bluuebunny/crossref_metadata_2025 huggingface dataset:
Pharmacokinetic Interaction between Amprenavir and Rifabutin or Rifampin in Healthy Males
Authors: Polk Ronald E. , Brophy Donald F. , Israel Debra S. , Patron Roberto , Sadler Brian M. , Chittick Gregory E. , Symonds William T. , Lou Yu , Kristoff Debbie , Stein D. S.
Published: July 2002

The objective of this study was to determine if there is a pharmacokinetic interaction when amprenavir is given with rifabutin or rifampin and to determine the effects of these drugs on the erythromycin breath test (ERMBT). Twenty-four healthy male subjects were randomized to one of two cohorts. All subjects received amprenavir (1,200 mg twice a day) for 4 days, followed by a 7-day washout period, followed by either rifabutin (300 mg once a day [QD]) (cohort 1) or rifampin (600 mg QD) (cohort 2) for 14 days. Cohort 1 then received amprenavir plus rifabutin for 10 days, and cohort 2 received amprenavir plus rifampin for 4 days. Serial plasma and urine samples for measurement of amprenavir, rifabutin, and rifampin and their 25-
O
-desacetyl metabolites, were measured by high-performance liquid chromatography. Rifabutin did not significantly affect amprenavir's pharmacokinetics. Amprenavir significantly increased the area under the curve at steady state (AUC
ss
) of rifabutin by 2.93-fold and the AUC
ss
of 25-
O
-desacetylrifabutin by 13.3-fold. Rifampin significantly decreased the AUC
ss
of amprenavir by 82%, but amprenavir had no effect on rifampin pharmacokinetics. Amprenavir decreased the results of the ERMBT by 83%. The results of the ERMBT after 2 weeks of rifabutin and rifampin therapy were increased 187 and 156%, respectively. Amprenavir plus rifampin was well tolerated. Amprenavir plus rifabutin was poorly tolerated, and 5 of 11 subjects discontinued therapy. Rifampin markedly increases the metabolic clearance of amprenavir, and coadministration is contraindicated. Amprenavir significantly decreases clearance of rifabutin and 25-
O
-desacetylrifabutin, and the combination is poorly tolerated. Amprenavir inhibits the ERMBT, and rifampin and rifabutin are equipotent inducers of the ERMBT.
Bioavailability and Preliminary Clinical Efficacy of Intrarectal Artesunate in Ghanaian Children with Moderate Malaria
Authors: Krishna Sanjeev , Planche Tim , Agbenyega Tsiri , Woodrow Charles , Agranoff Dan , Bedu-Addo George , Owusu-Ofori Alex K. , Appiah John Adabie , Ramanathan Surash , Mansor Sharif M. , Navaratnam Visweswaran
Published: July 2002

We report the first detailed pharmacokinetic assessment of intrarectal (i.r.) artesunate (ARS) in African children. Artesunate was given intravenously (i.v.; 2.4 mg/kg of body weight) and i.r. (10 or 20 mg/kg formulated as 50- or 200-mg suppositories [Rectocaps]) in a crossover study design to 34 Ghanaian children with moderate falciparum malaria. The median relative bioavailability of dihydroartemisinin (DHA), the active antimalarial metabolite of ARS, was higher in the low-dose i.r. group (10 mg/kg) than in the high-dose i.r. group (20 mg/kg) (58 versus 23%;
P
= 0.018). There was wide interpatient variation in the area under the concentration-time curve after i.r. ARS administration (up to 9-fold in the high-dose group and 20-fold in the low-dose group). i.r. administered ARS was more rapidly absorbed in the low-dose group than the high-dose group (median [range] absorption half-lives, 0.7 h [0.3 to 1.24 h] versus 1.1 h [0.6 to 2.7 h] [
P
= 0.023]. i.r. administered ARS was eliminated with a median (range) half-life of 0.8 h (0.4 to 2.7 h) (low-dose group and 0.9 h (0.1 to 2.5 h) (high-dose group) (
P
= 1). The fractional clearances of DHA were 3.9, 2.6, and 1.5 liters/kg/h for the 20-mg/kg, 10-mg/kg and i.v. groups, respectively (
P
= 0.001 and
P
= 0.06 for the high-and low-dose i.r. groups compared with the i.v. groups, respectively). The median volumes of distribution for DHA were 1.5 liters kg (20 mg/kg, i.r. group), 1.8 liters/kg (10 mg/kg, i.r. group), and 0.6 liters/kg (i.v. group) (
P
< 0.05 for both i.r. groups compared with the i.v. group). Parasite clearance kinetics were comparable in all treatment groups. i.r. administered ARS may be a useful alternative to parenterally administered ARS in the management of moderate childhood malaria and should be studied further.
Antibiotic Susceptibility Profiles of
Escherichia coli
Strains Lacking Multidrug Efflux Pump Genes
Authors: Sulavik Mark C. , Houseweart Chad , Cramer Christina , Jiwani Nilofer , Murgolo Nicholas , Greene Jonathan , DiDomenico Beth , Shaw Karen Joy , Miller George H. , Hare Roberta , Shimer George
Published: July 2002

The contribution of seven known and nine predicted genes or operons associated with multidrug resistance to the susceptibility of
Escherichia coli
W3110 was assessed for 20 different classes of antimicrobial compounds that include antibiotics, antiseptics, detergents, and dyes. Strains were constructed with deletions for genes in the major facilitator superfamily, the resistance nodulation-cell division family, the small multidrug resistance family, the ATP-binding cassette family, and outer membrane factors. The agar dilution MICs of 35 compounds were determined for strains with deletions for multidrug resistance (MDR) pumps. Deletions in
acrAB
or
tolC
resulted in increased susceptibilities to the majority of compounds tested. The remaining MDR pump gene deletions resulted in increased susceptibilities to far fewer compounds. The results identify which MDR pumps contribute to intrinsic resistance under the conditions tested and supply practical information useful for designing sensitive assay strains for cell-based screening of antibacterial compounds.
Genetic Characterization of Highly Fluoroquinolone-Resistant Clinical
Escherichia coli
Strains from China: Role of
acrR
Mutations
Authors: Wang Hui , Dzink-Fox Joann L. , Chen Minjun , Levy Stuart B.
Published: July 2002

