bluuebunny/crossref_metadata_2025 huggingface dataset:
In Vitro and In Vivo Potentiation of Artemisinin and Synthetic Endoperoxide Antimalarial Drugs by Metalloporphyrins
Authors: Benoit-Vical Françoise , Robert Anne , Meunier Bernard
Published: July 2002

The in vitro potentiation of artemisinin by synthetic manganese porphyrin complexes has been recently reported (F. Benoit-Vical, A. Robert, and B. Meunier, Antimicrob. Agents Chemother. 43:2555–2558, 1999). Since the activity of artemisinin and synthetic antimalarial endoperoxides is related to their interaction with heme (S. R. Meshnick, A. Thomas, A. Ranz, C. M. Xu, and H. Z. Pan, Mol. Biochem. Parasitol. 49:181–190, 1991), an improvement of their efficiency may be expected in the presence of a synthetic metalloporphyrin having the same activating role as endogenous heme. With the aim to boost the activity of antimalarial endoperoxide drugs, we were thus led to evaluate the in vitro and in vivo potentiation of natural and synthetic drugs of this family by a nontoxic and cheap metalloporphyrin. The potentiation of artemisinin, β-artemether, and arteflene (Ro 42-1611) by synthetic heme models is reported. In vitro studies on the chloroquine-resistant
Plasmodium falciparum
FcB1-Columbia strain indicate a synergistic effect of the manganese complex of
meso
-tetrakis(4-sulfonatophenylporphyrin) (Mn-TPPS) on the activity of artemisinin or β-artemether, whereas this heme model has no influence on the activity of arteflene. A significant synergistic effect on rodent malaria was also observed in vivo between artemisinin and Mn-TPPS using
Plasmodium vinckei petteri
strain.
Fluoroquinolone Resistance in Clinical Isolates of
Streptococcus pneumoniae
: Contributions of Type II Topoisomerase Mutations and Efflux to Levels of Resistance
Authors: Bast Darrin J. , Low Donald E. , Duncan Carla L. , Kilburn Laurie , Mandell Lionel A. , Davidson Ross J. , de Azavedo Joyce C. S.
Published: July 2002

We report on amino acid substitutions in the quinolone resistance-determining region of type II topisomerases and the prevalence of reserpine-inhibited efflux for 70 clinical isolates of
S. pneumoniae
for which the ciprofloxacin MIC is ≥4 μg/ml and 28 isolates for which the ciprofloxacin MIC is ≤2 μg/ml. The amino acid substitutions in ParC conferring low-level resistance (MICs, 4 to 8 μg/ml) included Phe, Tyr, and Ala for Ser-79; Asn, Ala, Gly, Tyr, and Val for Asp-83; Asn for Asp-78; and Pro for Ala-115. Isolates with intermediate-level (MICs, 16 to 32 μg/ml) and high-level (MICs, 64 μg/ml) resistance harbored substitutions of Phe and Tyr for Ser-79 or Asn and Ala for Asp-83 in ParC and an additional substitution in GyrA which included either Glu-85-Lys (Gly) or Ser-81-Phe (Tyr). Glu-85-Lys was found exclusively in isolates with high-level resistance. Efflux contributed primarily to low-level resistance in isolates with or without an amino acid substitution in ParC. The impact of amino acid substitutions in ParE was minimal, and no substitutions in GyrB were identified.
Effects of the Movement of Insertion Sequences on the Structure of VanA Glycopeptide Resistance Elements in
Enterococcus faecium
Authors: Darini Ana L. C. , Palepou Marie-France I. , Woodford Neil
Published: July 2002

A Tn
1546
-related element with IS
1216V
at position 8839 underwent a structural change after storage of the host strain of
Enterococcus faecium
at 4°C. The element acquired IS
1542
at position 3932, nucleotides 8732 to 8831 were deleted, and the first 3417 nucleotides were lost and replaced by an inverted copy of the IS
1216V–vanY–vanZ
-inverted-repeat block from the 3′ end. Insertion sequence movement is likely to play a key role in the evolution of VanA resistance elements.
In Vitro Activities of Daptomycin, Arbekacin, Vancomycin, and Gentamicin Alone and/or in Combination against Glycopeptide Intermediate-Resistant
Staphylococcus aureus
in an Infection Model
Authors: Akins Ronda L. , Rybak Michael J.
Published: July 2002

Daptomycin, a lipopeptide antibiotic, has broad activity against gram-positive organisms, similar to vancomycin; however, its mechanism of action differs, resulting in interference with cell membrane transport and a more rapid bactericidal activity. In light of increasing need for alternative treatments against intermediate-resistant
Staphylococcus aureus
, there is revitalized interest in this antibiotic. We, therefore, evaluated the activity of daptomycin alone or in combination in an in vitro infection model against two glycopeptide intermediate-resistant
S. aureus
(GISA) isolates. Newly designed regimens of daptomycin at 4 and 6 mg/kg of body weight every 24 h (q24h) were compared to the previous regimen of 3 mg/kg q12h. Daptomycin MICs and minimal bactericidal concentrations (MBCs) (MIC/MBC) for Mu-50, HIP5836 (992), and MRSA-67 were 0.5/1.0, 0.5/1.0, and 0.125/0.5 μg/ml, respectively. MICs and MBCs of arbekacin for the three strains were 2.0/8.0, 0.125/0.5, and 0.125/0.25 μg/ml, respectively. Vancomycin and gentamicin MICs and MBCs for the three strains were 8.0/8.0, 8.0/8.0, and 0.5/1.0 μg/ml and 128/128, 0.5/1.0, and 0.25/0.5 μg/ml, respectively. Our experience with daptomycin in an in vitro infection model has shown significant kill against the two GISA strains (Mu-50 and 992) (
P
< 0.03). We also noted that kill was related to a total dose effect for 992, in which simulated daptomycin in vivo dosages of 6 mg/kg q24h and 3 mg/kg q12h produced similar kill and 4 mg/kg q24h resulted in significant regrowth (
P
≤ 0.05). Combination therapy with arbekacin resulted in synergistic activity against Mu-50. Daptomycin area under the concentration-time curve/MIC and
C
max
/MIC ranges for GISA isolates were 80 to 116 and 6 to 12, respectively, and ranges for MRSA-67 were 320 to 461 and 24 to 48, respectively, and appeared to have an association with kill (i.e., decreased CFU/milliliter) at 24 and 48 h. Therefore, these experiments suggest that daptomycin alone or in combination could provide an alternative for the treatment of GISA.
Susceptibility Testing of
Malassezia
Species Using the Urea Broth Microdilution Method
Authors: Nakamura Yuka , Kano Rui , Murai Tae , Watanabe Shinichi , Hasegawa Atsuhiko
Published: July 2002

