bluuebunny/crossref_metadata_2025 huggingface dataset:
Clinical Efficacy of Intravenous followed by Oral Azithromycin Monotherapy in Hospitalized Patients with Community-Acquired Pneumonia
Authors: Plouffe Joseph , Schwartz Douglas B. , Kolokathis Antonia , Sherman Bruce W. , Arnow Paul M. , Gezon John A. , Suh Byungse , Anzuetto Antonio , Greenberg Richard N. , Niederman Michael , Paladino Joseph A. , Ramirez Julio A. , Inverso Jill , Knirsch Charles A. ,   the Azithromycin Intravenous Clinical Trials Group
Published: July 2002

The purpose of this study was to evaluate intravenous (i.v.) azithromycin followed by oral azithromycin as a monotherapeutic regimen for community-acquired pneumonia (CAP). Two trials of i.v. azithromycin used as initial monotherapy in hospitalized CAP patients are summarized. Clinical efficacy is reported from an open-label randomized trial of azithromycin compared to cefuroxime with or without erythromycin. Bacteriologic and clinical efficacy results are also presented from a noncomparative trial of i.v. azithromycin that was designed to give additional clinical experience with a larger number of pathogens. Azithromycin was administered to 414 patients: 202 and 212 in the comparative and noncomparative trials, respectively. The comparator regimen was used as treatment for 201 patients; 105 were treated with cefuroxime alone and 96 were given cefuroxime plus erythromycin. In the comparative trial, clinical outcome data were available for 268 evaluable patients with confirmed CAP at the 10- to 14-day visit, with 106 (77%) of the azithromycin patients cured or improved and 97 (74%) of the comparator patients cured or improved. Mean i.v. treatment duration and mean total treatment duration (i.v. and oral) for the clinically evaluable patients were significantly (
P
< 0.05) shorter for the azithromycin group (3.6 days for the i.v. group and 8.6 days for the i.v. and oral group) than for the evaluable patients given cefuroxime plus erythromycin (4.0 days for the i.v. group and 10.3 days for the i.v. and oral group). The present comparative study demonstrates that initial therapy with i.v. azithromycin for hospitalized patients with CAP is associated with fewer side effects and is equal in efficacy to a 1993 American Thoracic Society-suggested regimen of cefuroxime plus erythromycin when the erythromycin is deemed necessary by clinicians.
In Vitro Activity of Trovafloxacin Compared to Those of Five Antimicrobials against Mycoplasmas Including
Mycoplasma hominis
and
Ureaplasma urealyticum
Fluoroquinolone-Resistant Isolates That Have Been Genetically Characterized
Authors: Bebear C. M. , Renaudin H. , Charron A. , Gruson D. , Lefrancois M. , Bebear C.
Published: July 2002

The in vitro activity of trovafloxacin against 125 strains of
Mycoplasma
species and
Ureaplasma urealyticum
, including fluoroquinolone-susceptible and fluoroquinolone-resistant species, was compared to those of other fluoroquinolones, doxycycline, and erythromycin. The MIC at which 90% of isolates are inhibited for all fluoroquinolone-susceptible strains was 0.25 μg/ml. Whatever the associated mutations, trovafloxacin exhibited greater activity than the other fluoroquinolones tested against fluoroquinolone-resistant
Mycoplasma hominis
and
U. urealyticum
isolates.
Pharmacokinetics of the New Ketolide Telithromycin (HMR 3647) Administered in Ascending Single and Multiple Doses
Authors: Namour F. , Wessels D. H. , Pascual M. H. , Reynolds D. , Sultan E. , Lenfant B.
Published: July 2002

Telithromycin (HMR 3647) is a novel ketolide antimicrobial with good activity against both common and atypical respiratory pathogens, including many resistant strains. This randomized, three-period crossover study determined the dose proportionality of telithromycin pharmacokinetics after single and multiple dosing in healthy subjects. In each treatment period, subjects received a single oral dose of 400, 800 or 1,600 mg of telithromycin followed 4 days later by the same dose once daily for 7 days. Blood and urine samples were taken throughout the study for determination of pharmacokinetic parameters for telithromycin and RU 76363, its main metabolite. Telithromycin and RU 76363 achieved steady state within 2 to 3 days of once-daily dosing. A slight accumulation of telithromycin was observed after 7 days of therapy, with values of the area under the concentration-time curve from 0 to 24 h approximately 1.5 times higher than those achieved with the single dose. The pharmacokinetics of telithromycin and RU 76363 deviated moderately from dose proportionality. At a dose of 800 mg/day, telithromycin attained mean maximal and trough plasma concentrations of 2.27 and 0.070 mg/liter respectively. Elimination was biphasic; initial and terminal half-lives were 2.87 and 9.81 h for the 800-mg dose. Study medication was well tolerated, although adverse events tended to be more frequent at the 1,600-mg dose. This study showed that telithromycin was generally well tolerated and suggests that a once-daily 800-mg oral dose of telithromycin maintains an effective concentration in plasma for the treatment of respiratory tract infections involving the key respiratory pathogens.
β-Lactamase Inhibitors Derived from Single-Domain Antibody Fragments Elicited in the
Camelidae
Authors: Conrath Katja E. , Lauwereys Marc , Galleni Moreno , Matagne André , Frère Jean-Marie , Kinne Jörg , Wyns Lode , Muyldermans Serge
Published: July 2002

Small, soluble single-domain fragments derived from the unique variable region of dromedary heavy-chain antibodies (VHHs) against enzymes are known to be potent inhibitors. The immunization of dromedaries with the TEM-1 and BcII β-lactamases has lead to the isolation of such single-domain antibody fragments specifically recognizing and inhibiting those β-lactamases. Two VHHs were isolated that inhibit TEM-1 and one BcII inhibiting VHH was identified. All inhibitory VHHs were tight-binding inhibitors. The 50% inhibitory concentrations were determined for all inhibitors and they were all in the same range as the enzyme concentration used in the assay. Addition of the VHHs to the TEM-1 β-lactamase, expressed on the surface of bacteria, leads to a higher ampicillin sensitivity of the bacteria. This innovative strategy could generate multiple potent inhibitors for all types of β-lactamases.
Activity of Ertapenem (MK-0826) versus
Enterobacteriaceae
with Potent β-Lactamases
Authors: Livermore David M. , Oakton Karen J. , Carter Michael W. , Warner Marina
Published: July 2002