The genetic basis for fluoroquinolone resistance was examined in 30 high-level fluoroquinolone-resistant
Escherichia coli
clinical isolates from Beijing, China. Each strain also demonstrated resistance to a variety of other antibiotics. PCR sequence analysis of the quinolone resistance-determining region of the topoisomerase genes (
gyrA/B, parC
) revealed three to five mutations known to be associated with fluoroquinolone resistance. Western blot analysis failed to demonstrate overexpression of MarA, and Northern blot analysis did not detect overexpression of
soxS
RNA in any of the clinical strains. The AcrA protein of the AcrAB multidrug efflux pump was overexpressed in 19 of 30 strains of
E. coli
tested, and all 19 strains were tolerant to organic solvents. PCR amplification of the complete
acrR
(regulator/repressor) gene of eight isolates revealed amino acid changes in four isolates, a 9-bp deletion in another, and a 22-bp duplication in a sixth strain. Complementation with a plasmid-borne wild-type
acrR
gene reduced the level of AcrA in the mutants and partially restored antibiotic susceptibility 1.5- to 6-fold. This study shows that mutations in
acrR
are an additional genetic basis for fluoroquinolone resistance.
Association of Amino Acid Substitutions in Penicillin-Binding Protein 3 with β-Lactam Resistance in β-Lactamase-Negative Ampicillin-Resistant
Haemophilus influenzae
Authors: Ubukata Kimiko , Shibasaki Yumi , Yamamoto Kentarou , Chiba Naoko , Hasegawa Keiko , Takeuchi Yasuo , Sunakawa Keisuke , Inoue Matsuhisa , Konno Masatoshi
Published: July 2002

The affinity of [
3
H]benzylpenicillin for penicillin-binding protein (PBP) 3A was reduced in 25 clinical isolates of β-lactamase-negative ampicillin (AMP)-resistant (BLNAR)
Haemophilus influenzae
for which the AMP MIC was ≥1.0 μg/ml. The affinities of PBP 3B and PBP 4 were also reduced in some strains. The sequences of the
ftsI
gene encoding the transpeptidase domain of PBP 3A and/or PBP 3B and of the
dacB
gene encoding PBP 4 were determined for these strains and compared to those of AMP-susceptible Rd strains. The BLNAR strains were classified into three groups on the basis of deduced amino acid substitutions in the
ftsI
gene, which is thought to be involved in septal peptidoglycan synthesis. His-517, near the conserved Lys-Thr-Gly (KTG) motif, was substituted for Arg-517 in group I strains (
n
= 9), and Lys-526 was substituted for Asn-526 in group II strains (
n
= 12). In group III strains (
n
= 4), three residues (Met-377, Ser-385, and Leu-389), positioned near the conserved Ser-Ser-Asn (SSN) motif, were replaced with Ile, Thr, and Phe, respectively, in addition to the replacement with Lys-526. The MICs of cephem antibiotics with relatively high affinities for PBP 3A and PBP 3B were higher than those of AMP and meropenem for group III strains. The MICs of β-lactams for
H. influenzae
transformants into which the
ftsI
gene from BLNAR strains was introduced were as high as those for the donors, and PBP 3A and PBP 3B showed decreased affinities for β-lactams. There was no clear relationship between 7-bp deletions in the
dacB
gene and AMP susceptibility. Even though mutations in another gene(s) may be involved in β-lactam resistance, these data indicate that mutations in the
ftsI
gene are the most important for development of resistance to β-lactams in BLNAR strains.
Trifluoromethionine, a Prodrug Designed against Methionine γ-Lyase-Containing Pathogens, Has Efficacy In Vitro and In Vivo against
Trichomonas vaginalis
Authors: Coombs Graham H. , Mottram Jeremy C.
Published: July 2002

Methionine γ-lyase, the enzyme which catalyzes the single-step conversion of methionine to α-ketobutyrate, ammonia, and methanethiol, is highly active in many anaerobic pathogenic microorganisms but has no counterpart in mammals. This study tested the hypothesis that this pathogen-specific enzyme can be exploited as a drug target by prodrugs that are exclusively activated by it. Trifluoromethionine was confirmed as such a prodrug and shown to be highly toxic in vitro to the anaerobic protozoan parasite
Trichomonas vaginalis
, to anaerobic bacteria containing methionine γ-lyase, and to
Escherichia coli
expressing the trichomonad gene. The compound also has exceptional activity against the parasite growing in vivo, with a single dose preventing lesion formation in five of the six mice challenged. These findings suggest that trifluoromethionine represents a lead compound for a novel class of anti-infective drugs with potential as chemotherapeutic agents against a range of prokaryotic and eukaryotic anaerobic pathogens.
Pharmacokinetic and Pharmacodynamic Parameters for Antimicrobial Effects of Cefotaxime and Amoxicillin in an In Vitro Kinetic Model
Authors: Gustafsson I. , Löwdin E. , Odenholt I. , Cars O.
Published: July 2002

