bluuebunny/crossref_metadata_2025 huggingface dataset:
RNA Polymerase Inhibitors with Activity against Rifampin-Resistant Mutants of
Staphylococcus aureus
Authors: O'Neill Alexander , Oliva Brunello , Storey Christopher , Hoyle Anthony , Fishwick Colin , Chopra Ian
Published: July 2002

A collection of rifampin-resistant mutants of
Staphylococcus aureus
with characterized RNA polymerase β-subunit (
rpoB
) gene mutations was cross-screened against a number of other RNA polymerase inhibitors to correlate susceptibility with specific
rpoB
genotypes. The
rpoB
mutants were cross-resistant to streptolydigin and sorangicin A. In contrast, thiolutin, holomycin, corallopyronin A, and ripostatin A retained activity against the
rpoB
mutants. The second group of inhibitors may be of interest as drug development candidates.
Characterization of a Divergent
vanD
-Type Resistance Element from the First Glycopeptide-Resistant Strain of
Enterococcus faecium
Isolated in Brazil
Authors: Dalla Costa Libera M. , Reynolds Peter E. , Souza Helena A. P. H. M. , Souza Dilair C. , Palepou Marie-France I. , Woodford Neil
Published: July 2002

Enterococcus faecium
10/96A from Brazil was resistant to vancomycin (MIC, 256 μg/ml) but gave no amplification products with primers specific for known
van
genotypes. A 2,368-bp fragment of a
van
cluster contained one open reading frame encoding a peptide with 83% amino acid identity to VanH
D
, and a second encoding a
d
-alanine-
d
-lactate ligase with 83 to 85% identity to VanD. The divergent glycopeptide resistance phenotype was designated VanD4.
A Population Pharmacokinetic Model for Vancomycin in Pediatric Patients and Its Predictive Value in a Naive Population
Authors: Lamarre Patrice , Lebel Denis , Ducharme Murray P.
Published: July 2002

The objectives of this study were to (i) construct a population pharmacokinetic (PK) model able to describe vancomycin (VAN) concentrations in serum in pediatric patients, (ii) determine VAN PK parameters in this population, and (iii) validate the predictive ability of this model in a naive pediatric population. Data used in this study were obtained from 78 pediatric patients (under 18 years old). PK analyses were performed using compartmental methods. The most appropriate model was chosen based on the evaluation of pertinent graphics and calculation of the Akaike information criterion test. The population PK analysis was performed using an iterative two-stage method. A two-compartment PK model using age, sex, weight, and serum creatinine as covariates was determined to be the most appropriate one to describe serum VAN concentrations. The quality of fit was very good, and the distribution of weighted residuals was found to be homoscedastic (Wilcoxon signed rank test). Fitted population PK parameters (mean ± standard deviation) were as follows: central clearance (0.1 ± 0.05 liter/h/kg), central volume of distribution (0.27 ± 0.07 liter/kg), peripheral volume of distribution (0.16 ± 0.07 liter/kg), and distributional clearance (0.16 ± 0.07 liter/kg). The predictive ability of the developed model (including the above-mentioned covariates) was evaluated in a naive population of 19 pediatric patients. The predictability was very good. Precision (±95% confidence interval [CI]) (peak, 4.1 [±1.4], and trough, 2.2 [±0.7]) and bias (±95% CI) (peak, −0.58 [±2.2], and trough, 0.63 [±1.1] mg/liter) were significantly (
P
< 0.05) superior to those obtained using a conventional method (precision [±95% CI]: peak, 8.03 [±2.46], and trough, 2.7 [±0.74]; bias: peak, −7.1 [±2.9], and trough, −1.35 [±1.2] mg/liter). We propose the use of this population PK model to optimize VAN clinical therapies in our institution and others with similar patient population characteristics.
A Double-Blind Placebo-Controlled Study of an Infusion of Lexipafant (Platelet-Activating Factor Receptor Antagonist) in Patients with Severe Sepsis
Authors: Suputtamongkol Yupin , Intaranongpai Sunanta , Smith Michael D. , Angus Brian , Chaowagul Wipada , Permpikul Chairat , Simpson Julie A. , Leelarasamee Amorn , Curtis Lloyd , White Nicholas J.
Published: July 2002

Platelet-activating factor (PAF) is a potent endogenous proinflammatory mediator implicated in the pathogenesis of septic shock. A double-blind randomized placebo-controlled trial of an intravenous PAF receptor antagonist (lexipafant) was conducted with 131 adult Thai patients with suspected severe sepsis (66 of whom had positive blood cultures). Detailed serial clinical, biochemical, and cytokine measurements were performed. Lexipafant treatment was well tolerated. The 28-day mortality in the lexipafant group (61.4%) was similar to that in the placebo group (62.6%). There was also no evidence that lexipafant affected clinical or biochemical measures of disease severity or the profile of sequentially measured plasma cytokine levels. PAF may not have an important role in the pathogenesis of severe sepsis.
Horizontal Transfer of
parC
and
gyrA
in Fluoroquinolone-Resistant Clinical Isolates of
Streptococcus pneumoniae
Authors: Ferrándiz María José , Fenoll Asunción , Liñares Josefina , De La Campa Adela G.
Published: July 2002

We have analyzed genetically three clinical isolates (3180, 3870, and 1244) of
Streptococcus pneumoniae
with high-level ciprofloxacin resistance. Isolates 3180 and 3870 were atypical because of their insolubility in deoxycholate. However, they hybridized specifically with pneumococcal autolysin and pneumolysin gene probes and have typical pneumococcal
atpC
and
atpA
gene sequences. Analysis of the complete sequences of the
parC
and
gyrA
genes revealed total variations of 8 and 8.7% (isolate 3180) and 7.4 and 3.6% (isolate 3870), respectively, compared to the wild-type strain R6 sequence. The variations observed between the sequences of R6 and isolate 1244 were less than 0.9%. The structure of the
gyrA
and
parC
genes from isolates 3180 and 3870 was organized in sequence blocks that show different levels of divergence, suggesting a pattern of recombination. These results are evidence for recombination at the fluoroquinolone target genes in clinical isolates of
S. pneumoniae
. The genetically related viridans group streptococci could act as a reservoir for fluoroquinolone resistance genes.
Role of P Glycoprotein in the Course and Treatment of
Encephalitozoon
Microsporidiosis
Authors: Leitch Gordon J. , Scanlon Mary , Shaw Andrew , Visvesvara Govinda S.
Published: July 2002