A urea broth microdilution method to assay the susceptibilities of seven
Malassezia
species was developed. This method indicated the same sensitivities as the agar plate dilution method for isolates of
Malassezia furfur
,
M. pachydermatis
,
M. slooffiae
, and
M. sympodialis
.
Analysis of Ciprofloxacin Activity against
Streptococcus pneumoniae
after 10 Years of Use in the United States
Authors: Sahm Daniel F. , Peterson Dan E. , Critchley Ian A. , Thornsberry Clyde
Published: July 2002

As the most commonly used fluoroquinolone in the United States since 1987, ciprofloxacin has exerted the greatest selective pressure on
S. pneumoniae
and provides a valuable marker to evaluate the actual and potential emergence of fluoroquinolone resistance in this species. Analysis of susceptibility results obtained with 5,640 strains collected from throughout the United States showed that only 16 (0.3%) of the isolates demonstrated MICs of ≥4 μg/ml. The prevalence of this phenotype was significantly higher (
P
< 0.05) among penicillin-resistant populations, among isolates from patients >64 years old, and among respiratory isolates. However, >99% of strains had MICs of <4 μg/ml regardless of the risk group examined, and the MIC population distributions were the same for each risk group. These findings demonstrate that the phenotype of a MIC of ≥4 μg/ml remains uncommon after 10 years of ciprofloxacin use; however, these findings are no reason to become complacent with regard to appropriate use of fluoroquinolones and the need to carefully track resistance trends. Equally important is careful analysis of data that result from surveillance in terms of risk factors and other associated trends so that resistance and susceptibility, and their consequences, are neither over- nor underestimated.
Inducible Azole Resistance Associated with a Heterogeneous Phenotype in
Candida albicans
Authors: Marr Kieren A. , Lyons Christopher N. , Ha Kien , Rustad Tige R. , White Theodore C.
Published: July 2002

The development of azole resistance in
Candida albicans
is most problematic in patients with AIDS who receive long courses of drug for therapy or prevention of oral candidiasis. Recently, the rapid development of resistance was noted in other immunosuppressed patients who developed disseminated candidiasis despite fluconazole prophylaxis. One of these series of
C. albicans
isolates became resistant, with an associated increase in mRNA specific for a
CDR
ATP-binding cassette transporter efflux pump (K. A. Marr, C. N. Lyons, T. R. Rustad, R. A. Bowden, and T. C. White, Antimicrob. Agents Chemother. 42:2584–2589, 1998). Here we study this series of
C. albicans
isolates further and examine the mechanism of azole resistance in a second series of
C. albicans
isolates that caused disseminated infection in a recipient of bone marrow transplantation. The susceptible isolates in both series become resistant to fluconazole after serial growth in the presence of drug, while the resistant isolates in both series become susceptible after serial transfer in the absence of drug. Population analysis of the inducible, transiently resistant isolates reveals a heterogeneous population of fluconazole-susceptible and -resistant cells. We conclude that the rapid development of azole resistance occurs by a mechanism that involves selection of a resistant clone from a heterogeneous population of cells.
High Therapeutic Index of Factor C Sushi Peptides: Potent Antimicrobials against
Pseudomonas aeruginosa
Authors: Yau Yin Hoe , Ho Bow , Tan Nguan Soon , Ng Miang Lon , Ding Jeak Ling
Published: July 2002

Factor C protein isolated from the horseshoe crab,
Carcinoscorpius rotundicauda
, has endotoxin binding capability. Synthetic peptides of 34 amino acids based on the sequence of two regions of factor C (Sushi 1 and Sushi 3) as well as their corresponding mutants exhibited activities against 30 clinical isolates of
Pseudomonas aeruginosa
. Collectively, all four peptides demonstrated exceptionally effective bactericidal activity against
P. aeruginosa
with 90% minimal bactericidal concentrations (MBC
90
s) in the range of 0.06 to 0.25 μg/ml (16 to 63 nM). Viable bacteria were reduced by 90% after 7 min and were totally eradicated within 40 to 50 min. These peptides are minimally hemolytic against both rabbit and human erythrocytes even at concentrations up to 1,600-fold their MBC
90
s. Both in vitro and in vivo studies indicate that cytotoxic effects are small even at 1,000-fold their MBC
90
s. Furthermore, the Sushi peptides are tolerant of high-salt and adverse pH conditions. These findings demonstrate the promising therapeutic potential of the Sushi peptides.
Compartmental Pharmacokinetics and Tissue Distribution of the Antifungal Echinocandin Lipopeptide Micafungin (FK463) in Rabbits
Authors: Groll Andreas H. , Mickiene Diana , Petraitis Vidmantas , Petraitiene Ruta , Ibrahim Khalid H. , Piscitelli Stephen C. , Bekersky Ihor , Walsh Thomas J.
Published: July 2002