An in vitro kinetic model was used to study the relation between pharmacokinetic and pharmacodynamic (PK-PD) parameters for antimicrobial effect, e.g., the time above MIC (T>MIC), maximum concentration in serum (
C
max
), and area under the concentration-time curve (AUC).
Streptococcus pyogenes
and
Escherichia coli
were exposed to cefotaxime, and the activity of amoxicillin against four strains of
Streptococcus pneumoniae
with different susceptibilities to penicillin was studied. The drug elimination rate varied so that the T>MIC ranged from 20 to 100% during 24 h, while the AUC and/or the initial concentration (
C
max
) were kept constant. For
S. pyogenes
and
E. coli
, the maximal antimicrobial effect (
E
max
) at 24 h occurred when the antimicrobial concentration exceeded the MIC for 50 and 80% of the strains tested, respectively. The penicillin-susceptible pneumococci (MIC, 0.03 mg/liter) and the penicillin-intermediate strain (MIC, 0.25 mg/liter) showed maximal killing by amoxicillin at a T>MIC of 50%. For a strain for which the MIC was 2 mg/liter,
C
max
needed to be increased to achieve the
E
max
. Under the condition that
C
max
was 10 times the MIC,
E
max
was obtained at a T>MIC of 60%, indicating that
C
max
, in addition to T>MIC, may be an important parameter for antimicrobial effect on moderately penicillin-resistant pneumococci. For the strain for which the MIC was 4 mg/liter, the reduction of bacteria varied from −0.4 to −3.6 log
10
CFU/ml at a T>MIC of 100%, despite an initial antimicrobial concentration of 10 times the MIC. Our studies have shown that the in vitro kinetic model is a useful complement to animal models for studying the PK-PD relationship for antimicrobial effect of antibiotics.
Multiple Interactions of Cimetidine and Probenecid with Valaciclovir and Its Metabolite Acyclovir
Authors: De Bony F. , Tod M. , Bidault R. , On N. T. , Posner J. , Rolan P.
Published: July 2002

The effects of probenecid and cimetidine on the pharmacokinetics of valaciclovir and its metabolite acyclovir have been investigated. Twelve healthy male volunteers participated in this open single-dose study with a four-way-crossover randomized and balanced design. At the first of four administrations, volunteers in four groups received 1 g of valaciclovir alone, valaciclovir with 1 g of probenecid, valaciclovir with 800 mg of cimetidine, or valaciclovir with a combination of probenecid and cimetidine. At three subsequent administrations, drug regimens were alternated among groups so that each group received each regimen. Probenecid and cimetidine increased the mean maximum concentrations in serum (
C
max
) of valaciclovir by 23 and 53% and the areas under the concentration-time curves (AUC) for valaciclovir by 22 and 73%, respectively; probenecid and cimetidine also increased the mean acyclovir
C
max
by 22 and 8% and its AUC by 48 and 27%, respectively. The combination had a greater effect than either drug alone. Their effects may be due to competitive inhibition of membrane transport of valaciclovir and acyclovir in the liver and kidney. Neither cimetidine nor probenecid affected the absorption of valaciclovir. Both probe drugs reduced the rate of valaciclovir metabolism but not its extent. These pharmacokinetic modifications did not affect the tolerability of valaciclovir.
Genetic and Biochemical Diversity among Isolates of
Paenibacillus alvei
Cultured from Australian Honeybee (
Apis mellifera
) Colonies
Authors: Djordjevic Steven P. , Forbes Wendy A. , Smith Lisa A. , Hornitzky Michael A.
Published: July 2002

Twenty-five unique
Cfo
I-generated whole-cell DNA profiles were identified in a study of 30
Paenibacillus alvei
isolates cultured from honey and diseased larvae collected from honeybee (
Apis mellifera
) colonies in geographically diverse areas in Australia. The fingerprint patterns were highly variable and readily discernible from one another, which highlighted the potential of this method for tracing the movement of isolates in epidemiological studies. 16S rRNA gene fragments (length, 1,416 bp) for all 30 isolates were enzymatically amplified by PCR and subjected to restriction analysis with
Dra
I,
Hin
fI,
Cfo
I,
Alu
I,
Fok
I, and
Rsa
I. With each enzyme the restriction profiles of the 16S rRNA genes from all 30 isolates were identical (one restriction fragment length polymorphism [RFLP] was observed in the
Hin
fI profile of the 16S rRNA gene from isolate 17), which confirmed that the isolates belonged to the same species. The restriction profiles generated by using
Dra
I,
Fok
I, and
Hin
fI differentiated
P. alvei
from the phylogenetically closely related species
Paenibacillus macerans
and
Paenibacillus macquariensis
. Alveolysin gene fragments (length, 1,555 bp) were enzymatically amplified from some of the
P. alvei
isolates (19 of 30 isolates), and RFLP were detected by using the enzymes
Cfo
I,
Sau
3AI, and
Rsa
I. Extrachromosomal DNA ranging in size from 1 to 10 kb was detected in 17 of 30 (57%)
P. alvei
whole-cell DNA profiles. Extensive biochemical heterogeneity was observed among the 28
P. alvei
isolates examined with the API 50CHB system. All of these isolates were catalase, oxidase, and Voges-Proskauer positive and nitrate negative, and all produced acid when glycerol, esculin, and maltose were added. The isolates produced variable results for 16 of the 49 biochemical tests; negative reactions were recorded in the remaining 30 assays. The genetic and biochemical heterogeneity in
P. alvei
isolates may be a reflection of adaptation to the special habitats in which they originated.
Molecular Ecological Analysis of the Succession and Diversity of Sulfate-Reducing Bacteria in the Mouse Gastrointestinal Tract
Authors: Deplancke B. , Hristova K. R. , Oakley H. A. , McCracken V. J. , Aminov R. , Mackie R. I. , Gaskins H. R.
Published: July 2002