Ertapenem (MK-0826; L-749,345), a new carbapenem with a long serum half-life, was tested, in vitro, against β-lactamase-producing bacteria. The new compound had a MIC at which 90% of the isolates were inhibited of 0.06 μg/ml for extended-spectrum β-lactamase (ESBL)-producing klebsiellas, compared with 0.5 μg/ml for imipenem, 16 μg/ml for cefepime, and >128 μg/ml for ceftazidime and piperacillin-tazobactam. MICs of ertapenem for AmpC-derepressed mutant
Enterobacteriaceae
were 0.015 to 0.5 μg/ml, whereas imipenem MICs were 0.25 to 1 μg/ml and those of cefepime were 0.5 to 4 μg/ml, and resistance to ceftazidime and piperacillin-tazobactam was generalized. Despite this good activity, the MICs of ertapenem for ESBL-positive klebsiellas mostly were two- to fourfold above those for ESBL-negative strains, and the MICs for AmpC-hyperproducing
Enterobacter cloacae
and
Citrobacter freundii
mutants exceeded those for the corresponding AmpC-basal mutants. These differentials did not increase when the inoculum was raised from 10
4
to 10
6
CFU/spot, contraindicating significant lability. Carbapenemase producers were also tested. The IMP-1 metallo-β-lactamase conferred substantial ertapenem resistance (MIC, 128 μg/ml) in a porin-deficient
Klebsiella pneumoniae
strain, whereas a MIC of 6 μg/ml was recorded for its porin-expressing revertant. SME-1 carbapenemase was associated with an ertapenem MIC of 2 μg/ml for
Serratia marcescens
S6, compared with <0.03 μg/ml for
Serratia
strains lacking this enzyme. In summary, ertapenem had good activity against strains with potent β-lactamases, except for those with known carbapenemases.
Biochemical-Genetic Characterization of the Chromosomally Encoded Extended-Spectrum Class A β-Lactamase from
Rahnella aquatilis
Authors: Bellais Samuel , Poirel Laurent , Fortineau Nicolas , Decousser Jean W. , Nordmann Patrice
Published: July 2002

From whole-cell DNA of a clinical isolate of the enterobacterial species
Rahnella aquatilis
, a β-lactamase gene was cloned that encoded a chromosomally encoded Ambler class A enzyme, RAHN-1. RAHN-1, with a pI of 7.2, shares 76, 73, and 71% amino acid identity with the extended-spectrum β-lactamase of chromosomal origin from
Serratia fonticola
and with the plasmid-mediated β-lactamases CTX-M-2 and CTX-M-1, respectively. The hydrolysis spectrum of the clavulanic acid-inhibited RAHN-1 was expanded to cephalosporins such as cefuroxime, cefotaxime, and ceftriaxone, but not to ceftazidime. Its expression was not inducible.
Antimicrobial Susceptibility Testing of Clinical Isolates of
Bordetella pertussis
from Northern California: Report from the SENTRY Antimicrobial Surveillance Program
Authors: Gordon Kelley A. , Fusco Judy , Biedenbach Douglas J. , Pfaller Michael A. , Jones Ronald N.
Published: July 2002

Reports of an increased clinical incidence of pertussis and the development of resistance by
Bordetella pertussis
to erythromycin prompted the collection and testing of recent clinical isolates from patients in northern California against a range of antimicrobial agents by the Etest (AB BIODISK, Solna, Sweden) method. All isolates were fully susceptible to all eight agents tested (MIC, ≤0.38 μg/ml), including newer fluoroquinolones, such as gatifloxacin (MIC of which 90% of the isolates tested are inhibited, 0.006 μg/ml), which may be used in cases of adolescent or adult pertussis. Continued surveillance of
B. pertussis
isolates appears to be a prudent practice.
Inhibition of Cytochrome P450 (CYP450) Isoforms by Isoniazid: Potent Inhibition of CYP2C19 and CYP3A
Authors: Desta Zeruesenay , Soukhova Nadia V. , Flockhart David A.
Published: July 2002

Isoniazid (INH) remains the most safe and cost-effective drug for the treatment and prophylaxis of tuberculosis. The use of INH has increased over the past years, largely as a result of the coepidemic of human immunodeficiency virus infection. It is frequently given chronically to critically ill patients who are coprescribed multiple medications. The ability of INH to elevate the concentrations in plasma and/or toxicity of coadministered drugs, including those of narrow therapeutic range (e.g., phenytoin), has been documented in humans, but the mechanisms involved are not well understood. Using human liver microsomes (HLMs), we tested the inhibitory effect of INH on the activity of common drug-metabolizing human cytochrome P450 (CYP450) isoforms using isoform-specific substrate probe reactions. Incubation experiments were performed at a single concentration of each substrate probe at itsKmvalue with a range of INH concentrations. CYP2C19 and CYP3A were inhibited potently by INH in a concentration-dependent manner. At 50 μM INH (∼6.86 μg/ml), the activities of these isoforms decreased by ∼40%. INH did not show significant inhibition (<10% at 50 μM) of other isoforms (CYP2C9, CYP1A2, and CYP2D6). To accurately estimate the inhibition constants (Kivalues) for each isoform, four concentrations of INH were incubated across a range of five concentrations of specific substrate probes. The meanKivalues (± standard deviation) for the inhibition of CYP2C19 by INH in HLMs and recombinant human CYP2C19 were 25.4 ± 6.2 and 13 ± 2.4 μM, respectively. INH showed potent noncompetitive inhibition of CYP3A (Ki= 51.8 ± 2.5 to 75.9 ± 7.8 μM, depending on the substrate used). INH was a weak noncompetitive inhibitor of CYP2E1 (Ki= 110 ± 33 μM) and a competitive inhibitor of CYP2D6 (Ki= 126 ± 23 μM), but the meanKivalues for the inhibition of CYP2C9 and CYP1A2 were above 500 μM. Inhibition of one or both CYP2C19 and CYP3A isoforms is the likely mechanism by which INH slows the elimination of coadministered drugs, including phenytoin, carbamazepine, diazepam, triazolam, and primidone. Slow acetylators of INH may be at greater risk for adverse drug interactions, as the degree of inhibition was concentration dependent. These data provide a rational basis for understanding drug interaction with INH and predict that other drugs metabolized by these two enzymes may also interact.
In Vivo Effect of α
1
-Acid Glycoprotein on Pharmacokinetics of Amprenavir, a Human Immunodeficiency Virus Protease Inhibitor
Authors: Sadler Brian M. , Gillotin Catherine , Lou Yu , Stein Daniel S.
Published: July 2002