The plasma pharmacokinetics and tissue distribution of the novel antifungal echinocandin-like lipopeptide micafungin (FK463) were investigated in healthy rabbits. Cohorts of three animals each received micafungin at 0.5, 1, and 2 mg/kg of body weight intravenously once daily for a total of 8 days. Serial plasma samples were collected on days 1 and 7, and tissue samples were obtained 30 min after the eighth dose. Drug concentrations were determined by validated high-performance liquid chromatographic methods. Plasma drug concentration data were fit to a two-compartment pharmacokinetic model, and pharmacokinetic parameters were estimated using weighted nonlinear least-square regression analysis. Micafungin demonstrated linear plasma pharmacokinetics without changes in total clearance and dose-normalized area under the concentration-time curve from 0 h to infinity. After administration of single doses to the rabbits, mean peak plasma drug concentrations ranged from 7.62 μg/ml at 0.5 mg/kg to 16.8 μg/ml at 2 mg/kg, the area under the concentration-time curve from 0 to 24 h ranged from 5.66 to 21.79 μg · h/ml, the apparent volume of distribution at steady state ranged from 0.296 to 0.343 liter/kg, and the elimination half-life ranged from 2.97 to 3.20 h, respectively. No significant changes in pharmacokinetic parameters and no accumulation was noted after multiple dosing. Mean tissue micafungin concentrations 30 min after the last of eight daily doses were highest in the lung (2.26 to 11.76 μg/g), liver (2.05 to 8.82 μg/g), spleen (1.87 to 9.05 μg/g), and kidney (1.40 to 6.12 μg/g). While micafungin was not detectable in cerebrospinal fluid, the concentration in brain tissue ranged from 0.08 to 0.18 μg/g. These findings indicate linear disposition of micafungin at dosages of 0.5 to 2 mg/kg and achievement of potentially therapeutic drug concentrations in plasma and tissues that are common sites of invasive fungal infections.
Rapid and Simple Phenotypic Assay for Drug Susceptibility of Human Immunodeficiency Virus Type 1 Using CCR5-Expressing HeLa/CD4
+
Cell Clone 1-10 (MAGIC-5)
Authors: Hachiya Atsuko , Aizawa-Matsuoka Saori , Tanaka Mari , Takahashi Yukiko , Ida Setsuko , Gatanaga Hiroyuki , Hirabayashi Yoshihiro , Kojima Asato , Tatsumi Masashi , Oka Shinichi
Published: July 2002

We describe a rapid and simple novel phenotypic assay for drug susceptibility of human immunodeficiency virus type-1 (HIV-1) using a CCR5-expressing HeLa/CD4
+
cell clone 1-10 (MAGIC-5). MAGIC-5 cells produced large amounts of HIV-1 in culture supernatants, which enabled us to perform the phenotypic resistance assay. Determination of HIV-1 susceptibility to various protease inhibitors (PI) and nucleoside reverse transcriptase inhibitors was completed within 15 days in T-cell-tropic (X4) and macrophage-tropic (R5) viruses using fresh plasma samples containing at least 10
4
copies/ml. The nucleotide sequence of the envelope V3 region of HIV-1 in plasma was almost identical to that of the virus isolated by MAGIC-5 cells, suggesting a lack of selection bias in our assay. The assay variability was confined to within five-fold in all drugs examined. Accordingly, we used a 10-fold increase in the 50% inhibitory concentration as the cutoff value for viral resistance in the present assay. HIV-1 resistant to lamivudine, which was not detected by conventional genotypic assays, was isolated. In HIV-1 with PI-associated primary amino acid substitutions, our assay showed that drug resistance profiles correlated well with previously reported genotypic-assay data. Furthermore, our assay provided comprehensive results regarding PI resistance in the presence of multiple mutations. The novel assay successfully quantified the level of resistance of clinical HIV-1 isolates to a battery of anti-HIV drugs, indicating its clinical usefulness, particularly in patients who failed to respond to antiretroviral chemotherapy.
Characterization of OXA-25, OXA-26, and OXA-27, Molecular Class D β-Lactamases Associated with Carbapenem Resistance in Clinical Isolates of
Acinetobacter baumannii
Authors: Afzal-Shah Mariya , Woodford Neil , Livermore David M.
Published: July 2002

Carbapenem resistance in
Acinetobacter
spp. is increasingly being associated with OXA-type β-lactamases with weak hydrolytic activity against imipenem and meropenem. Such enzymes were characterized from
Acinetobacter
isolates collected in Belgium, Kuwait, Singapore, and Spain. The isolates from Spain and Belgium had novel class D β-lactamases that were active against carbapenems. These were designated OXA-25 and OXA-26, respectively, and had >98% amino acid homology with each other and with the OXA-24 enzyme recently described by others from an
Acinetobacter
isolate collected elsewhere in Spain. The isolate from Singapore had OXA-27 β-lactamase, another novel class D type with only 60% homology to OXA-24, -25, and -26, but with 99% homology to OXA-23 (ARI-1), described previously from an
Acinetobacter baumannii
isolate collected in Scotland. Sequence data were not obtained for the carbapenem-hydrolyzing OXA enzyme from the isolate from Kuwait; nevertheless, the enzyme was phenotypically similar to OXA-25 and -26. The enzymes OXA-23, -24, -25, -26, and -27 retained the STFK and SXV motifs typical of class D β-lactamases, but the YGN motif was altered to FGN. The KTG motif was retained by OXA-27 and -23 but was replaced by KSG in OXA-24, -25, and -26. OXA-25 and -26 enzymes were strongly active against oxacillin, but unusually for an OXA-type β-lactamase, OXA-27 had apparently weak activity, although measurement was complicated by biphasic kinetics. None of the new enzymes was transmissible to
Escherichia coli
recipients. Many
Acinetobacter
isolates are multiresistant to other antibiotics, and the emergence of class D enzymes with carbapenem-hydrolyzing activity is a disturbing development for antimicrobial chemotherapy.
Comparative Toxicities and Pharmacokinetics of Intrathecal Lipid (Amphotericin B Colloidal Dispersion) and Conventional Deoxycholate Formulations of Amphotericin B in Rabbits
Authors: Clemons Karl V. , Sobel Raymond A. , Williams Paul L. , Stevens David A.
Published: July 2002