Intestinal sulfate-reducing bacteria (SRB) growth and resultant hydrogen sulfide production may damage the gastrointestinal epithelium and thereby contribute to chronic intestinal disorders. However, the ecology and phylogenetic diversity of intestinal dissimilatory SRB populations are poorly understood, and endogenous or exogenous sources of available sulfate are not well defined. The succession of intestinal SRB was therefore compared in inbred C57BL/6J mice using a PCR-based metabolic molecular ecology (MME) approach that targets a conserved region of subunit A of the adenosine-5′-phosphosulfate (APS) reductase gene. The APS reductase-based MME strategy revealed intestinal SRB in the stomach and small intestine of 1-, 4-, and 7-day-old mice and throughout the gastrointestinal tract of 14-, 21-, 30-, 60-, and 90-day-old mice. Phylogenetic analysis of APS reductase amplicons obtained from the stomach, middle small intestine, and cecum of neonatal mice revealed that
Desulfotomaculum
spp. may be a predominant SRB group in the neonatal mouse intestine. Dot blot hybridizations with SRB-specific 16S ribosomal DNA (rDNA) probes demonstrated SRB colonization of the cecum and colon pre- and postweaning and colonization of the stomach and small intestine of mature mice only. The 16S rDNA hybridization data further demonstrated that SRB populations were most numerous in intestinal regions harboring sulfomucin-containing goblet cells, regardless of age. Reverse transcriptase PCR analysis demonstrated APS reductase mRNA expression in all intestinal segments of 30-day-old mice, including the stomach. These results demonstrate for the first time widespread colonization of the mouse intestine by dissimilatory SRB and evidence of spatial-specific SRB populations and sulfomucin patterns along the gastrointestinal tract.
Lack of Production of Electron-Shuttling Compounds or Solubilization of Fe(III) during Reduction of Insoluble Fe(III) Oxide by
Geobacter metallireducens
Authors: Nevin Kelly P. , Lovley Derek R.
Published: July 2002

Studies with the dissimilatory Fe(III)-reducing microorganism
Geobacter metallireducens
demonstrated that the common technique of separating Fe(III)-reducing microorganisms and Fe(III) oxides with semipermeable membranes in order to determine whether the Fe(III) reducers release electron-shuttling compounds and/or Fe(III) chelators is invalid. This raised doubts about the mechanisms for Fe(III) oxide reduction by this organism. However, several experimental approaches indicated that
G. metallireducens
does not release electron-shuttling compounds and does not significantly solubilize Fe(III) during Fe(III) oxide reduction. These results suggest that
G. metallireducens
directly reduces insoluble Fe(III) oxide.
Surface of Lactic Acid Bacteria: Relationships between Chemical Composition and Physicochemical Properties
Authors: Boonaert Christophe J. P. , Rouxhet Paul G.
Published: July 2002

The surface chemical composition and physicochemical properties (hydrophobicity and zeta potential) of two lactic acid bacteria,
Lactococcus lactis
subsp.
lactis
bv. diacetilactis and
Lactobacillus helveticus
, have been investigated using cells harvested in exponential or stationary growth phase. The surface composition determined by X-ray photoelectron spectroscopy (XPS) was converted into a molecular composition in terms of proteins, polysaccharides, and hydrocarbonlike compounds. The concentration of the last was always below 15% (wt/wt), which is related to the hydrophilic character revealed by water contact angles of less than 30°. The surfaces of
L. lactis
cells had a polysaccharide concentration about twice that of proteins. The S-layer of
L. helveticus
was either interrupted or crossed by polysaccharide-rich compounds; the concentration of the latter was higher in the stationary growth phase than in the exponential growth phase. Further progress was made in the interpretation of XPS data in terms of chemical functions by showing that the oxygen component at 531.2 eV contains a contribution of phosphate in addition to the main contribution of the peptide link. The isoelectric points were around 2 and 3, and the electrophoretic mobilities above pH 5 (ionic strength, 1 mM) were about −3.0 × 10
−8
and −0.6 × 10
−8
m
2
s
−1
V
−1
for
L. lactis
and
L. helveticus
, respectively. The electrokinetic properties of the latter reveal the influence of carboxyl groups, while the difference between the two strains is related to a difference between N/P surface concentration ratios, reflecting the relative exposure of proteins and phosphate groups at the surface.
Absence of a Putative Mannose-Specific Phosphotransferase System Enzyme IIAB Component in a Leucocin A-Resistant Strain of
Listeria monocytogenes
, as Shown by Two-Dimensional Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis
Authors: Ramnath M. , Beukes M. , Tamura K. , Hastings J. W.
Published: July 2002

Leucocin A is a class IIa bacteriocin produced by
Leuconostoc
spp. that has previously been shown to inhibit the growth of
Listeria monocytogenes
. A spontaneous resistant mutant of
L. monocytogenes
was isolated and found to be resistant to leucocin A at levels in excess of 2 mg/ml. The mutant showed no significant cross-resistance to nontype IIa bacteriocins including nisaplin and ESF1-7GR. However, there were no inhibition zones found on a lawn of the mutant when challenged with an extract containing 51,200 AU of pediocin PA-2 per ml as determined by a simultaneous assay on the sensitive wild-type strain. DNA and protein analysis of the resistant and susceptible strains were carried out using silver-stained amplified fragment length polymorphism (ssAFLP) and one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), respectively. Two-dimensional SDS-PAGE clearly showed a 35-kDa protein which was present in the sensitive but absent from the resistant strain. The N-terminal end of the 35-kDa protein was sequenced and found to have an 83% homology to the mannose-specific phosphotransferase system enzyme IIAB of
Streptococcus salivarius
.
Identification of the Dimerization Domain of Dehalogenase IVa ofBurkholderia cepaciaMBA4
Authors: Tsang Jimmy S. H. , Pang Benjamin C. M.
Published: July 2002

