bluuebunny/crossref_metadata_2025_split huggingface dataset:
Functional Expression of
Candida albicans
Drug Efflux Pump Cdr1p in a
Saccharomyces cerevisiae
Strain Deficient in Membrane Transporters
Authors: Nakamura Kenjirou , Niimi Masakazu , Niimi Kyoko , Holmes Ann R. , Yates Jenine E. , Decottignies Anabelle , Monk Brian C. , Goffeau Andre , Cannon Richard D. 
Published: July 2002

ABSTRACT

Analysis of the transport functions of individual
Candida albicans
plasma membrane drug efflux pumps is hampered by the multitude of endogenous transporters. We have stably expressed
C. albicans
Cdr1p, the major pump implicated in multiple-drug-resistance phenotypes, from the genomic
PDR5
locus in a
Saccharomyces cerevisiae
mutant (AD1-8u
−
) from which seven major transporters of the ATP-binding cassette (ABC) family have been deleted. High-level expression of Cdr1p, under the control of the
S. cerevisiae PDR5
promoter and driven by
S. cerevisiae
Pdr1p transcriptional regulator mutation
pdr1-3
, was demonstrated by increased levels of mRNA transcription, increased levels of nucleoside triphosphatase activity, and immunodetection in plasma membrane fractions.
S. cerevisiae
AD1-8u
−
was hypersensitive to azole antifungals (the MICs at which 80% of cells were inhibited [MIC
80
s] were 0.625 μg/ml for fluconazole, <0.016 μg/ml for ketoconazole, and <0.016 μg/ml for itraconazole), whereas the strain (AD1002) that overexpressed
C. albicans
Cdr1p was resistant to azoles (MIC
80
s of fluconazole, ketoconazole, and itraconazole, 30, 0.5, and 4 μg/ml, respectively). Drug resistance correlated with energy-dependent drug efflux. AD1002 demonstrated resistance to a variety of structurally unrelated chemicals which are potential drug pump substrates. The controlled overexpression of
C. albicans
Cdr1p in an
S. cerevisiae
background deficient in other pumps allows the functional analysis of pumping specificity and mechanisms of a major ABC transporter involved in drug efflux from an important human pathogen.
Characterization of
emeA
, a
norA
Homolog and Multidrug Resistance Efflux Pump, in
Enterococcus faecalis
Authors: Jonas Brandie M. , Murray Barbara E. , Weinstock George M. 
Published: July 2002

ABSTRACT

We hypothesized that multidrug resistance efflux pumps (MDRs) may be contributing to the drug resistance of enterococci. We recently identified potential MDR-encoding genes in the
Enterococcus faecalis
V583 genome. Among the putative MDRs, we found a gene that encodes a NorA homolog and have characterized this enterococcal MDR in the present study. A mutant from which the enterococcal NorA homolog has been deleted has reduced resistance to several NorA substrates. Complementation of the deletion mutant with the wild-type gene verified the involvement of this enterococcal gene in resistance to ethidium bromide (EtBr) and norfloxacin. Known MDR inhibitors (reserpine, lansoprazole, and verapamil) inhibit the efflux of EtBr and norfloxacin in wild-type strain OG1RF. A fluorescence assay with EtBr allowed us to quantitate the efflux capability of the enterococcal NorA pump. On the basis of these results, we have named this enterococcal gene
emeA
(enterococcal multidrug resistance efflux).
Impact of the Order of Initiation of Fluconazole and Amphotericin B in Sequential or Combination Therapy on Killing of
Candida albicans
In Vitro and in a Rabbit Model of Endocarditis and Pyelonephritis
Authors: Louie Arnold , Kaw Pamela , Banerjee Partha , Liu Weiguo , Chen George , Miller Michael H. 
Published: July 2002

ABSTRACT

In vitro time-kill studies and a rabbit model of endocarditis and pyelonephritis were used to define the impact that the order of exposure of
Candida albicans
to fluconazole (FLC) and amphotericin B (AMB), as sequential and combination therapies, had on the susceptibility of
C. albicans
to AMB and on the outcome. The contribution of FLC-induced resistance to AMB for
C. albicans
also was assessed. In vitro, AMB monotherapy rapidly killed each of four
C. albicans
strains; FLC alone was fungistatic. Preincubation of these fungi with FLC for 18 h prior to exposure to AMB decreased their susceptibilities to AMB for 8 to >40 h. Induced resistance to AMB was transient, but the duration of resistance increased with the length of FLC preincubation. Yeast sequentially incubated with FLC followed by AMB plus FLC (FLC→AMB+FLC) showed fungistatic growth kinetics similar to that of fungi that were exposed to FLC alone. This antagonistic effect persisted for at least 24 h. Simultaneous exposure of
C. albicans
to AMB and FLC [AMB+FLC(simult)] demonstrated activity similar to that with AMB alone for AMB concentrations of ≥1 μg/ml; antagonism was seen using an AMB concentration of 0.5 μg/ml. The in vitro findings accurately predicted outcomes in our rabbit infection model. In vivo, AMB monotherapy and treatment with AMB for 24 h followed by AMB plus FLC (AMB→AMB+FLC) rapidly sterilized kidneys and cardiac vegetations. AMB+FLC(simult) and FLC→AMB treatments were slower in clearing fungi from infected tissues. FLC monotherapy and FLC→AMB+FLC were both fungistatic and were the least active regimens. No adverse interaction was observed between AMB and FLC for the AMB→FLC regimen. However, FLC→AMB treatment was slower than AMB alone in clearing fungi from tissues. Thus, our in vitro and in vivo studies both demonstrate that preexposure of
C. albicans
to FLC reduces fungal susceptibility to AMB. The length of FLC preexposure and whether AMB is subsequently used alone or in combination with FLC determine the duration of induced resistance to AMB.
Antiviral Activity of β-
l
-2′,3′-Dideoxy-2′,3′-Didehydro-5-Fluorocytidine in Woodchucks Chronically Infected with Woodchuck Hepatitis Virus
Authors: Le Guerhier F. , Pichoud C. , Jamard C. , Guerret S. , Chevallier M. , Peyrol S. , Hantz O. , King I. , Trépo C. , Cheng Y.-C. , Zoulim F. 
Published: July 2002