The lipid formulation of amphotericin B, Amphotec (ABCD), has not been used intrathecally. After a single intrathecal dose or after four doses, conventionally formulated deoxycholate amphotericin B (AMB) (Fungizone) resulted in higher levels of amphotericin B in the cerebrospinal fluid of rabbits than did ABCD. Clinically and histologically, ABCD was about threefold less toxic than AMB after a single dose and 3- to 30-fold less toxic after multiple dosing. These data are encouraging for the potential use of ABCD as an intrathecal treatment.
Identification of β-Lactamase-Negative, Ampicillin-Resistant Strains of
Haemophilus influenzae
with Four Methods and Eight Media
Authors: Barry Arthur L. , Fuchs Peter C. , Brown Steven D.
Published: July 2002

A challenge set of 143 non-β-lactamase-producing strains of
Haemophilus influenzae
was tested for ampicillin susceptibility on two broth media and six agar media, using broth microdilution, agar dilution, disk diffusion, and E-test procedures. When β-lactamase-negative, ampicillin-resistant (BLNAR) strains were defined as those for which the ampicillin MIC was ≥4.0 μg/ml, 5 to 44% of our selected strains were BLNAR depending on the medium and/or test method used. If nonsusceptible strains for which ampicillin MICs were intermediate were included in the BLNAR category, 32 to 50% of our isolates would be considered BLNAR. These data emphasize the need for a standardized testing procedure and a universal definition of BLNAR strains before the clinical relevance of such strains can be evaluated. NCCLS dilution tests with haemophilus test medium broth or agar are preferred for testing ampicillin against
H. influenzae
.
Occurrence and Risk of Cochleotoxicity in Cystic Fibrosis Patients Receiving Repeated High-Dose Aminoglycoside Therapy
Authors: Mulheran M. , Degg C. , Burr S. , Morgan D. W. , Stableforth D. E.
Published: July 2002

Cystic fibrosis (CF) patients receive repeated courses of aminoglycoside therapy. These patients would consequently be expected to be more susceptible to cochleotoxicity, a recognized side effect with single courses of aminoglycoside therapy. The primary aim of this retrospective study was to establish the incidence and severity of auditory deficit in CF patients. Standard (0.25- to 8-kHz) and high-frequency (10- to 16-kHz) pure-tone audiometry was carried out in 70 CF patients, and the results were compared with the results from 91 control subjects. These subjects were further divided into pediatric and adult groups. Of 70 CF patients, 12 (1 pediatric) displayed hearing loss considered to be caused by repeated exposure to aminoglycosides. There was a nonlinear relationship between the courses of therapy received and the incidence of hearing loss. The severity of the loss did not appear to be related to the number of courses received. Assuming the risk of loss to be independent for each course, preliminary estimates of per course risk of hearing loss were less than 2%. Upon comparison with previous clinical studies and experimental work, these findings suggest that the incidence of cochleotoxicity in CF patients is considerably lower than would be expected, suggesting that the CF condition may confer protection against aminoglycoside cochleotoxicity.
CTX-M-1, CTX-M-3, and CTX-M-14 β-Lactamases from
Enterobacteriaceae
Isolated in France
Authors: Dutour C. , Bonnet R. , Marchandin H. , Boyer M. , Chanal C. , Sirot D. , Sirot J.
Published: July 2002

Six clinical CTX-M-producing isolates of the family
Enterobacteriaceae
were detected between 1999 and 2000 in different French hospitals. Two strains produced CTX-M-1 and CTX-M-3 and four strains produced CTX-M-14, a mutant Ala-231→Val of CTX-M-9. A putative transposable element, ISEcp-1, was located 43 bp upstream of all the
bla
CTX-M
genes. Two CTX-M-14-encoding plasmids exhibited similar restriction patterns. The CTX-M-1- and CTX-M-3-encoding plasmids were related to the CTX-M-1- and CTX-M-3-encoding plasmids previously reported in 1990 in France and in 1998 in Poland, respectively.
Rates and Extents of Antifungal Activities of Amphotericin B, Flucytosine, Fluconazole, and Voriconazole against
Candida lusitaniae
Determined by Microdilution, Etest, and Time-Kill Methods
Authors: Ernst Erika J. , Yodoi Keiko , Roling Ellen E. , Klepser Michael E.
Published: July 2002

Amphotericin B, flucytosine, fluconazole, and voriconazole alone and in combination were evaluated against isolates of
Candida lusitaniae
. MICs were determined by broth microdilution and Etest, and time-kill studies were conducted. Amphotericin B resulted in fungicidal activity against most isolates, whereas fluconazole, voriconazole, and flucytosine produced primarily fungistatic activities. The addition of flucytosine to amphotericin B resulted in a faster rate and greater extent of kill for isolates for which the MICs of amphotericin B were elevated.
Identification of Essential Residues in the Erm(B) rRNA Methyltransferase ofClostridium perfringens
Authors: Farrow Kylie A. , Lyras Dena , Polekhina Galina , Koutsis Katerina , Parker Michael W. , Rood Julian I.
Published: July 2002