Observations from early clinical pharmacology studies of amprenavir, an inhibitor of human immunodeficiency virus type 1 (HIV-1) protease that is highly bound to human plasma proteins (∼90%), showed the single-dose pharmacokinetics of amprenavir to be variable between and within individuals. A cross-study analysis of various demographic, laboratory, and clinical covariates was therefore performed. Differences in amprenavir pharmacokinetics could be due to variable concentrations in α
1
-acid glycoprotein (AAG), the predominant plasma protein to which amprenavir binds. Therefore, AAG was considered an important factor to study since the literature suggested that AAG levels vary by race, age, and weight and following trauma or infection, including HIV disease. Pooled data from three single-dose studies analyzed by stepwise linear regression indicated that AAG concentrations significantly correlated with age and race and that only AAG concentrations were a significant predictor of amprenavir apparent total clearance (CL/F). A significant inverse linear relationship was found between AAG and amprenavir CL/F. Compared to white subjects, black subjects had significantly lower AAG concentrations and therefore significantly higher amprenavir CL/F. Although AAG has a significant influence on the variability of total drug pharmacokinetics, unbound, or free, drug concentrations are not affected by AAG concentrations. Incorrect conclusions could be drawn on the pharmacokinetics of highly protein-bound drugs if AAG concentration is not included in the analysis.
Overexpression and Characterization of the Chromosomal Aminoglycoside 2′-
N
-Acetyltransferase of
Providencia stuartii
Authors: Franklin Kristyn , Clarke Anthony J.
Published: July 2002

The gene coding for aminoglycoside 2′-
N
-acetyltransferase Ia [AAC(2′)-Ia] from
Providencia stuartii
was amplified by PCR and cloned. The resulting construct, pACKF2, was transferred into
Escherichia coli
for overexpression of AAC(2′)-Ia as a fusion protein with an N-terminal hexa-His tag. The fusion protein was isolated and purified by affinity chromatography on Ni
2+
-nitrilotriacetic acid agarose and gel permeation chromatography on Superdex 75. Comparison of the specific activity of this enzyme with that of its enterokinase-digested derivative lacking the His tag indicated that the presence of the extra N-terminal peptide does not affect activity. The temperature and pH optima for activity of both forms of the 2′-
N
-acetyltransferase were 20°C and pH 6.0, respectively, while the enzymes were most stable at 15°C and pH 8.1. The Michaelis-Menten kinetic parameters for AAC(2′)-Ia at 20°C and pH 6.0 were determined using a series of aminoglycoside antibiotics possessing a 2′-amino group and a concentration of acetyl coenzyme A fixed at 10 times its
K

m

value of 8.75 μM. Under these conditions, gentamicin was determined to be the best substrate for the enzyme in terms of both
K

m

and
k
cat
/
K

m

values, whereas neomycin was the poorest. Comparison of the kinetic parameters obtained with the different aminoglycosides indicated that their hexopyranosyl residues provided the most important binding sites for AAC(2′)-Ia activity, while the enzyme exhibits greater tolerance further from these sites. No correlation was found between these kinetic parameters and MICs determined for
P. stuartii
PR50 expressing the 2′-
N
-acetyltransferase, suggesting that its true in vivo function is not as a resistance factor.
Natural Antibiotic Susceptibilities of
Edwardsiella tarda
,
E. ictaluri,
and
E. hoshinae
Authors: Stock Ingo , Wiedemann Bernd
Published: July 2002

The natural antibiotic susceptibilities to 71 antibiotics of 102
Edwardsiella
strains belonging to
E. tarda
(
n
= 42),
E. ictaluri
(
n
= 41), and
E.
hoshinae
(
n
= 19) were investigated. MICs were determined using a microdilution procedure according to NCCLS criteria and German standards. All edwardsiellae were naturally sensitive to tetracyclines, aminoglycosides, most β-lactams, quinolones, antifolates, chloramphenicol, nitrofurantoin, and fosfomycin.
Edwardsiella
species were naturally resistant to macrolides, lincosamides, streptogramins, glycopeptides, rifampin, fusidic acid, and oxacillin. Although slight species-dependent differences in natural susceptibilities to some antibiotics (e.g., macrolides and cefaclor) were seen, differences in natural susceptibility affecting clinical assessment criteria were only seen with benzylpenicillin. Whereas
E. tarda
was naturally resistant to benzylpenicillin,
E. hoshinae
was naturally sensitive. Natural sensitivity and resistance to this penicillin were found among the strains of
E. ictaluri.
The observed oxacillin sensitivity of
E. ictaluri
was attributed to the failure of the species to grow at higher salt concentrations found in oxacillin-containing microtiter plates. The present study describes a database concerning the natural susceptibility of
Edwardsiella
species to a wide range of antibiotics, which can be applied to validate forthcoming antibiotic susceptibility tests of these microorganisms.
Randomized, Double-Blind, Multicenter Study of Caspofungin versus Amphotericin B for Treatment of Oropharyngeal and Esophageal Candidiases
Authors: Arathoon Eduardo G. , Gotuzzo Eduardo , Noriega L. Miguel , Berman Rayanne S. , DiNubile Mark J. , Sable Carole A.
Published: July 2002