Encephalitozoon
microsporidia are obligate intracellular protozoan parasites that proliferate and differentiate within a parasitophorous vacuole inside host cells that are usually epithelial in nature. Isolates of the three species of the
Encephalitozoon
microsporidia,
E. cuniculi
,
E. hellem
, and
E. intestinalis
, were obtained from AIDS patients and cultured in green monkey (E6) kidney cells. Anti-P-glycoprotein (anti-Pgp) and anti-multidrug resistance-associated protein (anti-MRP) monoclonal antibodies were used to probe for multidrug resistance (MDR) pump epitopes and verapamil- or cyclosporin A- and probenecid-modulated intracellular calcein fluorescence were used to assess the expression of Pgp and MRP respectively in uninfected and infected cells. Pgp, but not MRP, was detected immunocytochemically and by verapamil- and cyclosporin A-potentiated intracellular fluorescence in both host cells and parasite developing stages. When an in vitro infection assay was employed, verapamil and cyclosporin A acted as chemosensitizing agents for the antiparasitic drug albendazole. These observations suggest that inhibiting host cell and perhaps parasite MDR pumps may increase the efficacy of antiparasitic agents in these and other microsporidia species.
Genetic Analysis of Multiple Loci Suggests that Mutations in the
Pneumocystis carinii
f. sp.
hominis
Dihydropteroate Synthase Gene Arose Independently in Multiple Strains
Authors: Ma Liang , Kovacs Joseph A.
Published: July 2002

To determine if mutations in the dihydropteroate synthase (DHPS) gene of
Pneumocystis carinii
f. sp.
hominis
arose in a single strain that was subsequently widely disseminated, we examined four genomic regions of 22
P. carinii
clinical isolates selected based on the absence or presence of mutations in the DHPS gene. By single-strand conformation polymorphism and DNA sequencing, we found varying genotypes for each of the four regions in isolates with DHPS mutations, suggesting that these mutations occurred independently in multiple strains of
P. carinii
. This suggests that exposure to sulfa will select for these mutations in diverse strains.
In Vitro and In Vivo Activities of Sitafloxacin against
Chlamydia
spp
Authors: Miyashita Naoyuki , Niki Yoshihito , Matsushima Toshiharu
Published: July 2002

The in vitro and in vivo antichlamydial activity of sitafloxacin was investigated. The MICs and minimal chlamydiacidal concentrations of sitafloxacin for various species of chlamydia ranged from 0.031 to 0.125 μg/ml. Sitafloxacin had an excellent therapeutic effect on experimental
Chlamydia psittaci
pneumonia and was more potent than tosufloxacin, ofloxacin, and ciproflxacin, although slightly less potent than sparfloxacin.
rho
Is Not Essential for Viability or Virulence in
Staphylococcus aureus
Authors: Washburn Robert S. , Marra Andrea , Bryant Alexander P. , Rosenberg Martin , Gentry Daniel R.
Published: July 2002

We have identified the gene for transcription termination factor Rho in
Staphylococcus aureus
. Deletion of
rho
in
S. aureus
reveals that it is not essential for viability or virulence. We also searched the available bacterial genomic sequences for homologs of Rho and found that it is broadly distributed and highly conserved. Exceptions include
Streptococcus pneumoniae
,
Streptococcus pyogenes
,
Mycoplasma genitalium, Mycoplasma pneumoniae, Ureaplasma urealyticum,
and
Synechocystis
sp. strain PCC6803, all of which appear not to possess a Rho homolog. Complementation studies indicate that
S. aureus
Rho possesses the same activity as
Escherichia coli
Rho and that the Rho inhibitor bicyclomycin is active against
S. aureus
Rho. Our results explain the lack of activity of bicyclomycin against many gram-positive bacteria and raise the possibility that the essentiality of
rho
may be the exception rather than the rule.
Efficacy of Teicoplanin-Gentamicin Given Once a Day on the Basis of Pharmacokinetics in Humans for Treatment of Enterococcal Experimental Endocarditis
Authors: López Pedro , Gavaldà Joan , Martin M. Teresa , Almirante Benito , Gomis Xavier , Azuaje Carlos , Borrell Nuria , Pou Leonor , Falcó Vicenç , Pigrau Carles , Pahissa Albert
Published: July 2002