Macrolide-lincosamide-streptogramin B resistance is widespread, with the determinants encoding resistance to antibiotics such as erythromycin being detected in many bacterial pathogens. Resistance is most commonly mediated by the production of an Erm protein, a 23S rRNA methyltransferase. We have undertaken a mutational analysis of the Erm(B) protein fromClostridium perfringenswith the objective of developing a greater understanding of the mechanism of action of this protein. A recombinant plasmid that carried theerm(B) gene was mutated by either in vitro hydroxylamine mutagenesis or passage through the mutator strain XL1-Red. Twenty-eight independently derived mutants were identified, nine of which had single point mutations in theerm(B) gene. These mutants produced stable but nonfunctional Erm(B) proteins, and all had amino acid changes within conserved methyltransferase motifs that were important for either substrate binding or catalysis. Modeling of theC. perfringensErm(B) protein confirmed that the point mutations all involved residues important for the structure and/or function of this rRNA methyltransferase. These regions of the protein therefore represent potential targets for the rational development of methyltransferase inhibitors.
Intrapulmonary Pharmacokinetics of Linezolid
Authors: Conte John E. , Golden Jeffrey A. , Kipps Juliana , Zurlinden Elisabeth
Published: July 2002

In this study, our objective was to determine the steady-state intrapulmonary concentrations and pharmacokinetic parameters of orally administered linezolid in healthy volunteers. Linezolid (600 mg every 12 h for a total of five doses) was administered orally to 25 healthy adult male subjects. Each subgroup contained five subjects, who underwent bronchoscopy and bronchoalveolar lavage (BAL) 4, 8, 12, 24, or 48 h after administration of the last dose. Blood was obtained for drug assay prior to administration of the first dose and fifth dose and at the completion of bronchoscopy and BAL. Standardized bronchoscopy was performed without systemic sedation. The volume of epithelial lining fluid (ELF) recovered was calculated by the urea dilution method, and the total number of alveolar cells (AC) was counted in a hemocytometer after cytocentrifugation. Linezolid was measured in plasma by a high-pressure liquid chromatography (HPLC) technique and in BAL specimens and AC by a combined HPLC-mass spectrometry technique. Areas under the concentration-time curves (AUCs) for linezolid in plasma, ELF, and AC were derived by noncompartmental analysis. Half-lives for linezolid in plasma, ELF, and AC were calculated from the elimination rate constants derived from a monoexponential fit of the means of the observed concentrations at each time point. Concentrations (means ± standard deviations) in plasma, ELF, and AC, respectively, were 7.3 ± 4.9, 64.3 ± 33.1, and 2.2 ± 0.6 μg/ml at the 4-h BAL time point and 7.6 ± 1.7, 24.3 ± 13.3, and 1.4 ± 1.3 μg/ml at the 12-h BAL time point. Linezolid concentrations in plasma, ELF, and AC declined monoexponentially, with half-lives of 6.9, 7.0, and 5.7 h, respectively. For a MIC of 4, the 12-h plasma AUC/MIC and maximum concentration/MIC ratios were 34.6 and 3.9, respectively, and the percentage of time the drug remained above the MIC for the 12-h dosing interval was 100%; the corresponding ratios in ELF were 120 and 16.1, respectively, and the percentage of time the drug remained above the MIC was 100%. The long plasma and intrapulmonary linezolid half-lives and the percentage of time spent above the MIC of 100% of the dosing interval provide a pharmacokinetic rationale for drug administration every 12 h and indicate that linezolid is likely to be an effective agent for the treatment of pulmonary infections.
Activity of Novispirin G10 againstPseudomonas aeruginosaIn Vitro and in Infected Burns
Authors: Steinstraesser Lars , Tack Brian F. , Waring Alan J. , Hong Teresa , Boo Lee M. , Fan Ming-Hui , Remick Daniel I. , Su Grace L. , Lehrer Robert I. , Wang Stewart C.
Published: July 2002

The emergence of multidrug-resistant microbes has serious implications for managing infection and sepsis and has stimulated efforts to develop alternative treatments, such as antimicrobial peptides. The objective of this study was to test a designer peptide, novispirin G10, against multidrug-resistant microorganisms. By two-stage radial diffusion assays, its activity against such organisms compared favorably with that of standard antibiotics and other antimicrobial peptides. It killed bacteria very rapidly, was nonhemolytic, and was relatively noncytotoxic. The peptide induced an immediate, massive efflux of potassium fromPseudomonas aeruginosa, suggesting that it altered the permeability of its inner membrane. The presence of human serum reduced but did not eliminate its activity. We tested the in vivo activity of novispirin G10 in rats with an infected, partial-thickness burn that covered 20% of their total body surface area. The burned area was seeded with 106CFU of a Silvadene-resistantP. aeruginosastrain, and 24 h later a single treatment with 0, 1, 3, or 6 mg of synthetic novispirin G10 (n= 16 at each concentration) per kg was given intradermally. Significant bacterial killing (P< 0.0001) was evident within 4 h in each peptide group compared to controls receiving vehicle. Antimicrobial peptides such as novispirin G10 may provide a useful alternative or adjunct to standard antibiotic agents in treating burns or other wound infections.
Adaptation of the Nisin-Controlled Expression System in
Lactobacillus plantarum
: a Tool To Study In Vivo Biological Effects
Authors: Pavan Sonia , Hols Pascal , Delcour Jean , Geoffroy Marie-Claude , Grangette Corinne , Kleerebezem Michiel , Mercenier Annick
Published: July 2002

The potential of lactic acid bacteria as live vehicles for the production and delivery of therapeutic molecules is being actively investigated today. For future applications it is essential to be able to establish dose-response curves for the targeted biological effect and thus to control the production of a heterologous biopeptide by a live lactobacillus. We therefore implemented in
Lactobacillus plantarum
NCIMB8826 the powerful nisin-controlled expression (NICE) system based on the autoregulatory properties of the bacteriocin nisin, which is produced by
Lactococcus lactis
. The original two-plasmid NICE system turned out to be poorly suited to
L. plantarum
. In order to obtain a stable and reproducible nisin dose-dependent synthesis of a reporter protein (β-glucuronidase) or a model antigen (the C subunit of the tetanus toxin, TTFC), the lactococcal
nisRK
regulatory genes were integrated into the chromosome of
L. plantarum
NCIMB8826. Moreover, recombinant
L. plantarum
producing increasing amounts of TTFC was used to establish a dose-response curve after subcutaneous administration to mice. The induced serum immunoglobulin G response was correlated with the dose of antigen delivered by the live lactobacilli.
Pacific Northwest Marine Sediments Contain Ammonia-Oxidizing Bacteria in the β Subdivision of theProteobacteria
Authors: Nold Stephen C. , Zhou Jizhong , Devol Allan H. , Tiedje James M.
Published: July 2002