Caspofungin is an antifungal agent of the novel echinocandin class. We investigated its efficacy, safety, and tolerability as therapy for oropharyngeal and/or esophageal candidiasis in a phase II dose-ranging study. Patients were randomized in a double-blind manner to receive either caspofungin acetate (35, 50, or 70 mg) or amphotericin B (0.5 mg/kg of body weight) intravenously once daily for 7 to 14 days. A favorable response required both complete resolution of symptoms and quantifiable improvement of mucosal lesions 3 to 4 days after discontinuation of study drug. Efficacy was assessed using a modified intent-to-treat analysis. No hypothesis testing of efficacy was planned or performed. Of 140 enrolled patients, 63% had esophageal involvement and 98% were infected with the human immunodeficiency virus (HIV) (median CD4 count, 30/mm
3
). A modestly higher proportion of patients in each of the caspofungin groups (74 to 91%) achieved favorable responses compared to amphotericin B recipients (63%), but there was considerable overlap in the 95% confidence intervals surrounding these point estimates. Similar trends were found in the subgroups with esophageal involvement, a history of fluconazole failure, and CD4 counts of ≤50/mm
3
. A smaller proportion of patients receiving any dose of caspofungin experienced drug-related adverse events compared to patients given standard doses of conventional amphotericin B (
P
< 0.01). Caspofungin provided a generally well-tolerated parenteral therapeutic option for HIV-infected patients with oropharyngeal and/or esophageal candidiasis in this study.
Levofloxacin Penetration into Epithelial Lining Fluid as Determined by Population Pharmacokinetic Modeling and Monte Carlo Simulation
Authors: Drusano G. L. , Preston S. L. , Gotfried M. H. , Danziger L. H. , Rodvold K. A.
Published: July 2002

Levofloxacin was administered orally to steady state to volunteers randomly in doses of 500 and 750 mg. Plasma and epithelial lining fluid (ELF) samples were obtained at 4, 12, and 24 h after the final dose. All data were comodeled in a population pharmacokinetic analysis employing BigNPEM. Penetration was evaluated from the population mean parameter vector values and from the results of a 1,000-subject Monte Carlo simulation. Evaluation from the population mean values demonstrated a penetration ratio (ELF/plasma) of 1.16. The Monte Carlo simulation provided a measure of dispersion, demonstrating a mean ratio of 3.18, with a median of 1.43 and a 95% confidence interval of 0.14 to 19.1. Population analysis with Monte Carlo simulation provides the best and least-biased estimate of penetration. It also demonstrates clearly that we can expect differences in penetration between patients. This analysis did not deal with inflammation, as it was performed in volunteers. The influence of lung pathology on penetration needs to be examined.
Molecular Evaluation of the Plasma Membrane Proton Pump fromAspergillus fumigatus
Authors: Burghoorn Henriette P. , Soteropoulos Patricia , Paderu Padmaja , Kashiwazaki Ryota , Perlin David S.
Published: July 2002

The gene encoding the plasma membrane proton pump (H+-ATPase) ofAspergillus fumigatus,PMA1, was characterized fromA. fumigatusstrain NIH 5233 and clinical isolate H11-20. An open reading frame of 3,109 nucleotides with two introns near the N terminus predicts a protein consisting of 989 amino acids with a molecular mass of approximately 108 kDa. The predictedA. fumigatusenzyme is 89 and 51% identical to H+- ATPases ofAspergillus nidulansandSaccharomyces cerevisiae, respectively. TheA. fumigatus PMA1is a typical member of the P-type ATPase family that contains 10 predicted transmembrane segments and conserved sequence motifs TGES, CSDKTGT, MLTGD, and GDGVN within the catalytic region. The enzyme represents 2% of the total plasma membrane protein, and it is characteristically inhibited by orthovanadate, with a 50% inhibitory concentration of ∼1.8 μM. H+-ATPases fromAspergillusspp. contain a highly acidic insertion region of 60 amino acids between transmembrane segments 2 and 3, which was confirmed for the membrane-assembled enzyme with a peptide-derived antibody. An increasingA. fumigatus PMA1copy number confers enhanced growth in low-pH medium, consistent with its role as a proton pump. These results provide support for the development of theA. fumigatusH+-ATPase as a potential drug discovery target.
Characterization of VariantSalmonellaGenomic Island 1 Multidrug Resistance Regions from Serovars Typhimurium DT104 and Agona
Authors: Boyd David , Cloeckaert Axel , Chaslus-Dancla Elisabeth , Mulvey Michael R.
Published: July 2002

Strains of multidrug-resistantSalmonella entericaserovar Typhimurium DT104 (DT104) andS. entericaserovar Agona (Agona) have been found to harborSalmonellagenomic island 1 (SGI1), a 43-kb genomic region that contains many of the drug resistance genes. Such strains are resistant to ampicillin (pse-1), chloramphenicol/florfenicol (floR), streptomycin/spectinomycin (aadA2), sulfonamides (sul1), and tetracycline [tet(G)] (commonly called the ACSSuT phenotype). All five resistance genes are found in a 13-kb multidrug resistance (MDR) region consisting of an unusual class I integron structure related to In4. We examined DT104 and Agona strains that exhibited other resistance phenotypes to determine if the resistance genes were associated with variant SGI1 MDR regions. All strains were found to harbor variant SGI1-like elements by using a combination of Southern hybridization, PCR mapping, and sequencing. Variant SGI1-like elements were found with MDR regions consisting of (i) an integron consisting of the SGI1 MDR region with the addition of a region containing a putative transposase gene (orf513) anddfrA10located between duplicatedqacEΔ1/sulIgenes (SGI1-A; ACSSuTTm); (ii) an integron with either anaadA2(SSu) or apse-1(ASu) cassette (SGI1-C and SGI1-B, respectively); (iii) an integron consisting of the SGI1-C MDR region plus an orf513/dfrA10region as in SGI1-A (SGI1-D; ASSuTm; ampicillin resistance due to a TEM β-lactamase); and (iv) an integron related to that in SGI1 but which contains a 10-kb inversion between two copies of IS6100, one which is inserted infloR(SGI1-E; ASSuT). We hypothesize that the MDR of SGI1 is subject to recombinational events that lead to the various resistance phenotypes in theSalmonellastrains in which it is found.
Comparative 16S rRNA Analysis of Lake Bacterioplankton Reveals Globally Distributed Phylogenetic Clusters Including an Abundant Group of Actinobacteria
Authors: Glöckner Frank Oliver , Zaichikov Evgeny , Belkova Natalia , Denissova Ludmilla , Pernthaler Jakob , Pernthaler Annelie , Amann Rudolf
Published: July 2002