Haloacid dehalogenases are enzymes that catalyze the hydrolytic removal of halogens from haloalkanoic acids. Dehalogenase IVa (DehIVa) fromBurkholderia cepaciaMBA4 and dehalogenase CI (DehCI) fromPseudomonassp. strain CBS3 exhibit 68% identity. Despite their similarity DehIVa is a dimeric enzyme while DehCI is a monomer. In this work, we describe the identification of the domain that confers the dimerization function of DehIVa. Recombinant DNA molecules were constructed by fusion of the respective dehalogenase geneshdlIVaanddehCI. When amino acids 73 to 89 of DehCI were replaced by amino acids 74 to 90 of DehIVa, the recombinant molecule migrated like that of DehIVa in a nondenaturing activity-stained gel. Similarly, when residues 73 to 89 of DehIVa were replaced by the corresponding residues of DehCI, the chimera migrated as a monomer. These 17 amino acid changes were able to determine the aggregation states of the molecules. The retention of the catalytic function in these chimeras indicated that the overall folding of these proteins was not affected. Site-directed mutagenesis onhdlIVahowever indicated that amino acids Phe58, Thr65, Leu78, and Phe92 of DehIVa are also important for the aggregation state of the protein. This indicates that the 17 residues are not sufficient for the dimerization of the protein.
Identification of
Mycobacterium ulcerans
in the Environment from Regions in Southeast Australia in Which It Is Endemic with Sequence Capture-PCR
Authors: Stinear Timothy , Davies John K. , Jenkin Grant A. , Hayman John A. , Oppedisano Frances , Johnson Paul D. R.
Published: July 2002

We recently described the use of PCR to identify the environmental source of
Mycobacterium ulcerans
during an outbreak of ulcerative disease that occurred in a localized region of southeast Australia. The PCR used was based on amplification of the
M. ulcerans
-specific insertion sequence, IS
2404
. In this study we developed a new test that is a substantial improvement over the original PCR method in terms of sensitivity, reliability, and ease of use. In the new method magnetic bead sequence capture-PCR is used to detect two
M. ulcerans
sequences (IS
2404
and IS
2606
) and total mycobacterial 16S ribosomal DNA. We used sequence capture-PCR to test water and plant material collected over a 12-month period during 1998 and 1999 from sites near the centers of two distinct foci of
M. ulcerans
infections. A golf course irrigation system in one area and a small shallow lake in another area repeatedly were PCR positive for
M. ulcerans
. Nearby sites and sites unrelated to the endemic areas were negative. Based on the PCR data, a most-probable-number method was used to estimate the concentration of
M. ulcerans
cells in positive samples from both regions. This procedure resulted in average concentrations of 0.5 cell per 100 ml of water and 40 cells per 100 g of detritus. Loss of the PCR signal coincided with a decrease in ulcerative disease in each area. These results provide further evidence that
M. ulcerans
may be transmitted from a point environmental source and demonstrate the utility of magnetic bead sequence capture-PCR for identification of nonculturable microbial pathogens in the environment.
Evaluation of Fluorescently Labeled Lectins for Noninvasive Localization of Extracellular Polymeric Substances in
Sphingomonas
Biofilms
Authors: Johnsen Anders R. , Hausner Martina , Schnell Annette , Wuertz Stefan
Published: July 2002

Three strains of
Sphingomonas
were grown as biofilms and tested for binding of five fluorescently labeled lectins (Con A-type IV-TRITC or -Cy5, Pha-E-TRITC, PNA-TRITC, UEA 1-TRITC, and WGA-Texas red). Only ConA and WGA were significantly bound by the biofilms. Binding of the five lectins to artificial biofilms made of the commercially available
Sphingomonas
extracellular polysaccharides was similar to binding to living biofilms. Staining of the living and artificial biofilms by ConA might be explained as binding of the lectin to the terminal mannosyl and terminal glucosyl residues in the polysaccharides secreted by
Sphingomonas
as well as to the terminal mannosyl residue in glycosphingolipids. Staining of the biofilms by WGA could only be explained as binding to the
Sphingomonas
glycosphingolipid membrane, binding to the cell wall, or nonspecific binding. Glycoconjugation of ConA and WGA with the target sugars glucose and
N
-acetylglucosamine, respectively, was used as a method for evaluation of the specificity of the lectins towards
Sphingomonas
biofilms and
Sphingomonas
polysaccharides. Our results show that the binding of lectins to biofilms does not necessarily prove the presence of specific target sugars in the extracellular polymeric substances (EPS) in biofilms. The lectins may bind to non-EPS targets or adhere nonspecifically to components of the biofilm matrix.
Applicability of an Arrhenius Model for the Combined Effect of Temperature and CO
2
Packaging on the Spoilage Microflora of Fish
Authors: Koutsoumanis Konstantinos P. , Taoukis Petros S. , Drosinos Eleftherios H. , Nychas George-John E.
Published: July 2002

The temperature behavior of the natural microflora on the Mediterranean fish red mullet (
Mullus barbatus
) was examined as a case study. The growth of the spoilage bacteria
Pseudomonas
spp.,
Shewanella putrefaciens
,
Brochothrix thermosphacta
, and lactic acid bacteria was modeled as a function of temperature and the concentration of carbon dioxide in modified atmosphere packaging. Combined models were developed and comparatively assessed based on polynomial, Belehradek, and Arrhenius equations. The activation energy parameter of the Arrhenius model,

E
A

, was independent of the packaging atmosphere and ranged from 75 to 85 kJ/mol for the different bacteria, whereas the preexponential constant decreased exponentially with the packaging CO
2
concentration. We evaluated the applicability of the models developed by using experimental bacterial growth rates obtained from 42 independent experiments performed with three Mediterranean fish species and growth rates predicted from the models under the same temperature and packaging conditions. The accuracy factor and bias factor were used as statistical tools for evaluation, and the developed Arrhenius model and the Belehradek model were judged satisfactory overall.
Mycophenolic Acid in Silage
Authors: Schneweis Isabell , Meyer Karsten , Hörmansdorfer Stefan , Bauer Johann
Published: July 2002