The diversity of ammonia-oxidizing bacteria in aquatic sediments was studied by retrieving ammonia monooxygenase and methane monooxygenase gene sequences. Methanotrophs dominated freshwater sediments, while β-proteobacterial ammonia oxidizers dominated marine sediments. These results suggest that γ-proteobacteria such asNitrosococcus oceaniare minor members of marine sediment ammonia-oxidizing communities.
Molecular Characterization of Bacterial Populations in Petroleum-Contaminated Groundwater Discharged from Underground Crude Oil Storage Cavities
Authors: Watanabe Kazuya , Watanabe Kanako , Kodama Yumiko , Syutsubo Kazuaki , Harayama Shigeaki
Published: July 2002

Petroleum-contaminated groundwater discharged from underground crude oil storage cavities (cavity groundwater) harbored more than 10
6
microorganisms ml
−1
, a density 100 times higher than the densities in groundwater around the cavities (control groundwater). To characterize bacterial populations growing in the cavity groundwater, 46 PCR-amplified almost full-length 16S ribosomal DNA (rDNA) fragments were cloned and sequenced, and 28 different sequences were obtained. All of the sequences were affiliated with the
Proteobacteria
; 25 sequences (43 clones) were affiliated with the epsilon subclass, 2 were affiliated with the beta subclass, and 1 was affiliated with the delta subclass. Two major clusters (designated clusters 1 and 2) were found for the epsilon subclass proteobacterial clones; cluster 1 (25 clones) was most closely related to
Thiomicrospira denitrificans
(88% identical in nucleotide sequence), while cluster 2 (11 clones) was closely related to
Arcobacter
spp. Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified partial 16S rDNA fragments showed that one band was detected most strongly in cavity groundwater profiles independent of storage oil type and season. The sequence of this major band was identical to the sequences of most of the cluster 1 clones. Fluorescence in situ hybridization (FISH) indicated that the cluster 1 population accounted for 12 to 24% of the total bacterial population. This phylotype was not detected in the control groundwater by DGGE and FISH analyses. These results indicate that the novel members of the epsilon subclass of the
Proteobacteria
grow as major populations in the petroleum-contaminated cavity groundwater.
Development of Catechol 2,3-Dioxygenase-Specific Primers for Monitoring Bioremediation by Competitive Quantitative PCR
Authors: Mesarch Matthew B. , Nakatsu Cindy H. , Nies Loring
Published: July 2002

Benzene, toluene, xylenes, phenol, naphthalene, and biphenyl are among a group of compounds that have at least one reported pathway for biodegradation involving catechol 2,3-dioxygenase enzymes. Thus, detection of the corresponding catechol 2,3-dioxygenase genes can serve as a basis for identifying and quantifying bacteria that have these catabolic abilities. Primers that can successfully amplify a 238-bp catechol 2,3-dioxygenase gene fragment from eight different bacteria are described. The identities of the amplicons were confirmed by hybridization with a 238-bp catechol 2,3-dioxygenase probe. The detection limit was 10
2
to 10
3
gene copies, which was lowered to 10
0
to 10
1
gene copies by hybridization. Using the dioxygenase-specific primers, an increase in catechol 2,3-dioxygenase genes was detected in petroleum-amended soils. The dioxygenase genes were enumerated by competitive quantitative PCR with a 163-bp competitor that was amplified using the same primers. Target and competitor sequences had identical amplification kinetics. Potential PCR inhibitors that could coextract with DNA, nonamplifying DNA, soil factors (humics), and soil pollutants (toluene) did not impact enumeration. Therefore, this technique can be used to accurately and reproducibly quantify catechol 2,3-dioxygenase genes in complex environments such as petroleum-contaminated soil. Direct, non-cultivation-based molecular techniques for detecting and enumerating microbial pollutant-biodegrading genes in environmental samples are powerful tools for monitoring bioremediation and developing field evidence in support of natural attenuation.
Diversity of
Salmonella
Strains Isolated from the Aquatic Environment as Determined by Serotyping and Amplification of the Ribosomal DNA Spacer Regions
Authors: Baudart Julia , Lemarchand Karine , Brisabois Anne , Lebaron Philippe
Published: July 2002

Salmonella
species are pathogenic bacteria often detected in sewage, freshwater, marine coastal water, and groundwater.
Salmonella
spp. can survive for long periods in natural waters, and the persistence of specific and epidemic strains is of great concern in public health. However, the diversity of species found in the natural environment remains unknown. The aim of this study was to investigate the diversity of
Salmonella
strains isolated from different natural aquatic systems within a Mediterranean coastal watershed (river, wastewater, and marine coastal areas). A total of 574 strains isolated from these natural environments were identified by both conventional serotyping and the ribosomal spacer-heteroduplex polymorphism (RS-HP) method (M. A. Jensen and N. Straus, PCR Methods Appl. 3:186–194, 1993). More than 40 different serotypes were found, and some serotypes probably mobilized from widespread animal-rearing activities were detected only during storm events. These serotypes may be good indicators of specific contamination sources. Furthermore, the RS-HP method based on the PCR amplification of the intergenic spacer region between the 16S and 23S rRNA genes can produce amplicon profiles allowing the discrimination of species at both serotype and intraserotype levels. This method represents a powerful tool that could be used for rapid typing of
Salmonella
isolates.
Construction and Evaluation of a Novel Bifunctional
N
-Carbamylase–
d
-Hydantoinase Fusion Enzyme
Authors: Kim Geun-Joong , Lee Dong-Eun , Kim Hak-Sung
Published: July 2002