In a search for cosmopolitan phylogenetic clusters of freshwater bacteria, we recovered a total of 190 full and partial 16S ribosomal DNA (rDNA) sequences from three different lakes (Lake Gossenköllesee, Austria; Lake Fuchskuhle, Germany; and Lake Baikal, Russia). The phylogenetic comparison with the currently available rDNA data set showed that our sequences fall into 16 clusters, which otherwise include bacterial rDNA sequences of primarily freshwater and soil, but not marine, origin. Six of the clusters were affiliated with the α, four were affiliated with the β, and one was affiliated with the γ subclass of the
Proteobacteria
; four were affiliated with the
Cytophaga-Flavobacterium-Bacteroides
group; and one was affiliated with the class
Actinobacteria
(formerly known as the high-G+C gram-positive bacteria). The latter cluster (hgcI) is monophyletic and so far includes only sequences directly retrieved from aquatic environments. Fluorescence in situ hybridization (FISH) with probes specific for the hgcI cluster showed abundances of up to 1.7 × 10
5
cells ml
−1
in Lake Gossenköllesee, with strong seasonal fluctuations, and high abundances in the two other lakes investigated. Cell size measurements revealed that
Actinobacteria
in Lake Gossenköllesee can account for up to 63% of the bacterioplankton biomass. A combination of phylogenetic analysis and FISH was used to reveal 16 globally distributed sequence clusters and to confirm the broad distribution, abundance, and high biomass of members of the class
Actinobacteria
in freshwater ecosystems.
Coliform Bacteria and Nitrogen Fixation in Pulp and Paper Mill Effluent Treatment Systems
Authors: Gauthier Francis , Neufeld Josh D. , Driscoll Brian T. , Archibald Frederick S.
Published: July 2002

The majority of pulp and paper mills now biotreat their combined effluents using activated sludge. On the assumption that their wood-based effluents have negligible fixed N, and that activated-sludge microorganisms will not fix significant N, these mills routinely spend large amounts adding ammonia or urea to their aeration tanks (bioreactors) to permit normal biomass growth. N
2
fixation in seven Eastern Canadian pulp and paper mill effluent treatment systems was analyzed using acetylene reduction assays, quantitative nitrogenase (
nifH
) gene probing, and bacterial isolations. In situ N
2
fixation was undetectable in all seven bioreactors but was present in six associated primary clarifiers. One primary clarifier was studied in greater detail. Approximately 50% of all culturable cells in the clarifier contained
nifH
, of which >90% were
Klebsiella
strains. All primary-clarifier coliform bacteria growing on MacConkey agar were identified as klebsiellas, and all those probed contained
nifH
. In contrast, analysis of 48 random coliform isolates from other mill water system locations showed that only 24 (50%) possessed the
nifH
gene, and only 13 (27%) showed inducible N
2
-fixing activity. Thus, all the pulp and paper mill primary clarifiers tested appeared to be sites of active N
2
fixation (0.87 to 4.90 mg of N liter
−1
day
−1
) and a microbial community strongly biased toward this activity. This may also explain why coliform bacteria, especially klebsiellas, are indigenous in pulp and paper mill water systems.
Biochemical and Genetic Characterization of Coagulin, a New Antilisterial Bacteriocin in the Pediocin Family of Bacteriocins, Produced by
Bacillus coagulans
I
4
Authors: Le Marrec Claire , Hyronimus Bertrand , Bressollier Philippe , Verneuil Bernard , Urdaci Maria C.
Published: July 2002

A plasmid-linked antimicrobial peptide, named coagulin, produced by
Bacillus coagulans
I
4
has recently been reported (B. Hyronimus, C. Le Marrec and M. C. Urdaci, J. Appl. Microbiol. 85:42–50, 1998). In the present study, the complete, unambiguous primary amino acid sequence of the peptide was obtained by a combination of both N-terminal sequencing of purified peptide and the complete sequence deduced from the structural gene harbored by plasmid I
4
. Data revealed that this peptide of 44 residues has an amino acid sequence similar to that described for pediocins AcH and PA-1, produced by different
Pediococcus acidilactici
strains and 100% identical. Coagulin and pediocin differed only by a single amino acid at their C terminus. Analysis of the genetic determinants revealed the presence, on the pI
4
DNA, of the entire 3.5-kb operon of four genes described for pediocin AcH and PA-1 production. No extended homology was observed between pSMB74 from
P. acidilactici
and pI
4
when analyzing the regions upstream and downstream of the operon. An oppositely oriented gene immediately dowstream of the bacteriocin operon specifies a 474-amino-acid protein which shows homology to Mob-Pre (plasmid recombination enzyme) proteins encoded by several small plasmids extracted from gram-positive bacteria. This is the first report of a pediocin-like peptide appearing naturally in a non-lactic acid bacterium genus.
Physiological Study of
Lactobacillus delbrueckii
subsp.
bulgaricus
Strains in a Novel Chemically Defined Medium
Authors: Chervaux Christian , Ehrlich S. Dusko , Maguin Emmanuelle
Published: July 2002