We examined 233 silage samples and found that molds were present in 206 samples with counts between 1 × 10
3
and 8.9 × 10
7
(mean, 4.7 × 10
6
) CFU/g. Mycophenolic acid, a metabolite of
Penicillium roqueforti
, was detected by liquid chromatography-mass spectrometry in 74 (32%) of these samples at levels ranging from 20 to 35,000 (mean, 1,400) μg/kg. This compound has well-known immunosuppressive properties, so feeding with contaminated silage may promote the development of infectious diseases in livestock.
Effects of High Pressure on Survival and Metabolic Activity of
Lactobacillus plantarum
TMW1.460
Authors: Ulmer Helge M. , Gänzle Michael G. , Vogel Rudi F.
Published: July 2002

The application of high pressure (HP) for food preservation requires insight into mechanisms of HP-mediated cell injury and death. The HP inactivation in model beer of
Lactobacillus plantarum
TMW1.460, a beer-spoiling organism, was investigated at pressures ranging from 200 to 600 MPa. Surviving cells were characterized by determination of (i) cell viability and sublethal injury, (ii) membrane permeability to the fluorescent dyes propidium iodide (PI) and ethidium bromide (EB), (iii) metabolic activity with tetrazolium salts, and (iv) the activity of HorA, an ATP binding cassette-type multidrug resistance transporter conferring resistance to hop compounds. HP inactivation curves exhibited a shoulder, an exponential inactivation phase, and pronounced tailing caused by a barotolerant fraction of the population, about 1 in 10
6
cells. During exponential inactivation, more than 99.99% of cells were sublethally injured; however, no sublethal injury was detected in the barotolerant fraction of the culture. Sublethally injured cells were metabolically active, and loss of metabolic activity corresponded to the decrease of cell viability. Membrane damage measured by PI uptake occurred later than cell death, indicating that dye exclusion may be used as a fail-safe method for preliminary characterization of HP inactivation. An increase of membrane permeability to EB and a reduction of HorA activity were observed prior to the loss of cell viability, indicating loss of hop resistance of pressurized cells. Even mild HP treatments thus abolished the ability of cells to survive under adverse conditions.
Molecular Characterization of
Lactobacillus plantarum
Genes for β-Ketoacyl-Acyl Carrier Protein Synthase III (
fabH
) and Acetyl Coenzyme A Carboxylase (
accBCDA
), Which Are Essential for Fatty Acid Biosynthesis
Authors: Kiatpapan Pornpimon , Kobayashi Hajime , Sakaguchi Maki , Ono Hisayo , Yamashita Mitsuo , Kaneko Yoshinobu , Murooka Yoshikatsu
Published: July 2002

Genes for subunits of acetyl coenzyme A carboxylase (ACC), which is the enzyme that catalyzes the first step in the synthesis of fatty acids in
Lactobacillus plantarum
L137, were cloned and characterized. We identified six potential open reading frames, namely,
manB
,
fabH
,
accB
,
accC
,
accD
, and
accA
, in that order. Nucleotide sequence analysis suggested that
fabH
encoded β-ketoacyl-acyl carrier protein synthase III, that the
accB
,
accC
,
accD
, and
accA
genes encoded biotin carboxyl carrier protein, biotin carboxylase, and the β and α subunits of carboxyltransferase, respectively, and that these genes were clustered. The organization of
acc
genes was different from that reported for
Escherichia coli
, for
Bacillus subtilis
, and for
Pseudomonas aeruginosa. E. coli accB
and
accD
mutations were complemented by the
L. plantarum accB
and
accD
genes, respectively. The predicted products of all five genes were confirmed by using the T7 expression system in
E. coli
. The gene product of
accB
was biotinylated in
E. coli
. Northern and primer extension analyses demonstrated that the five genes in
L. plantarum
were regulated polycistronically in an
acc
operon.
Characterization of a Highly Thermostable Alkaline Phosphatase from the EuryarchaeonPyrococcus abyssi
Authors: Zappa Sébastien , Rolland Jean-Luc , Flament Didier , Gueguen Yannick , Boudrant Joseph , Dietrich Jacques
Published: July 2002

This work reports the first isolation and characterization of an alkaline phosphatase (AP) from a hyperthermophilic archaeon. An AP gene fromPyrococcus abyssi, a euryarchaeon isolated from a deep-sea hydrothermal vent, was cloned and the enzyme expressed inEscherichia coli. Analysis of the sequence showed conservation of the active site and structural elements of theE. coliAP. The recombinant AP was purified and characterized. Monomeric and homodimeric active forms were detected, with a monomer molecular mass of 54 kDa. Apparent optimum pH and temperature were estimated at 11.0 and 70°C, respectively. Thus far,P. abyssiAP has been demonstrated to be the most thermostable AP, with half-lives at 100 and 105°C of 18 and 5 h, respectively. Enzyme activity was found to be dependent on divalent cations: metal ion chelators inhibited activity, whereas the addition of exogenous Mg(II), Zn(II), and Co(II) increased activity. The enzyme was inhibited by inorganic phosphate, but not by molybdate and vanadate. Strong inhibitory effects were observed in the presence of thiol-reducing agents, although cysteine residues of theP. abyssiAP were not found to be incorporated within intra- or interchain disulfide bonds. In addition,P. abyssiAP was demonstrated to dephosphorylate linear DNA fragments with dephosphorylation efficiencies of 93.8 and 84.1% with regard to cohesive and blunt ends, respectively.
Diversity of
Geotrichum candidum
Strains Isolated from Traditional Cheesemaking Fabrications in France
Authors: Marcellino N. , Beuvier E. , Grappin R. , Guéguen M. , Benson D. R.
Published: July 2002