With the aim of investigating home therapy for enterococcal endocarditis, we compared the efficacy of teicoplanin combined with gentamicin given once a day or in three daily doses (t.i.d.) with the standard treatment, ampicillin plus gentamicin administered t.i.d., for treating experimental enterococcal endocarditis. The antibiotics were administered by using “human-like pharmacokinetics” (H-L), i.e, pharmacokinetics like those in humans, that simulated the profiles of these drugs in human serum. Animals with catheter-induced endocarditis were infected intravenously with 10
8
CFU of
Enterococcus faecalis
EF91 (MICs and MBCs of ampicillin, gentamicin, and teicoplanin, 0.5 and 32, 16 and 32, and 0.5 and 1 μg/ml, respectively) and were treated for 3 days with ampicillin H-L at 2 g every 4 h plus gentamicin H-L at 1 mg/kg every 8 h, or teicoplanin H-L at 10 mg/kg every 24 h, alone or combined with gentamicin, administered at dose of H-L at 1 mg/kg every 8 h or H-L at 4.5 mg/kg every 24 h. The results of therapy for experimental endocarditis due to EF91 showed that teicoplanin alone was as effective as ampicillin alone in reducing the bacterial load (
P
> 0.05). The combination of ampicillin or teicoplanin with gentamicin was more effective than the administration of both drugs alone in reducing the log
10
CFU/gram of aortic vegetation (
P
< 0.01 and
P
< 0.05, respectively). Teicoplanin plus gentamicin H-L at 4.5 mg/kg, both administered every 24 h, showed an efficacy equal to the “gold standard,” ampicillin plus gentamicin H-L at 1 mg/kg t.i.d. (
P
> 0.05). Increasing the interval of administration of gentamicin to a single daily dose combined with teicoplanin resulted in a reduction of bacteria in the vegetations equivalent to that achieved with the recommended regimen of ampicillin plus thrice-daily gentamicin in the treatment of experimental endocarditis due to
E. faecalis
. Teicoplanin plus gentamicin, both administered once a day, may be useful home therapy for selected cases of enterococcal endocarditis.
Multidrug Efflux Pumps: Expression Patterns and Contribution to Antibiotic Resistance in
Pseudomonas aeruginosa
Biofilms
Authors: De Kievit Teresa R. , Parkins Michael D. , Gillis Richard J. , Srikumar Ramakrishnan , Ceri Howard , Poole Keith , Iglewski Barbara H. , Storey Douglas G.
Published: July 2002

Pseudomonas aeruginosa
biofilms are intrinsically resistant to antimicrobial chemotherapies. At present, very little is known about the physiological changes that occur during the transition from the planktonic to biofilm mode of growth. The resistance of
P. aeruginosa
biofilms to numerous antimicrobial agents that are substrates subject to active efflux from planktonic cells suggests that efflux pumps may substantially contribute to the innate resistance of biofilms. In this study, we investigated the expression of genes associated with two multidrug resistance (MDR) efflux pumps, MexAB-OprM and MexCD-OprJ, throughout the course of biofilm development. Using fusions to
gfp
, we were able to analyze spatial and temporal expression of
mexA
and
mexC
in the developing biofilm. Remarkably, expression of
mexAB-oprM
and
mexCD-oprJ
was not upregulated but rather decreased over time in the developing biofilm. Northern blot analysis confirmed that these pumps were not hyperexpressed in the biofilm. Furthermore, spatial differences in
mexAB-oprM
and
mexCD-oprJ
expression were observed, with maximal activity occurring at the biofilm substratum. Using a series of MDR mutants, we assessed the contribution of the MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY efflux pumps to
P. aeruginosa
biofilm resistance. These analyses led to the surprising discovery that the four characterized efflux pumps do not play a role in the antibiotic-resistant phenotype of
P. aeruginosa
biofilms.
Growth and Development of Tetracycline-Resistant
Chlamydia suis
Authors: Lenart J. , Andersen A. A. , Rockey D. D.
Published: July 2002

Tetracycline (TET) is a front-line antibiotic for the treatment of chlamydial infections in both humans and animals, and the emergence of TET-resistant (Tet
r
)
Chlamydia
is of significant clinical importance. Recently, several Tet
r
chlamydial strains have been isolated from swine (
Sus scrofa)
raised in production facilities in Nebraska. Here, the intracellular development of two Tet
r
strains, R19 and R27, is characterized through the use of tissue culture and immunofluorescence. The strains grow in concentrations of up to 4 μg of TET/ml, while a TET-sensitive (Tet
s
) swine strain (S45) and a strain of the human serovar L2 (LGV-434) grow in up to 0.1 μg of TET/ml. Although inclusions form in the presence of TET, many contain large aberrant reticulate bodies (RBs) that do not differentiate into infectious elementary bodies. The percentage of inclusions containing typical developmental forms decreases with increasing TET concentrations, and at 3 μg of TET/ml 100% of inclusions contain aberrant RBs. However, upon removal of TET the aberrant RBs revert to typical RBs, and a productive developmental cycle ensues. In addition, inclusions were found that contained both
C. suis
R19 and
Chlamydia trachomatis
L2 after sequential infection, demonstrating that two biologically distinct chlamydial strains could both develop within a single inclusion.
Pharmacodynamics of Gatifloxacin against
Streptococcus pneumoniae
in an In Vitro Pharmacokinetic Model: Impact of Area under the Curve/MIC Ratios on Eradication
Authors: Lister Philip D.
Published: July 2002

Previous studies have demonstrated that fluoroquinolone area under the curve (AUC)/MIC ratios of 30 to 50 are sufficient to eradicate pneumococci from in vitro pharmacokinetic models (IVPMs). However, more systematic studies of the impact of AUC/MIC ratios on the antipneumococcal activities of fluoroquinolones are needed. In the present study, a two-compartment IVPM was used to evaluate the impact of AUC/MIC ratios on the pharmacodynamics of gatifloxacin against four strains of
Streptococcus pneumoniae
. Gatifloxacin MICs were 0.4 to 1 μg/ml, whereas levofloxacin MICs were 1.8 to 3.2 μg/ml. Since both peak concentration/MIC (peak/MIC) and AUC/MIC ratios affect fluoroquinolone pharmacodynamics, logarithmic-phase cultures (5 × 10
7
CFU/ml) were exposed to gatifloxacin at constant peak/MIC ratios of 2:1 to 3:1 at 0 and 24 h, elimination half-lives were varied to provide a range of AUC/MIC ratios, and changes in viable counts were measured over 30 h. As a comparison, levofloxacin was evaluated at similar peak/MIC ratios and at AUC/MIC ratios of 30 to 38. For each strain, killing rates through 4 to 8 h were similar since peak/MIC ratios were kept constant. However, continued killing and eradication were observed only when gatifloxacin AUC/MIC ratios were 27 to 48. Levofloxacin also provided eradication. In contrast, substantial regrowth was observed in most experiments when gatifloxacin AUC/MIC ratios were 9 to 24. These data provide further support that fluoroquinolone AUC/MIC ratios of approximately 30 or higher can be sufficient for eradication of pneumococci from IVPMs. Furthermore, the overall impact of the AUC/MIC ratio was not influenced by the strain evaluated or its susceptibility to gatifloxacin. Further studies with other fluoroquinolones and pneumococci that exhibit wider ranges of susceptibilities are warranted. In addition, similar studies with higher peak/MIC ratios are needed to better define the impact of AUC/MIC ratios and peak/MIC ratios on the antipneumococcal pharmacodynamics of fluoroquinolones.
Molecular Analysis of Rifampin Resistance in
Bacillus anthracis
and
Bacillus cereus
Authors: Vogler Amy J. , Busch Joseph D. , Percy-Fine Stephanie , Tipton-Hunton Christine , Smith Kimothy L. , Keim Paul
Published: July 2002