ABSTRACT

The
l
-nucleoside analog β-
l
-2′,3′-dideoxy-2′,3′-didehydro-5-fluorocytidine (β-
l
-Fd4C) was first shown to exhibit potent activity against hepatitis B virus (HBV) in tissue culture and then to significantly inhibit viral spread during acute infection in the duck HBV model (F. Le Guerhier et al., Antimicrob. Agents Chemother. 44:111–122, 2000). We have therefore examined its antiviral activity in a mammalian model of chronic HBV infection, the woodchuck chronically infected with woodchuck hepatitis virus (WHV). Side-by-side comparison of β-
l
-Fd4C and lamivudine administered intraperitoneally during short-term and long-term protocols demonstrated a more profound inhibition of viremia in β-
l
-Fd4C-treated groups. Moreover, β-
l
-Fd4C induced a marked inhibition of intrahepatic viral DNA synthesis compared with that induced by lamivudine. Nevertheless, covalently closed circular (CCC) DNA persistence explained the lack of clearance of infected hepatocytes expressing viral antigens and the relapse of WHV replication after drug withdrawal. Liver histology showed a decrease in the inflammatory activity of chronic hepatitis in woodchucks receiving β-
l
-Fd4C. An electron microscopy study showed the absence of ultrastructural changes of hepatic mitochondria, biliary canaliculi, and bile ducts. However, a loss of weight was observed in all animals, whatever the treatment, as was a transient skin pigmentation in all woodchucks during β-
l
-Fd4C treatment. There was no evidence that lamivudine or β-
l
-Fd4C could prevent the development of hepatocellular carcinoma with the protocols used. These results indicate that β-
l
-Fd4C exhibits a more potent antiviral effect than lamivudine in the WHV model but was not able to eradicate CCC DNA and infected cells from the liver at the dosage and with the protocol used.
Enhanced Expression of the Multidrug Efflux Pumps AcrAB and AcrEF Associated with Insertion Element Transposition in
Escherichia coli
Mutants Selected with a Fluoroquinolone
Authors: Jellen-Ritter A. S. , Kern W. V. 
Published: July 2002

ABSTRACT

The development of fluoroquinolone resistance in
Escherichia coli
may be associated with mutations in regulatory gene loci such as
marRAB
that lead to increased multidrug efflux, presumably through activation of expression of the AcrAB multidrug efflux pump. We found that multidrug-resistant (MDR) phenotypes with enhanced efflux can also be selected by fluoroquinolones from
marRAB
- or
acrAB
-inactivated
E. coli
K-12 strains having a single mutation in the quinolone-resistance-determining region of
gyrA
. Mutant 3-AG100MKX, obtained from a
mar
knockout strain after two selection steps, showed enhanced expression of
acrB
in a reverse transcriptase PCR associated with insertion of IS
186
into the AcrAB repressor gene
acrR
. In vitro selection experiments with
acrAB
knockout strains yielded MDR mutants after a single step. Enhanced efflux in these mutants was due to increased expression of
acrEF
and associated with insertion of IS2 into the upstream region of
acrEF
, presumably creating a hybrid promoter. These observations confirm the importance of efflux-associated nontarget gene mutations and indicate that transposition of genetic elements may have a role in the development of fluoroquinolone resistance in
E. coli
.
Selection and Characterization of Varicella-Zoster Virus Variants Resistant to (
R
)-9-[4-Hydroxy-2-(Hydroxymethy)Butyl]Guanine
Authors: Ng Teresa I. , Shi Yan , Huffaker H. Janette , Kati Warren , Liu Yaya , Chen Chih-Ming , Lin Zhen , Maring Clarence , Kohlbrenner William E. , Molla Akhteruzzaman 
Published: July 2002