A fully enzymatic process employing two sequential enzymes,
d
-hydantoinase and
N
-carbamylase, is a typical case requiring combined enzyme activity for the production of
d
-amino acids. To test the possibility of generating a bifunctional fusion enzyme, we constructed a fusion protein via end-to-end fusion of a whole gene that encodes an intact protein at the N terminus of the
d
-hydantoinase. Firstly, maltose-binding protein (MBP) gene of
E. coli
was fused with
d
-hydantoinase gene from
Bacillus stearothermophilus
SD1, and the properties of the resulting fusion protein (MBP-HYD) were compared with those of native
d
-hydantoinase. Gel filtration and kinetic analyses clearly demonstrated that the typical characteristics of
d
-hydantoinase are maintained even in a fusion state. Based on this result, we constructed an artificial fusion enzyme composed of the whole length of
N
-carbamylase (304 amino acids [aa]) from
Agrobacterim radiobacter
NRRL B11291 and
d
-hydantoinase (471 aa). The fusion enzyme (CAB-HYD) was functionally expressed with an expected molecular mass of 86 kDa and efficiently converted exogenous hydantoin derivatives to the
d
-amino acids. A related
d
-hydantoinase (HYD1) gene from
Bacillus thermocatenulatus
GH2 was also fused with the
N
-carbamylase gene at its N terminus. The resulting enzyme (CAB-HYD1) was bifunctional as expected and showed better performance than the CAB-HYD fusion enzyme. The conversion of hydantoin derivatives to corresponding amino acids by the fusion enzymes was much higher than that by the separately expressed enzymes, and comparable to that by the coexpressed enzymes. Thus, the fusion enzyme might be useful as a potential biocatalyst for the production of nonnatural amino acids.
trans-o
-Hydroxybenzylidenepyruvate Hydratase-Aldolase as a Biocatalyst
Authors: Eaton Richard W.
Published: July 2002

The hydratase-aldolase-catalyzed conversion of
trans-o
-hydroxybenzylidenepyruvate to salicylaldehyde and pyruvate is an intermediate reaction in the conversion of naphthalene to salicylate by bacteria. Here, a variety of aromatic aldehydes and some nonaromatic aldehydes together with pyruvate have been shown to be substrates for aldol condensations catalyzed by this enzyme in extracts of the recombinant strain
Escherichia coli
JM109(pRE701). Some of the products of these reactions were also compared as substrates in the opposite (hydration-aldol cleavage) reaction.
Differentiation of Chitinase-Active and Non-Chitinase-Active Subpopulations of a Marine Bacterium during Chitin Degradation
Authors: Baty Ace M. , Eastburn Callie C. , Diwu Zhenjun , Techkarnjanaruk Somkiet , Goodman Amanda E. , Geesey Gill G.
Published: July 2002

The ability of marine bacteria to adhere to detrital particulate organic matter and rapidly switch on metabolic genes in an effort to reproduce is an important response for bacterial survival in the pelagic marine environment. The goal of this investigation was to evaluate the relationship between chitinolytic gene expression and extracellular chitinase activity in individual cells of the marine bacterium
Pseudoalteromonas
sp. strain S91 attached to solid chitin. A green fluorescent protein reporter gene under the control of the
chiA
promoter was used to evaluate
chiA
gene expression, and a precipitating enzyme-linked fluorescent probe, ELF-97–
N
-acetyl-β-
d
-glucosaminide, was used to evaluate extracellular chitinase activity among cells in the bacterial population. Evaluation of
chiA
expression and ELF-97 crystal location at the single-cell level revealed two physiologically distinct subpopulations of S91 on the chitin surface: one that was chitinase active and remained associated with the surface and another that was non-chitinase active and released daughter cells into the bulk aqueous phase. It is hypothesized that the surface-associated, non-chitinase-active population is utilizing chitin degradation products that were released by the adjacent chitinase-active population for cell replication and dissemination into the bulk aqueous phase.
Real-Time Measurements of the Interaction between Single Cells of
Listeria monocytogenes
and Nisin on a Solid Surface
Authors: Budde Birgitte Bjørn , Jakobsen Mogens
Published: July 2002

A method to obtain real-time measurements of the interactions between nisin and single cells of
Listeria monocytogenes
on a solid surface was developed. This method was based on fluorescence ratio-imaging microscopy and measurements of changes in the intracellular pH (pH
i
) of carboxyfluorescein succinimidyl ester-stained cells during exposure to nisin. Immobilized cells were placed in a chamber mounted on a microscope and attached to a high-precision peristaltic pump which allowed rapid changes in the nisin concentration. In the absence of nisin, the pH
i
of
L. monocytogenes
was almost constant (approximately pH 8.0) and independent of the external pH in the pH range from 5.0 to 9.0. In the presence of nisin, dissipation of the pH gradient (ΔpH) was observed, and this dissipation was both time and nisin concentration dependent. The dissipation of ΔpH resulted in cell death, as determined by the number of CFU. In the model system which we used the immobilized cells were significantly more resistant to nisin than the planktonic cells. The kinetics of ΔpH dissipation for single cells revealed a variable lag phase depending on the nisin concentration, which was followed by a very rapid decrease in pH
i
within 1 to 2 min. The differences in nisin sensitivity between single cells in a
L. monocytogenes
population were insignificant for cells grown to the stationary phase in a liquid laboratory substrate, but differences were observed for cells grown on an agar medium under similar conditions, which resulted in some cells having increased resistance to nisin.
Reduction of Technetium(VII) by
Desulfovibrio fructosovorans
Is Mediated by the Nickel-Iron Hydrogenase
Authors: De Luca Gilles , de Philip Pascale , Dermoun Zorah , Rousset Marc , Verméglio André
Published: July 2002