Rifampin-resistant mutants were selected from UV-light-treated
Bacillus cereus
(20 mutants) and attenuated
B. anthracis
(23 mutants). In addition, spontaneous rifampin-resistant mutants were also isolated in attenuated
B. anthracis
(22 mutants). The rifampin resistance clusters of the
rpoB
gene were sequenced for all 65 mutants. Mutations associated with resistance were consistent with those from other bacteria, though two novel changes were observed. The spontaneous rate of resistance was estimated at 1.57 × 10
−9
mutations/generation by a Luria-Delbrück fluctuation test.
Efficacy of Ravuconazole in Treatment of Systemic Murine Histoplasmosis
Authors: Clemons K. V. , Martinez M. , Calderon L. , Stevens D. A.
Published: July 2002

Ravuconazole (RCZ) was evaluated for efficacy in comparison to fluconazole (FCZ) and itraconazole (ITZ) in murine models of disseminated histoplasmosis. All regimens tested prolonged survival (
P
< 0.05 to 0.0001). At equivalent doses of 50 mg/kg of body weight, RCZ and ITZ were equally effective and RCZ was more effective than FCZ (
P
= 0.02). Clearance of fungal burden from the livers and spleens of mice showed RCZ and ITZ at doses of 50 mg/kg to be efficacious but not curative. These data indicate that RCZ should be studied further.
Kinetic Analysis of Wild-Type and YMDD Mutant Hepatitis B Virus Polymerases and Effects of Deoxyribonucleotide Concentrations on Polymerase Activity
Authors: Gaillard Richard K. , Barnard Jennifer , Lopez Vincent , Hodges Paula , Bourne Eric , Johnson Lance , Allen Marchelle I. , Condreay Patrick , Miller Wayne H. , Condreay Lynn D.
Published: July 2002

Mutations in the YMDD motif of the hepatitis B virus (HBV) DNA polymerase result in reduced susceptibility of HBV to inhibition by lamivudine, at a cost in replication fitness. The mechanisms underlying the effects of YMDD mutations on replication fitness were investigated using both a cell-based viral replication system and an in vitro enzyme assay to examine wild-type (wt) and YMDD-mutant polymerases. We calculated the affinities of wt and YMDD-mutant polymerases for each natural deoxyribonucleoside triphosphate (dNTP) and determined the intracellular concentrations of each dNTP in HepG2 cells under conditions that support HBV replication. In addition, inhibition constants for lamivudine triphosphate were determined for wt and YMDD-mutant polymerases. Relative to wt HBV polymerase, each of the YMDD-mutant polymerases showed increased apparent

K
m

values for the natural dNTP substrates, indicating decreased affinities for these substrates, as well as increased

K
i

values for lamivudine triphosphate, indicating decreased affinity for the drug. The effect of the differences in apparent

K
m

values between YMDD-mutant polymerase and wt HBV polymerase could be masked by high levels of dNTP substrates (>20 μM). However, assays using dNTP concentrations equivalent to those measured in HepG2 cells under physiological conditions showed decreased enzymatic activity of YMDD-mutant polymerases relative to wt polymerase. Therefore, the decrease in replication fitness of YMDD-mutant HBV strains results from the lower affinities (increased

K
m

values) of the YMDD-mutant polymerases for the natural dNTP substrates and physiological intracellular concentrations of dNTPs that are limiting for the replication of YMDD-mutant HBV strains.
Nonparametric Population Pharmacokinetic Analysis of Amikacin in Neonates, Infants, and Children
Authors: Tréluyer J. M. , Merlé Y. , Tonnelier S. , Rey E. , Pons G.
Published: July 2002

The therapeutic and toxic effects of amikacin are known to depend on its concentration in plasma, but the pharmacokinetics of this drug in neonates, infants, and children and the influences of clinical and biological variables have been only partially assessed. Therapeutic drug monitoring data collected from 155 patients (49 neonates, 77 infants, and 29 children) receiving amikacin were analyzed by a nonparametric population-based approach, the nonparametric maximum-likelihood method. We assessed the effects of gestational and postnatal age, weight, Apgar score, and plasma creatinine and urea concentrations on pharmacokinetic parameters. There is no specific formulation of amikacin for neonates and infants. We therefore used an error model to account for errors due to dilution during preparation of the infusion. The covariates that reduced the variance of clearance from plasma and the volume of distribution by more than 10% were postnatal age (43 and 28%, respectively) and body weight (30.4 and 17.4%, respectively). The expected reduction of clearance was about 10% for the plasma creatinine concentration. The other covariates studied (Apgar scores, plasma urea concentration, gestational age, sex) were found to have little effect. Simulations showed that a smaller percentage of patients had a maximum concentration in plasma/MIC ratio greater than 8 with a regimen of 7.5 mg/kg of body weight twice daily than with a regimen of 15 mg/kg once a day for MICs of 1 to 8 mg/liter.
High Prevalence of KatG Ser315Thr Substitution among Isoniazid-Resistant
Mycobacterium tuberculosis
Clinical Isolates from Northwestern Russia, 1996 to 2001
Authors: Mokrousov Igor , Narvskaya Olga , Otten Tatiana , Limeschenko Elena , Steklova Lidia , Vyshnevskiy Boris
Published: July 2002

