively. Compared with the activities of the wild type towards these polycyclic aromatic hydrocarbons, those of the mutant were improved by up to 4 orders of magnitude. The coupling efficiencies of the mutant towards naphthalene, fluorene, acenaphthene, acenaphthylene, and 9-methylanthracene were 11, 26, 5.4, 15, and 3.2%, respectively, which were also improved several to hundreds fold. The high activities of the mutant towards polycyclic aromatic hydrocarbons indicate the potential of engineering P450 BM-3 for the biodegradation of these compounds in the environment.