The diversity of French fungus-ripened cheeses is due partly to the succession of fungi that colonize the cheese during ripening.
Geotrichum candidum
appears in the early stages of ripening on soft cheeses such as Camembert and semihard cheeses such as St. Nectaire and Reblochon. Its lipases and proteases promote flavor development, and its aminopeptidases reduce bitterness imparted by low-molecular-weight peptides in cheese. We assessed the genetic diversity of
G. candidum
strains by using random amplification of polymorphic DNA (RAPD)-PCR correlated with phenotypic tests for carbon assimilation and salt tolerance. Strains were isolated from milk, curd, and cheese collected in seven major cheesemaking regions of France. Sixty-four isolates were characterized. We found high genetic diversity of
G. candidum
even within the same cheesemaking regions. Strains did not group according to region. All of the strains from the Haute-Savoie were able to assimilate lactate as the sole source of carbon, while lactate assimilation varied among strains from the Auvergne. Strains varied in
d
-mannitol assimilation, and none used citrate as the sole source of carbon. Yeast-like colony morphology predominated in Reblochon, while all of the strains isolated from St. Nectaire were filamentous. The RAPD-PCR technique readily differentiated
Geotrichum fragrans
isolated from milk and curd in a St. Nectaire cheesemaking facility. This study reveals an enormous diversity of
G. candidum
that has been empirically selected through the centuries by the cheesemakers of France.
Variation of Microbial Communities in Soil, Rhizosphere, and Rhizoplane in Response to Crop Species, Soil Type, and Crop Development
Authors: Wieland Gabriele , Neumann Regine , Backhaus Horst
Published: July 2002

We investigated the influence of plant species, soil type, and plant development time on the shaping of microbial communities in soil and in association with roots. The sample group consisted of a total of 32 microcosms in three habitats: soil, rhizosphere, and rhizoplane. Communities were represented by the patterns of a sequence-specific separation of rRNA target sequences. Effects of experimental parameters were classified by a cluster analysis of pattern similarities. The type of plant species (clover, bean, or alfalfa) had the greatest effect in plant-associated habitats and also affected soil patterns. Plant development had a minor habitat-dependent effect that was partly obscured by replicate variation. The results stress the applicability of biased community representations in an analysis of induced variation.
Impact of Dilution on Microbial Community Structure and Functional Potential: Comparison of Numerical Simulations and Batch Culture Experiments
Authors: Franklin Rima B. , Garland Jay L. , Bolster Carl H. , Mills Aaron L.
Published: July 2002

A series of microcosm experiments was performed using serial dilutions of a sewage microbial community to inoculate a set of batch cultures in sterile sewage. After inoculation, the dilution-defined communities were allowed to regrow for several days and a number of community attributes were measured in the regrown assemblages. Based upon a set of numerical simulations, community structure was expected to differ along the dilution gradient; the greatest differences in structure were anticipated between the undiluted–low-dilution communities and the communities regrown from the very dilute (more than 10
−4
) inocula. Furthermore, some differences were expected among the lower-dilution treatments (e.g., between undiluted and 10
−1
) depending upon the evenness of the original community. In general, each of the procedures used to examine the experimental community structures separated the communities into at least two, often three, distinct groups. The groupings were consistent with the simulated dilution of a mixture of organisms with a very uneven distribution. Significant differences in community structure were detected with genetic (amplified fragment length polymorphism and terminal restriction fragment length polymorphism), physiological (community level physiological profiling), and culture-based (colony morphology on R2A agar) measurements. Along with differences in community structure, differences in community size (acridine orange direct counting), composition (ratio of sewage medium counts to R2A counts, monitoring of each colony morphology across the treatments), and metabolic redundancy (i.e., generalist versus specialist) were also observed, suggesting that the differences in structure and diversity of communities maintained in the same environment can be manifested as differences in community organization and function.
Cell Cycle Regulation by Light in
Prochlorococcus
Strains
Authors: Jacquet Stéphan , Partensky Frédéric , Marie Dominique , Casotti Raffaella , Vaulot Daniel
Published: July 2002

The effect of light on the synchronization of cell cycling was investigated in several strains of the oceanic photosynthetic prokaryote
Prochlorococcus
using flow cytometry. When exposed to a light-dark (L-D) cycle with an irradiance of 25 μmol of quanta · m
−2
s
−1
, the low-light-adapted strain SS 120 appeared to be better synchronized than the high-light-adapted strain PCC 9511. Submitting L-D-entrained populations to shifts (advances or delays) in the timing of the “light on” signal translated to corresponding shifts in the initiation of the S phase, suggesting that this signal is a key parameter for the synchronization of population cell cycles. Cultures that were shifted from an L-D cycle to continuous irradiance showed persistent diel oscillations of flow-cytometric signals (light scatter and chlorophyll fluorescence) but with significantly reduced amplitudes and a phase shift. Complete darkness arrested most of the cells in the G
1
phase of the cell cycle, indicating that light is required to trigger the initiation of DNA replication and cell division. However, some cells also arrested in the S phase, suggesting that cell cycle controls in
Prochlorococcus
spp. are not as strict as in marine
Synechococcus
spp. Shifting
Prochlorococcus
cells from low to high irradiance translated quasi-instantaneously into an increase of cells in both the S and G
2
phases of the cell cycle and then into faster growth, whereas the inverse shift induced rapid slowing of the population growth rate. These data suggest a close coupling between irradiance levels and cell cycling in
Prochlorococcus
spp.
In Situ Accessibility of
Escherichia coli
23S rRNA to Fluorescently Labeled Oligonucleotide Probes
Authors: Fuchs Bernhard M. , Syutsubo Kazuaki , Ludwig Wolfgang , Amann Rudolf
Published: July 2002