A total of 204 isoniazid (INH)-resistant strains of
Mycobacterium tuberculosis
isolated from different patients in the northwestern region of Russia from 1996 to 2001 were screened by a PCR-restriction fragment length polymorphism (RFLP) assay. This assay uses
Hap
II cleavage of an amplified fragment of the
katG
gene to detect the transversion 315AGC→ACC (Ser→Thr), which is associated with INH resistance. This analysis revealed a 93.6% prevalence of the
katG
S315T mutation in strains from patients with both newly and previously diagnosed cases of tuberculosis (TB). This mutation was not found in any of 57 INH-susceptible isolates included in the study. The specificity of the assay was 100%; all isolates that contained the S315T mutation were classified as resistant by a culture-based susceptibility testing method. The Beijing genotype, defined by IS
6110
-RFLP analysis and the spacer oligonucleotide typing (spoligotyping) method, was found in 60.3% of the INH-resistant strains studied. The
katG
S315T shift was more prevalent among Beijing genotype strains than among non-Beijing genotype strains: 97.8 versus 84.6%, respectively, for all isolates, including those from patients with new and previously diagnosed cases, isolated from 1999 to 2001 and 100.0 versus 86.5%, respectively, for isolates from patients with new cases isolated from 1996 to 2001. The design of this PCR-RFLP assay allows the rapid and unambiguous identification of the
katG
315ACC mutant allele. The simplicity of the assay permits its implementation into routine practice in clinical microbiology laboratories in regions with a high incidence of TB where this mutation is predominant, including northwestern Russia.
Bactericidal Activity of Increasing Daily and Weekly Doses of Moxifloxacin in Murine Tuberculosis
Authors: Yoshimatsu Tetsuyuki , Nuermberger Eric , Tyagi Sandeep , Chaisson Richard , Bishai William , Grosset Jacques
Published: July 2002

Moxifloxacin (MXF) is a new 8-methoxyquinolone with potent activity against
Mycobacterium tuberculosis
and a half-life of 9 to 12 h in humans. Previous in vivo studies using daily doses of 100 mg/kg of body weight have demonstrated bactericidal activity comparable to that of isoniazid (INH) in a murine model of tuberculosis (TB). Recent pharmacokinetic data suggest that MXF may have been underadministered in these studies and that a 400-mg/kg dose in mice better approximates the area under the concentration-time curve obtained in humans after a 400-mg oral dose. Therefore, the bactericidal activity of MXF in doses up to 400 mg/kg given daily or weekly for 28 days was assessed in mice infected intravenously with 5 × 10
6
CFU of
M. tuberculosis
. INH was used as a positive control. After 3 days of daily therapy, the CFU counts from splenic homogenates for mice treated with MXF in doses of 100 to 400 mg/kg/day were lower than those from pretreatment controls. No significant differences in CFU counts were seen when mice receiving INH or MXF at 50 mg/kg/day were compared to pretreatment controls. After 28 days of therapy, dose-dependent reductions in CFU counts in splenic homogenates were seen for daily MXF therapy. The maximum bactericidal effect was seen with daily doses of 400 mg/kg, which resulted in a reduction in CFU counts of 1 log
10
greater than that with INH treatment, although the difference was not statistically significant. CFU counts from lung homogenates after 28 days of therapy were significantly lower in all treatment groups than in untreated controls. The weekly administration of MXF in doses ranging from 50 to 400 mg/kg resulted in no significant bactericidal activity. Mice receiving daily MXF doses of 200 and 400 mg/kg/day failed to gain weight and appeared ill after 28 days of therapy, findings suggestive of drug toxicity. In conclusion, MXF has dose-dependent bactericidal activity against
M. tuberculosis
in the mouse when given in doses up to 400 mg/kg, where its pharmacokinetic profile better approximates that of standard human dosages. Combination regimens which take advantage of the enhanced pharmacodynamic profile of MXF at these doses have the potential to shorten the course of antituberculous therapy or allow more intermittent (i.e., once-weekly) therapy and should be evaluated in the mouse model of TB.
Chromosome-Encoded Class D β-Lactamase OXA-23 in
Proteus mirabilis
Authors: Bonnet R. , Marchandin H. , Chanal C. , Sirot D. , Labia R. , De Champs C. , Jumas-Bilak E. , Sirot J.
Published: July 2002

Ten nonrepetitive
Proteus mirabilis
isolates, which were collected over 4 years (1996 to 1999) at the teaching hospital of Clermont-Ferrand, France, produced class D carbapenemase OXA-23. MICs of imipenem were 0.25 to 0.5 μg/ml for these clinical isolates. Molecular typing revealed that the 10
P. mirabilis
isolates originated from the same clonal strain. Hybridization of I-
Ceu
I-generated chromosome fragments with a
bla
OXA-23
probe showed that the gene was chromosome encoded in the
P. mirabilis
strain.
Inactivation of
Escherichia coli
and
Listeria innocua
in Milk by Combined Treatment with High Hydrostatic Pressure and the Lactoperoxidase System
Authors: García-Graells Cristina , Valckx Caroline , Michiels Chris W.
Published: July 2002