We developed a chemically defined medium called milieu proche du lait (MPL), in which 22
Lactobacillus delbrueckii
subsp.
bulgaricus
(
L. bulgaricus
) strains exhibited growth rates ranging from 0.55 to 1 h
−1
. MPL can also be used for cultivation of other lactobacilli and
Streptococcus thermophilus
. The growth characteristics of
L. bulgaricus
in MPL containing different carbon sources were determined, including an initial characterization of the phosphotransferase system transporters involved. For the 22 tested strains, growth on lactose was faster than on glucose, mannose, and fructose. Lactose concentrations below 0.4% were limiting for growth. We isolated 2-deoxyglucose-resistant mutants from strains CNRZ397 and ATCC 11842. CNRZ397-derived mutants were all deficient for glucose, fructose, and mannose utilization, indicating that these three sugars are probably transported via a unique mannose-specific-enzyme-II-like transporter. In contrast, mutants of ATCC 11842 exhibited diverse phenotypes, suggesting that multiple transporters may exist in that strain. We also developed a protein labeling method and verified that exopolysaccharide production and phage infection can occur in MPL. The MPL medium should thus be useful in conducting physiological studies of
L. bulgaricus
and other lactic acid bacteria under well controlled nutritional conditions.
A Tetrodotoxin-Producing
Vibrio
Strain, LM-1, from the Puffer Fish
Fugu vermicularis radiatus
Authors: Lee Myoung-Ja , Jeong Dong-Youn , Kim Woo-Seong , Kim Hyun-Dae , Kim Cheorl-Ho , Park Won-Whan , Park Yong-Ha , Kim Kyung-Sam , Kim Hyung-Min , Kim Dong-Soo
Published: July 2002

Identification of tetrodotoxin (TTX) and its derivatives produced from a
Vibrio
strain in the intestine of the puffer fish
Fugu vermicularis radiatus
was performed by thin-layer chromatography, electrophoresis, high-performance liquid chromatography, and gas chromatography-mass spectrometry, together with a mouse bioassay for toxicity. It was demonstrated that the isolated bacterium produced TTX, 4-epi-TTX, and anhTTX during cultivation, suggesting that
Vibrio
strains are responsible for the toxification of the puffer fish.
Microbiological Transformation of Enrofloxacin by the Fungus
Mucor ramannianus
Authors: Parshikov Igor A. , Freeman James P. , Lay Jackson O. , Beger Richard D. , Williams Anna J. , Sutherland John B.
Published: July 2002

Enrofloxacin metabolism by
Mucor ramannianus
was investigated as a model for the biotransformation of veterinary fluoroquinolones. Cultures grown in sucrose-peptone broth were dosed with enrofloxacin. After 21 days, 22% of the enrofloxacin remained. Three metabolites were identified: enrofloxacin
N
-oxide (62% of the total absorbance),
N
-acetylciprofloxacin (8.0%), and desethylene-enrofloxacin (3.5%).
Overexpression of Protein Disulfide Isomerase DsbC Stabilizes Multiple-Disulfide-Bonded Recombinant Protein Produced and Transported to the Periplasm in
Escherichia coli
Authors: Kurokawa Yoichi , Yanagi Hideki , Yura Takashi
Published: July 2002

Dsb proteins (DsbA, DsbB, DsbC, and DsbD) catalyze formation and isomerization of protein disulfide bonds in the periplasm of
Escherichia coli
. By using a set of Dsb coexpression plasmids constructed recently, we analyzed the effects of Dsb overexpression on production of horseradish peroxidase (HRP) isozyme C that contains complex disulfide bonds and tends to aggregate when produced in
E. coli
. When transported to the periplasm, HRP was unstable but was markedly stabilized upon simultaneous overexpression of the set of Dsb proteins (DsbABCD). Whereas total HRP production increased severalfold upon overexpression of at least disulfide-bonded isomerase DsbC, maximum transport of HRP to the periplasm seemed to require overexpression of all DsbABCD proteins, suggesting that excess Dsb proteins exert synergistic effects in assisting folding and transport of HRP. Periplasmic production of HRP also increased when calcium, thought to play an essential role in folding of nascent HRP polypeptide, was added to the medium with or without Dsb overexpression. These results suggest that Dsb proteins and calcium play distinct roles in periplasmic production of HRP, presumably through facilitating correct folding. The present Dsb expression plasmids should be useful in assessing and dissecting periplasmic production of proteins that contain multiple disulfide bonds in
E. coli
.
Development of a Combined Selection and Enrichment PCR Procedure for
Clostridium botulinum
Types B, E, and F and Its Use To Determine Prevalence in Fecal Samples from Slaughtered Pigs
Authors: Dahlenborg Maria , Borch Elisabeth , Rådström Peter
Published: July 2002

A specific and sensitive combined selection and enrichment PCR procedure was developed for the detection of
Clostridium botulinum
types B, E, and F in fecal samples from slaughtered pigs. Two enrichment PCR assays, using the DNA polymerase
rTth,
were constructed. One assay was specific for the type B neurotoxin gene, and the other assay was specific for the type E and F neurotoxin genes. Based on examination of 29 strains of
C. botulinum
, 16 strains of other
Clostridium
spp., and 48 non-
Clostridium
strains, it was concluded that the two PCR assays detect
C. botulinum
types B, E, and F specifically. Sample preparation prior to the PCR was based on heat treatment of feces homogenate at 70°C for 10 min, enrichment in tryptone-peptone-glucose-yeast extract broth at 30°C for 18 h, and DNA extraction. The detection limits after sample preparation were established as being 10 spores per g of fecal sample for nonproteolytic type B, and 3.0 × 10
3
spores per g of fecal sample for type E and nonproteolytic type F with a detection probability of 95%. Seventy-eight pig fecal samples collected from slaughter houses were analyzed according to the combined selection and enrichment PCR procedure, and 62% were found to be PCR positive with respect to the type B neurotoxin gene. No samples were positive regarding the type E and F neurotoxin genes, indicating a prevalence of less than 1.3%. Thirty-four (71%) of the positive fecal samples had a spore load of less than 4 spores per g. Statistical analysis showed that both rearing conditions (outdoors and indoors) and seasonal variation (summer and winter) had significant effects on the prevalence of
C. botulinum
type B, whereas the effects of geographical location (southern and central Sweden) were less significant.
Concentrations of Copper Thought To Be Toxic to
Escherichia coli
Can Induce the Viable but Nonculturable Condition
Authors: Grey Brian , Steck Todd R.
Published: July 2002