One of the main causes of failure of fluorescence in situ hybridization with rRNA-targeted oligonucleotides, besides low cellular ribosome content and impermeability of cell walls, is the inaccessibility of probe target sites due to higher-order structure of the ribosome. Analogous to a study on the 16S rRNA (B. M. Fuchs, G. Wallner, W. Beisker, I. Schwippl, W. Ludwig, and R. Amann, Appl. Environ. Microbiol. 64:4973–4982, 1998), the accessibility of the 23S rRNA of
Escherichia coli
DSM 30083
T
was studied in detail with a set of 184 CY3-labeled oligonucleotide probes. The probe-conferred fluorescence was quantified flow cytometrically. The brightest signal resulted from probe 23S-2018, complementary to positions 2018 to 2035. The distribution of probe-conferred cell fluorescence in six arbitrarily set brightness classes (classes I to VI, 100 to 81%, 80 to 61%, 60 to 41%, 40 to 21%, 20 to 6%, and 5 to 0% of the brightness of 23S-2018, respectively) was as follows: class I, 3%; class II, 21%; class III, 35%; class IV, 18%; class V, 16%; and class VI, 7%. A fine-resolution analysis of selected areas confirmed steep changes in accessibility on the 23S RNA to oligonucleotide probes. This is similar to the situation for the 16S rRNA. Indeed, no significant differences were found between the hybridization of oligonucleotide probes to 16S and 23S rRNA. Interestingly, indications were obtained of an effect of the type of fluorescent dye coupled to a probe on in situ accessibility. The results were translated into an accessibility map for the 23S rRNA of
E. coli
, which may be extrapolated to other bacteria. Thereby, it may contribute to a better exploitation of the high potential of the 23S rRNA for identification of bacteria in the future.
Community Structure and Activity Dynamics of Nitrifying Bacteria in a Phosphate-Removing Biofilm
Authors: Gieseke Armin , Purkhold Ulrike , Wagner Michael , Amann Rudolf , Schramm Andreas
Published: July 2002

The microbial community structure and activity dynamics of a phosphate-removing biofilm from a sequencing batch biofilm reactor were investigated with special focus on the nitrifying community. O
2
, NO
2
−
, and NO
3
−
profiles in the biofilm were measured with microsensors at various times during the nonaerated-aerated reactor cycle. In the aeration period, nitrification was oxygen limited and restricted to the first 200 μm at the biofilm surface. Additionally, a delayed onset of nitrification after the start of the aeration was observed. Nitrate accumulating in the biofilm in this period was denitrified during the nonaeration period of the next reactor cycle. Fluorescence in situ hybridization (FISH) revealed three distinct ammonia-oxidizing populations, related to the
Nitrosomonas europaea, Nitrosomonas oligotropha,
and
Nitrosomonas communis
lineages. This was confirmed by analysis of the genes coding for 16S rRNA and for ammonia monooxygenase (
amoA
). Based upon these results, a new 16S rRNA-targeted oligonucleotide probe specific for the
Nitrosomonas oligotropha
lineage was designed. FISH analysis revealed that the first 100 μm at the biofilm surface was dominated by members of the
N. europaea
and the
N. oligotropha
lineages, with a minor fraction related to
N. communis
. In deeper biofilm layers, exclusively members of the
N. oligotropha
lineage were found. This separation in space and a potential separation of activities in time are suggested as mechanisms that allow coexistence of the different ammonia-oxidizing populations. Nitrite-oxidizing bacteria belonged exclusively to the genus
Nitrospira
and could be assigned to a 16S rRNA sequence cluster also found in other sequencing batch systems.
Comparative Analysis of Methane-Oxidizing Archaea and Sulfate-Reducing Bacteria in Anoxic Marine Sediments
Authors: Orphan V. J. , Hinrichs K.-U. , Ussler W. , Paull C. K. , Taylor L. T. , Sylva S. P. , Hayes J. M. , Delong E. F.
Published: July 2002

The oxidation of methane in anoxic marine sediments is thought to be mediated by a consortium of methane-consuming archaea and sulfate-reducing bacteria. In this study, we compared results of rRNA gene (rDNA) surveys and lipid analyses of archaea and bacteria associated with methane seep sediments from several different sites on the Californian continental margin. Two distinct archaeal lineages (ANME-1 and ANME-2), peripherally related to the order
Methanosarcinales
, were consistently associated with methane seep marine sediments. The same sediments contained abundant
13
C-depleted archaeal lipids, indicating that one or both of these archaeal groups are members of anaerobic methane-oxidizing consortia.
13
C-depleted lipids and the signature 16S rDNAs for these archaeal groups were absent in nearby control sediments. Concurrent surveys of bacterial rDNAs revealed a predominance of δ-proteobacteria, in particular, close relatives of
Desulfosarcina variabilis
. Biomarker analyses of the same sediments showed bacterial fatty acids with strong
13
C depletion that are likely products of these sulfate-reducing bacteria. Consistent with these observations, whole-cell fluorescent in situ hybridization revealed aggregations of ANME-2 archaea and sulfate-reducing
Desulfosarcina
and
Desulfococcus
species. Additionally, the presence of abundant
13
C-depleted ether lipids, presumed to be of bacterial origin but unrelated to ether lipids of members of the order
Desulfosarcinales
, suggests the participation of additional bacterial groups in the methane-oxidizing process. Although the
Desulfosarcinales
and ANME-2 consortia appear to participate in the anaerobic oxidation of methane in marine sediments, our data suggest that other bacteria and archaea are also involved in methane oxidation in these environments.