We have studied inactivation of four strains each of
Escherichia coli
and
Listeria innocua
in milk by the combined use of high hydrostatic pressure and the lactoperoxidase-thiocyanate-hydrogen peroxide system as a potential mild food preservation method. The lactoperoxidase system alone exerted a bacteriostatic effect on both species for at least 24 h at room temperature, but none of the strains was inactivated. Upon high-pressure treatment in the presence of the lactoperoxidase system, different results were obtained for
E. coli
and
L. innocua
. For none of the
E. coli
strains did the lactoperoxidase system increase the inactivation compared to a treatment with high pressure alone. However, a strong synergistic interaction of both treatments was observed for
L. innocua
. Inactivation exceeding 7 decades was achieved for all strains with a mild treatment (400 MPa, 15 min, 20°C), which in the absence of the lactoperoxidase system caused only 2 to 5 decades of inactivation depending on the strain. Milk as a substrate was found to have a considerable effect protecting
E. coli
and
L. innocua
against pressure inactivation and reducing the effectiveness of the lactoperoxidase system under pressure on
L. innocua
. Time course experiments showed that
L. innocua
counts continued to decrease in the first hours after pressure treatment in the presence of the lactoperoxidase system.
E. coli
counts remained constant for at least 24 h, except after treatment at the highest pressure level (600 MPa, 15 min, 20°C), in which case, in the presence of the lactoperoxidase system, a transient decrease was observed, indicating sublethal injury rather than true inactivation.
Zoospore Homing and Infection Events: Effects of the Biocontrol Bacterium
Burkholderia cepacia
AMMDR1 on Two Oomycete Pathogens of Pea (
Pisum sativum
L.)
Authors: Heungens K. , Parke J. L.
Published: July 2002

Burkholderia cepacia
AMMDR1 is a biocontrol agent that protects pea and sweet corn seeds from Pythium damping-off in field experiments. The goal of this work was to understand the effect of
B. cepacia
AMMDR1 on
Pythium aphanidermatum
and
Aphanomyces euteiches
zoospore homing events and on infection of pea seeds or roots. In vitro,
B. cepacia
AMMDR1 caused zoospore lysis, prevented cyst germination, and inhibited germ tube growth of both oomycetes.
B. cepacia
AMMDR1 also reduced the attractiveness of seed exudates to
Pythium
zoospores to nondetectable levels. However, when present at high levels on seeds,
B. cepacia
AMMDR1 had little net effect on zoospore attraction, probably because it also enhanced seed exudation. Seed-applied
B. cepacia
AMMDR1 dramatically reduced the incidence of infection by
Pythium
zoospores in situ compared with an antibiosis-deficient Tn
5
mutant strain. This mutant strain also decreased
Pythium
infection incidence to some extent, but only when the pathogen inoculum potential was low.
B. cepacia
AMMDR1 did not affect attraction of
Aphanomyces
zoospores or Aphanomyces root rot incidence. These results suggest that
B. cepacia
AMMDR1 controls
P. aphanidermatum
largely through antibiosis, but competition for zoospore-attracting compounds can contribute to the effect. Differences in suppression of
Aphanomyces
and
Pythium
are discussed in relation to differences in the ecology of the two pathogens.
Degradation ofn-Hexadecane and Its Metabolites byPseudomonas aeruginosaunder Microaerobic and Anaerobic Denitrifying Conditions
Authors: Chayabutra Chawala , Ju Lu-Kwang
Published: July 2002

A strategy for sequential hydrocarbon bioremediation is proposed. The initial O2-requiring transformation is effected by aerobic resting cells, thus avoiding a high oxygen demand. The oxygenated metabolites can then be degraded even under anaerobic conditions when supplemented with a highly water-soluble alternative electron acceptor, such as nitrate. To develop the new strategy, some phenomena were studied by examiningPseudomonas aeruginosafermentation. The effects of dissolved oxygen (DO) concentration onn-hexadecane biodegradation were investigated first. Under microaerobic conditions, the denitrification rate decreased as the DO concentration decreased, implying that the O2-requiring reactions were rate limiting. The effects of different nitrate and nitrite concentrations were examined next. When cultivated aerobically in tryptic soy broth supplemented with 0 to 0.35 g of NO2−-N per liter, cells grew in all systems, but the lag phase was longer in the presence of higher nitrite concentrations. However, under anaerobic denitrifying conditions, even 0.1 g of NO2−-N per liter totally inhibited cell growth. Growth was also inhibited by high nitrate concentrations (>1 g of NO3−-N per liter). Cells were found to be more sensitive to nitrate or nitrite inhibition under denitrifying conditions than under aerobic conditions. Sequential hexadecane biodegradation byP. aeruginosawas then investigated. The initial fermentation was aerobic for cell growth and hydrocarbon oxidation to oxygenated metabolites, as confirmed by increasing dissolved total organic carbon (TOC) concentrations. The culture was then supplemented with nitrate and purged with nitrogen (N2). Nitrate was consumed rapidly initially. The live cell concentration, however, also decreased. The aqueous-phase TOC level decreased by about 40% during the initial active period but remained high after this period. Additional experiments confirmed that only about one-half of the derived TOC was readily consumable under anaerobic denitrifying conditions.
Survival of
Bifidobacterium longum
Immobilized in Calcium Alginate Beads in Simulated Gastric Juices and Bile Salt Solution
Authors: Lee Ki-Yong , Heo Tae-Ryeon
Published: July 2002

Bifidobacterium longum
KCTC 3128 and HLC 3742 were independently immobilized (entrapped) in calcium alginate beads containing 2, 3, and 4% sodium alginate. When the bifidobacteria entrapped in calcium alginate beads were exposed to simulated gastric juices and a bile salt solution, the death rate of the cells in the beads decreased proportionally with an increase in both the alginate gel concentration and bead size. The initial cell numbers in the beads affected the numbers of survivors after exposure to these solutions; however, the death rates of the viable cells were not affected. Accordingly, a mathematical model was formulated which expressed the influences of several parameters (gel concentration, bead size, and initial cell numbers) on the survival of entrapped bifidobacteria after sequential exposure to simulated gastric juices followed by a bile salt solution. The model proposed in this paper may be useful for estimating the survival of bifidobacteria in beads and establishing optimal entrapment conditions.
Selective Accumulation May Account for Shellfish-Associated Viral Illness
Authors: Burkhardt William , Calci Kevin R.
Published: July 2002