We have determined that concentrations of copper considered to be toxic can induce a fraction of a population of
Escherichia coli
to enter the viable but nonculturable (VBNC) condition. Copper-induced VBNC cells could be resuscitated for up to 2 weeks after entering the VBNC state.
Isolation from Agricultural Soil and Characterization of a
Sphingomonas
sp. Able To Mineralize the Phenylurea Herbicide Isoproturon
Authors: Sørensen Sebastian R. , Ronen Zeev , Aamand Jens
Published: July 2002

A soil bacterium (designated strain SRS2) able to metabolize the phenylurea herbicide isoproturon, 3-(4-isopropylphenyl)-1,1-dimethylurea (IPU), was isolated from a previously IPU-treated agricultural soil. Based on a partial analysis of the 16S rRNA gene and the cellular fatty acids, the strain was identified as a
Sphingomonas
sp. within the α-subdivision of the proteobacteria. Strain SRS2 was able to mineralize IPU when provided as a source of carbon, nitrogen, and energy. Supplementing the medium with a mixture of amino acids considerably enhanced IPU mineralization. Mineralization of IPU was accompanied by transient accumulation of the metabolites 3-(4-isopropylphenyl)-1-methylurea, 3-(4-isopropylphenyl)-urea, and 4-isopropyl-aniline identified by high-performance liquid chromatography analysis, thus indicating a metabolic pathway initiated by two successive N-demethylations, followed by cleavage of the urea side chain and finally by mineralization of the phenyl structure. Strain SRS2 also transformed the dimethylurea-substituted herbicides diuron and chlorotoluron, giving rise to as-yet-unidentified products. In addition, no degradation of the methoxy-methylurea-substituted herbicide linuron was observed. This report is the first characterization of a pure bacterial culture able to mineralize IPU.
Characterization of Acetic Acid Bacteria in Traditional Acetic Acid Fermentation of Rice Vinegar (Komesu) and Unpolished Rice Vinegar (Kurosu) Produced in Japan
Authors: Nanda Kumiko , Taniguchi Mariko , Ujike Satoshi , Ishihara Nobuhiro , Mori Hirotaka , Ono Hisayo , Murooka Yoshikatsu
Published: July 2002

Bacterial strains were isolated from samples of Japanese rice vinegar (komesu) and unpolished rice vinegar (kurosu) fermented by the traditional static method. Fermentations have never been inoculated with a pure culture since they were started in 1907. A total of 178 isolates were divided into groups A and B on the basis of enterobacterial repetitive intergenic consensus-PCR and random amplified polymorphic DNA fingerprinting analyses. The 16S ribosomal DNA sequences of strains belonging to each group showed similarities of more than 99% with
Acetobacter pasteurianus
. Group A strains overwhelmingly dominated all stages of fermentation of both types of vinegar. Our results indicate that appropriate strains of acetic acid bacteria have spontaneously established almost pure cultures during nearly a century of komesu and kurosu fermentation.
Chromatic Adaptation in Marine
Synechococcus
Strains
Authors: Palenik Brian
Published: July 2002

Characterization of two genetically distinct groups of marine
Synechococcus
sp. strains shows that one, but not the other, increases its phycourobilin/phycoerythrobilin chromophore ratio when growing in blue light. This ability of at least some marine
Synechococcus
strains to chromatically adapt may help explain their greater abundance in particular ocean environments than cyanobacteria of the genus
Prochlorococcus
.
Identification and Characterization of Integron-Mediated Antibiotic Resistance among Shiga Toxin-Producing
Escherichia coli
Isolates
Authors: Zhao Shaohua , White David G. , Ge Beilei , Ayers Sherry , Friedman Sharon , English Linda , Wagner David , Gaines Stuart , Meng Jianghong
Published: July 2002

A total of 50 isolates of Shiga toxin-producing
Escherichia coli
(STEC), including 29 O157:H7 and 21 non-O157 STEC strains, were analyzed for antimicrobial susceptibilities and the presence of class 1 integrons. Seventy-eight (
n
= 39) percent of the isolates exhibited resistance to two or more antimicrobial classes. Multiple resistance to streptomycin, sulfamethoxazole, and tetracycline was most often observed. Class 1 integrons were identified among nine STEC isolates, including serotypes O157:H7, O111:H11, O111:H8, O111:NM, O103:H2, O45:H2, O26:H11, and O5:NM. The majority of the amplified integron fragments were 1 kb in size with the exception of one
E. coli
O111:H8 isolate which possessed a 2-kb amplicon. DNA sequence analysis revealed that the integrons identified within the O111:H11, O111:NM, O45:H2, and O26:H11 isolates contained the
aadA
gene encoding resistance to streptomycin and spectinomycin. Integrons identified among the O157:H7 and O103:H2 isolates also possessed a similar
aadA
gene. However, DNA sequencing revealed only 86 and 88% homology, respectively. The 2-kb integron of the
E. coli
O111:H8 isolate contained three genes,
dfr
XII,
aadA
2, and a gene of unknown function,
orfF
, which were 86, 100, and 100% homologous, respectively, to previously reported gene cassettes identified in integrons found in
Citrobacter freundii
and
Klebsiella pneumoniae
. Furthermore, integrons identified among the O157:H7 and O111:NM strains were transferable via conjugation to another strain of
E. coli
O157:H7 and to several strains of
Hafnia alvei
. To our knowledge, this is the first report of integrons and antibiotic resistance gene cassettes in STEC, in particular
E. coli
O157:H7.
Phylogenetic Diversity of Bacterial and Archaeal Communities in the Anoxic Zone of the Cariaco Basin
Authors: Madrid Vanessa M. , Taylor Gordon T. , Scranton Mary I. , Chistoserdov Andrei Y.
Published: July 2002