Resting cells of the sulfate-reducing bacterium
Desulfovibrio fructosovorans
grown in the absence of sulfate had a very high Tc(VII)-reducing activity, which led to the formation of an insoluble black precipitate. The involvement of a periplasmic hydrogenase in Tc(VII) reduction was indicated (i) by the requirement for hydrogen as an electron donor, (ii) by the tolerance of this activity to oxygen, and (iii) by the inhibition of this activity by Cu(II). Moreover, a mutant carrying a deletion in the nickel-iron hydrogenase operon showed a dramatic decrease in the rate of Tc(VII) reduction. The restoration of Tc(VII) reduction by complementation of this mutation with nickel-iron hydrogenase genes demonstrated the specific involvement of the periplasmic nickel-iron hydrogenase in the mechanism in vivo. The Tc(VII)-reducing activity was also observed with cell extracts in the presence of hydrogen. Under these conditions, Tc(VII) was reduced enzymatically to soluble Tc(V) or precipitated to an insoluble black precipitate, depending on the chemical nature of the buffer used. The purified nickel-iron hydrogenase performed Tc(VII) reduction and precipitation at high rates. These series of genetic and biochemical approaches demonstrated that the periplasmic nickel-iron hydrogenase of sulfate-reducing bacteria functions as a Tc(VII) reductase. The role of cytochrome
c
3
in the mechanism is also discussed.
Isolation and Characterization of Intracellular Protein Inclusions Produced by the Entomopathogenic Bacterium
Photorhabdus luminescens
Authors: Bowen David J. , Ensign Jerald C.
Published: July 2002

Cells of the entomopathogenic bacterium
Photorhabdus luminescens
contain two types of morphologically distinct crystalline inclusion proteins. The larger rectangular inclusion (type 1) and a smaller bipyramid-shaped inclusion (type 2) were purified from cell lysates by differential centrifugation and isopycnic density gradient centrifugation. Both structures are composed of protein and are readily soluble at pH 11 and 4 in 1% sodium dodecyl sulfate (SDS) and in 8 M urea. Electrophoretic analysis reveals that each inclusion is composed of a single protein subunit with a molecular mass of 11,000 Da. The proteins differ in amino acid composition, protease digestion pattern, and immunological cross-reactivity. The protein inclusions are first visible in the cells at the time of late exponential growth. Western blot analyses showed that the proteins appeared in cells during mid- to late exponential growth. When at maximum size in stationary-phase cells, the proteins constitute 40% of the total cellular protein. The protein inclusions are not used during long-term starvation of the cells and were not toxic when injected into or fed to
Galleria mellonella
larvae.
DNA Sequence and Mutational Analysis of Rhizobitoxine Biosynthesis Genes in
Bradyrhizobium elkanii
Authors: Yasuta Tsuyoshi , Okazaki Shin , Mitsui Hisayuki , Yuhashi Ken-Ichi , Ezura Hiroshi , Minamisawa Kiwamu
Published: July 2002

We cloned and sequenced a cluster of genes involved in the biosynthesis of rhizobitoxine, a nodulation enhancer produced by
Bradyrhizobium elkanii
. The nucleotide sequence of the cloned 28.4-kb DNA region encompassing
rtxA
showed that several open reading frames (ORFs) were located downstream of
rtxA
. A large-deletion mutant of
B. elkanii
, USDA94Δ
rtx
::Ω1, which lacks
rtxA
, ORF1 (
rtxC
), ORF2, and ORF3, did not produce rhizobitoxine, dihydrorhizobitoxine, or serinol. The broad-host-range cosmid pLAFR1, which contains
rtxA
and these ORFs, complemented rhizobitoxine production in USDA94Δ
rtx
::Ω1. Further complementation experiments involving cosmid derivatives obtained by random mutagenesis with a kanamycin cassette revealed that at least
rtxA
and
rtxC
are necessary for rhizobitoxine production. Insertional mutagenesis of the N-terminal and C-terminal regions of
rtxA
indicated that
rtxA
is responsible for two crucial steps, serinol formation and dihydrorhizobitoxine biosynthesis. An insertional mutant of
rtxC
produced serinol and dihydrorhizobitoxine but no rhizobitoxine. Moreover, the
rtxC
product was highly homologous to the fatty acid desaturase of
Pseudomonas syringae
and included the copper-binding signature and eight histidine residues conserved in membrane-bound desaturase. This result suggested that
rtxC
encodes dihydrorhizobitoxine desaturase for the final step of rhizobitoxine production. In light of results from DNA sequence comparison, gene disruption experiments, and dihydrorhizobitoxine production from various substrates, we discuss the biosynthetic pathway of rhizobitoxine and its evolutionary significance in bradyrhizobia.
Poly(3-Hydroxybutyrate) Synthesis Genes in
Azotobacter
sp. Strain FA8
Authors: Pettinari M. Julia , Vázquez Gustavo J. , Silberschmidt Daniel , Rehm Bernd , Steinbüchel Alexander , Méndez Beatriz S.
Published: July 2002

Genes responsible for the synthesis of poly(3-hydroxybutyrate) (PHB) in
Azotobacter
sp. FA8 were cloned and analyzed. A PHB polymerase gene (
phbC
) was found downstream from genes coding for β-ketothiolase (
phbA
) and acetoacetyl-coenzyme A reductase (
phbB
). A PHB synthase mutant was obtained by gene inactivation and used for genetic studies. The
phbC
gene from this strain was introduced into
Ralstonia eutropha
PHB-4 (
phbC
-negative mutant), and the recombinant accumulated PHB when either glucose or octanoate was used as a source of carbon, indicating that this PHB synthase cannot incorporate medium-chain-length hydroxyalkanoates into PHB.