From 1991 through 1998, 1,266 cases of shellfish-related illnesses were attributed to Norwalk-like viruses. Seventy-eight percent of these illnesses occurred following consumption of oysters harvested from the Gulf Coast during the months of November through January. This study investigated the ability of eastern oysters (
Crassostrea virginica
) to accumulate indicator microorganisms (i.e., fecal coliforms,
Escherichia coli
,
Clostridium perfringens
, and F
+
coliphage) from estuarine water. One-week trials over a 1-year period were used to determine if these indicator organisms could provide insight into the seasonal occurrence of these gastrointestinal illnesses. The results demonstrate that oysters preferentially accumulated F
+
coliphage, an enteric viral surrogate, to their greatest levels from late November through January, with a concentration factor of up to 99-fold. However, similar increases in accumulation of the other indicator microorganisms were not observed. These findings suggest that the seasonal occurrence of shellfish-related illnesses by enteric viruses is, in part, the result of seasonal physiological changes undergone by the oysters that affect their ability to accumulate viral particles from estuarine waters.
Characterization of a Novel β-Galactosidase from
Bifidobacterium adolescentis
DSM 20083 Active towards Transgalactooligosaccharides
Authors: Van Laere Katrien M. J. , Abee Tjakko , Schols Henk A. , Beldman Gerrit , Voragen Alphons G. J.
Published: July 2002

This paper reports on the effects of both reducing and nonreducing transgalactooligosaccharides (TOS) comprising 2 to 8 residues on the growth of
Bifidobacterium adolescentis
DSM 20083 and on the production of a novel β-galactosidase (β-Gal II). In cells grown on TOS, in addition to the lactose-degrading β-Gal (β-Gal I), another β-Gal (β-Gal II) was detected and it showed activity towards TOS but not towards lactose. β-Gal II activity was at least 20-fold higher when cells were grown on TOS than when cells were grown on galactose, glucose, and lactose. Subsequently, the enzyme was purified from the cell extract of TOS-grown
B. adolescentis
by anion-exchange chromatography, adsorption chromatography, and size-exclusion chromatography. β-Gal II has apparent molecular masses of 350 and 89 kDa as judged by size-exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively, indicating that the enzyme is active in vivo as a tetramer. β-Gal II had an optimal activity at pH 6 and was not active below pH 5. Its optimum temperature was 35°C. The enzyme showed highest
V
max
values towards galactooligosaccharides with a low degree of polymerization. This result is in agreement with the observation that during fermentation of TOS, the di- and trisaccharides were fermented first. β-Gal II was active towards β-galactosyl residues that were 1→4, 1→6, 1→3, and 1↔1 linked, signifying its role in the metabolism of galactooligosaccharides by
B. adolescentis
.
Role of Molybdate and Other Transition Metals in the Accumulation of Protochelin by
Azotobacter vinelandii
Authors: Cornish Anthony S. , Page William J.
Published: July 2002

Both molybdate and iron are metals that are required by the obligately aerobic organism
Azotobacter vinelandii
to survive in the nutrient-limited conditions of its natural soil environment. Previous studies have shown that a high concentration of molybdate (1 mM) affects the formation of
A. vinelandii
siderophores such that the tricatecholate protochelin is formed to the exclusion of the other catecholate siderophores, azotochelin and aminochelin. It has been shown previously that molybdate combines readily with catecholates and interferes with siderophore function. In this study, we found that the manner in which each catecholate siderophore interacted with molybdate was consistent with the structure and binding potential of the siderophore. The affinity that each siderophore had for molybdate was high enough that stable molybdo-siderophore complexes were formed but low enough that the complexes were readily destabilized by Fe
3+
. Thus, competition between Fe
3+
and molybdate did not appear to be the primary cause of protochelin accumulation; in addition, we determined that protochelin accumulated in the presence of vanadate, tungstate, Zn
2+
, and Mn
2+
. We found that all five of these metal ions partially inhibited uptake of
55
Fe-protochelin and
55
Fe-azotochelin complexes. Also, each of these metal ions partially inhibited the activity of ferric reductase, an enzyme important in the deferration of ferric siderophores. Our results suggest that protochelin accumulates in the presence of molybdate because protochelin uptake and conversion into its component parts, azotochelin and aminochelin, are inhibited by interference with ferric reductase.
Phosphorylation of Nucleosides by the Mutated Acid Phosphatase from
Morganella morganii
Authors: Mihara Yasuhiro , Utagawa Takashi , Yamada Hideaki , Asano Yasuhisa
Published: July 2002

A novel nucleoside phosphorylation process using the food additive pyrophosphate as the phosphate source was investigated. The
Morganella morganii
gene encoding a selective nucleoside pyrophosphate phosphotransferase was cloned. It was identical to the
M. morganii
PhoC acid phosphatase gene. Sequential in vitro random mutagenesis was performed on the gene by error-prone PCR to construct a mutant library. The mutant library was introduced into
Escherichia coli
, and the transformants were screened for the production of 5′-IMP. One mutated acid phosphatase with an increased phosphotransferase reaction yield was obtained. With
E. coli
overproducing the mutated acid phosphatase, 101 g of 5′-IMP per liter (192 mM) was synthesized from inosine in an 88% molar yield. This improvement was achieved with two mutations, Gly to Asp at position 92 and Ile to Thr at position 171. A decreased

K
m

value for inosine was responsible for the increased productivity.
Enrichment of an Endosulfan-Degrading Mixed Bacterial Culture
Authors: Sutherland Tara D. , Horne Irene , Lacey Michael J. , Harcourt Rebecca L. , Russell Robyn J. , Oakeshott John G.
Published: July 2002