Microbial community samples were collected from the anoxic zone of the Cariaco Basin at depths of 320, 500, and 1,310 m on a November 1996 cruise and were used to construct 16S ribosomal DNA libraries. Of 60 nonchimeric sequences in the 320-m library, 56 belonged to the ɛ subdivision of the
Proteobacteria
(ɛ-
Proteobacteria
) and 53 were closely related to ectosymbionts of
Rimicaris exoculata
and
Alvinella pompejana
, which are referred to here as epsilon symbiont relatives (ESR). The 500-m library contained sequences affiliated with the fibrobacteria, the
Flexibacter-Cytophaga-Bacteroides
division, the division
Verrucomicrobia
, the division
Proteobacteria
, and the OP3 candidate division. The Proteobacteria included members of the γ, δ, ɛ and new candidate subdivisions, and γ-proteobacterial sequences were dominant (25.6%) among the proteobacterial sequences. As in the 320-m library, the majority of the ɛ-proteobacteria belonged to the ESR group. The genus
Fibrobacter
and its relatives were the second largest group in the library (23.6%), followed by the δ-proteobacteria and the ɛ-proteobacteria. The 1,310-m library had the greatest diversity; 59 nonchimeric clones in the library contained 30 unique sequences belonging to the planctomycetes, the fibrobacteria, the
Flexibacter-Cytophaga-Bacteroides
division, the
Proteobacteria
, and the OP3 and OP8 candidate divisions. The proteobacteria included members of new candidate subdivisions and the β, γ, δ, and ɛ-subdivisions. ESR sequences were still present in the 1,310-m library but in a much lower proportion (8.5%). One archaeal sequence was present in the 500-m library (2% of all microorganisms in the library), and eight archaeal sequences were present in the 1,310-m library (13.6%). All archaeal sequences fell into two groups; two clones in the 1,310-m library belonged to the kingdom
Crenarchaeota
and the remaining sequences in both libraries belonged to the kingdom
Euryarchaeota
. The latter group appears to be related to the Eel-TA1f2 sequence, which belongs to an archaeon suggested to be able to oxidize methane anaerobically. Based on phylogenetic inferences and measurements of dark CO
2
fixation, we hypothesized that (i) the ESR are autotrophic anaerobic sulfide oxidizers, (ii) sulfate reduction and fermentative metabolism may be carried out by a large number of bacteria in the 500- and 1,310-m libraries, and (iii) members of the
Euryarchaeota
found in relatively large numbers in the 1,310-m library may be involved in anaerobic methane oxidation. Overall, the composition of microbial communities from the Cariaco Basin resembles the compositions of communities from several anaerobic sediments, supporting the hypothesis that the Cariaco Basin water column is similar to anaerobic sediments.
Hydrolysis of 4-Hydroxybenzoic Acid Esters (Parabens) and Their Aerobic Transformation into Phenol by the Resistant
Enterobacter cloacae
Strain EM
Authors: Valkova Nelly , Lépine François , Valeanu Loredana , Dupont Maryse , Labrie Louisette , Bisaillon Jean-Guy , Beaudet Réjean , Shareck François , Villemur Richard
Published: July 2002

Enterobacter cloacae
strain EM was isolated from a commercial dietary mineral supplement stabilized by a mixture of methylparaben and propylparaben. It harbored a high-molecular-weight plasmid and was resistant to high concentrations of parabens. Strain EM was able to grow in liquid media containing similar amounts of parabens as found in the mineral supplement (1,700 and 180 mg of methyl and propylparaben, respectively, per liter or 11.2 and 1.0 mM) and in very high concentrations of methylparaben (3,000 mg liter
−1
, or 19.7 mM). This strain was able to hydrolyze approximately 500 mg of methyl-, ethyl-, or propylparaben liter
−1
(3 mM) in less than 2 h in liquid culture, and the supernatant of a sonicated culture, after a 30-fold dilution, was able to hydrolyze 1,000 mg of methylparaben liter
−1
(6.6 mM) in 15 min. The first step of paraben degradation was the hydrolysis of the ester bond to produce 4-hydroxybenzoic acid, followed by a decarboxylation step to produce phenol under aerobic conditions. The transformation of 4-hydroxybenzoic acid into phenol was stoichiometric. The conversion of approximately 500 mg of parabens liter
−1
(3 mM) to phenol in liquid culture was completed within 5 h without significant hindrance to the growth of strain EM, while higher concentrations of parabens partially inhibited its growth.
Requirement for Phosphoglucomutase in Exopolysaccharide Biosynthesis in Glucose- and Lactose-Utilizing
Streptococcus thermophilus
Authors: Levander Fredrik , Rådström Peter
Published: July 2002

To study the influence of phosphoglucomutase (PGM) activity on exopolysaccharide (EPS) synthesis in glucose- and lactose-growing
Streptococcus thermophilus
, a knockout PGM mutant and a strain with elevated PGM activity were constructed. The
pgmA
gene, encoding PGM in
S. thermophilus
LY03, was identified and cloned. The gene was functional in
Escherichia coli
and was shown to be expressed from its own promoter. The
pgmA
-deficient mutant was unable to grow on glucose, while the mutation did not affect growth on lactose. Overexpression of
pgmA
had no significant effect on EPS production in glucose-growing cells. Neither deletion nor overexpression of
pgmA
changed the growth or EPS production on lactose. Thus, the EPS precursors in lactose-utilizing
S. thermophilus
are most probably formed from the galactose moiety of lactose via the Leloir pathway, which circumvents the need for a functional PGM.