An endosulfan-degrading mixed bacterial culture was enriched from soil with a history of endosulfan exposure. Enrichment was obtained by using the insecticide as the sole source of sulfur. Chemical hydrolysis was minimized by using strongly buffered culture medium (pH 6.6), and the detergent Tween 80 was included to emulsify the insecticide, thereby increasing the amount of endosulfan in contact with the bacteria. No growth occurred in control cultures in the absence of endosulfan. Degradation of the insecticide occurred concomitant with bacterial growth. The compound was both oxidized and hydrolyzed. The oxidation reaction favored thealphaisomer and produced endosulfate, a terminal pathway product. Hydrolysis involved a novel intermediate, tentatively identified as endosulfan monoaldehyde on the basis of gas chromatography-mass spectrometry and chemical derivatization results. The accumulation and decline of metabolites suggest that the parent compound was hydrolyzed to the putative monoaldehyde, thereby releasing the sulfite moiety required for growth. The monoaldehyde was then oxidized to endosulfan hydroxyether and further metabolized to (a) polar product(s). The cytochrome P450 inhibitor, piperonyl butoxide, did not prevent endosulfan oxidation or the formation of other metabolites. These results suggest that this mixed culture is worth investigating as a source of endosulfan-hydrolyzing enzymes for use in enzymatic bioremediation of endosulfan residues.
Autocloning and Amplification of
LIP2
in
Yarrowia lipolytica
Authors: Pignède Georges , Wang Hui-Jie , Fudalej Franck , Seman Michel , Gaillardin Claude , Nicaud Jean-Marc
Published: July 2002

We synthesized a
Yarrowia lipolytica
strain overproducing lipase for industrial applications by using long terminal repeat (ζ) of the
Y. lipolytica
retrotransposon Ylt1 and an allele of
URA3
with a promoter deletion to construct JMP3. JMP3 is a derivative of plasmid pHSS6 carrying a
Not
I-
Not
I cassette which contains a defective
URA3
allele, a polylinker sequence, and the ζ region for targeting to multiple sites in the genome of the recipient. We inserted the
LIP2
gene (encoding extracellular lipase) under the control of the strong
POX2
promoter into JMP3 to generate JMP6. The pHSS6 region was removed by
Not
I digestion prior to transformation. Two
Y. lipolytica
strains transformed with the JMP6
LIP2
cassette had a mean of 10 integrated copies devoid of the
Escherichia coli
region, corresponding to an autocloning event. The copy number in the transformants was stable even after 120 generations in nonselective and lipase-inducing conditions. The resulting strains could produce 0.5 g of active lipase per liter in the supernatant, 40 times more than the single-copy strain with the
LIP2
promoter. This work provides a new expression system in
Y. lipolytica
that results in strains devoid of bacterial DNA and in strains producing a high level of lipase for industrial uses, waste treatment, and pancreatic insufficiency therapy.
Phylogenetic Specificity and Reproducibility and New Method for Analysis of Terminal Restriction Fragment Profiles of 16S rRNA Genes from Bacterial Communities
Authors: Dunbar John , Ticknor Lawrence O. , Kuske Cheryl R.
Published: July 2002

Terminal restriction fragment (TRF) analysis of 16S rRNA genes is an increasingly popular method for rapid comparison of microbial communities, but analysis of the data is still in a developmental stage. We assessed the phylogenetic resolution and reproducibility of TRF profiles in order to evaluate the limitations of the method, and we developed an essential analysis technique to improve the interpretation of TRF data. The theoretical phylogenetic resolution of TRF profiles was determined based on the specificity of TRFs predicted from 3,908 16S rRNA gene sequences. With sequences from the
Proteobacteria
or gram-positive division, as much as 73% of the TRFs were phylogenetically specific (representing strains from at most two genera). However, the fraction decreased when sequences from the two divisions were combined. The data show that phylogenetic inference will be most effective if TRF profiles represent only a single bacterial division or smaller group. The analytical precision of the TRF method was assessed by comparing nine replicate profiles of a single soil DNA sample. Despite meticulous care in producing the replicates, numerous small, irreproducible peaks were observed. As many as 85% of the 169 distinct TRFs found among the profiles were irreproducible (i.e., not present in all nine replicates). Substantial variation also occurred in the height of synonymous peaks. To make comparisons of microbial communities more reliable, we developed an analytical procedure that reduces variation and extracts a reproducible subset of data from replicate TRF profiles. The procedure can also be used with other DNA fingerprinting techniques for microbial communities or microbial genomes.
Development and Optimization of a Novel Immunomagnetic Separation- Bacteriophage Assay for Detection of
Salmonella enterica
Serovar Enteritidis in Broth
Authors: Favrin Stacy J. , Jassim Sabah A. , Griffiths Mansel W.
Published: July 2002

Salmonella
is the second-leading cause of food-borne illness in most developed countries, causing diarrhea, cramps, vomiting, and often fever. Many rapid methods are available for detection of
Salmonella
in foods, but these methods are often insensitive or expensive or require a high degree of technical ability to perform. In this paper we describe development and characterization of a novel assay that utilizes the normal infection cycle of bacteriophage SJ2 for detection of
Salmonella enterica
serovar Enteritidis in broth. The assay consists of four main stages: (i) capture and concentration of target cells by using immunomagnetic separation (IMS); (ii) infection of the target bacterium with phage; (iii) amplification and recovery of progeny phage; and (iv) assay of progeny phage on the basis of their effect on a healthy population of host cells (signal-amplifying cells). The end point of the assay can be determined by using either fluorescence or optical density measurements. The detection limit of the assay in broth is less than 10
4
CFU/ml, and the assay can be performed in 4 to 5 h. The results of this study demonstrate that the IMS-bacteriophage assay is a rapid, simple, and sensitive technique for detection of
Salmonella
serovar Enteritidis in broth cultures which can be applied to preenriched food samples.