ABSTRACT

(
R
)-9-[4-Hydroxy-2-(hydroxymethy)butyl]guanine (H2G) is a potent and selective inhibitor of herpesvirus replication. It is a nucleoside analog, and its triphosphate derivative (H2G-TP) is a competitive inhibitor of herpesvirus DNA polymerases. In this study, the antiviral activities of H2G and acyclovir (ACV) and the development of viral resistance to these agents were compared in varicella-zoster virus (VZV)-infected cells. In plaque reduction assays, the 50% effective concentration of H2G for VZV was 60- to 400-fold lower than that of ACV, depending on the virus strain and the cell line tested. The enhanced efficacy of H2G against VZV can be accounted for in part by the fact that the intaracellular H2G-TP level (>170 pmol/10
6
cells) is higher than the intracellular ACV-TP level (<1 pmol/10
6
cells). In addition, H2G-TP has extended half-lives of 3.9 and 8.6 h in VZV-infected MRC-5 and MeWo cells, respectively. To assess the emergence of H2G-resistant VZV in vitro, VZV was passaged in the presence of increasing concentrations of H2G. Earlier in the passage, when the concentration of H2G was relatively low, the predominant variant had the (A)76 deletion in the viral thymidine kinase (TK) gene. This mutant was identical to an ACV-resistant mutant generated in parallel experiments. However, higher concentrations of H2G appeared to favor a novel mutant, which had deletions of two consecutive nucleotides at positions 805 and 806 of the TK gene. All of these changes introduced frameshift mutations in the TK gene resulting in the expression of truncated polypeptides. H2G-resistant viruses were cross-resistant to ACV, and vice versa.
Uptake, Transport, and Delivery of Antimicrobial Agents by Human Polymorphonuclear Neutrophils
Authors: Mandell Gerald L. , Coleman Elizabeth 
Published: July 2002

ABSTRACT
Polymorphonuclear neutrophils (PMN) are attracted to sites of infection. They have the potential to deliver antimicrobial agents to these sites if the agents enter the cells and do not alter migration. Penicillin G did not enter cells and was not transported by PMN. We found that azithromycin, ciprofloxacin, levofloxacin, moxifloxacin, and telithromycin were concentrated in PMN and transported toward a chemoattractant. These antimicrobial agents were released from the PMN and inhibited the growth of bacteria on test plates.
In Vitro Activities of Moxifloxacin and Other Fluoroquinolones against
Mycoplasma pneumoniae
Authors: Hamamoto Kumiko , Shimizu Takashi , Fujimoto Naoyuki , Zhang Ye , Arai Sumio 
Published: July 2002

ABSTRACT

A total of 105 isolates of
Mycoplasma pneumoniae
were evaluated for susceptibility to moxifloxacin, sparfloxacin, levofloxacin, and ciprofloxacin. Moxifloxacin, a newly synthesized compound, showed the greatest activity. The MICs and MBCs at which 50 and 90% of isolates were affected were 0.15 (MIC
50
and MBC
50
) and 0.3 μg/ml (MIC
90
and MBC
90
) respectively. The results indicate that moxifloxacin might be promising an antimycoplasmal agent.
Biochemical Mechanism of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Resistance to Stavudine
Authors: Lennerstrand Johan , Stammers David K. , Larder Brendan A. 
Published: July 2002

ABSTRACTWe have found a close correlation between viral stavudine (d4T) resistance and resistance to d4T-triphosphate at the human immunodeficiency virus type 1 reverse transcriptase (RT) level. RT from site-directed mutants with 69S-XX codon insertions and/or conventional zidovudine resistance mutations seems to be involved in an ATP-dependent resistance mechanism analogous to pyrophosphorolysis, whereas the mechanism for RT with the Q151M or V75T mutation appears to be independent of added ATP for reducing binding to d4T-triphosphate.
Telithromycin Is Active against
Mycobacterium avium
in Mice despite Lacking Significant Activity in Standard In Vitro and Macrophage Assays and Is Associated with Low Frequency of Resistance during Treatment
Authors: Bermudez Luiz E. , Inderlied Clark B. , Kolonoski Peter , Wu Martin , Aralar Priscilla , Young Lowell S. 
Published: July 2002

ABSTRACT

The activity of telithromycin, a new ketolide, was evaluated in vitro and in vivo against
Mycobacterium avium
complex (MAC) strains. The MIC of telithromycin for several
M. avium
isolates obtained from the blood of AIDS patients ranged from 16 to >128 μg/ml (MIC at which 90% of isolates are inhibited, >128 μg/ml), and the compound did show activity in the macrophage system at concentrations greater than 8 or 16 μg/ml, but this was dependent on the MAC strain used. Telithromycin was then administered to mice infected with MAC strain 101 for 4 weeks at doses of 100, 200, or 400 mg/kg of body weight/day. Treatment with 100 and 200 mg/kg/day was bacteriostatic, but at 400 mg/kg/day telithromycin was bactericidal for MAC strains. The frequency of the emergence of resistance to telithromycin was low despite prolonged usage (12 weeks). This study demonstrates that telithromycin is active in vivo against MAC and warrants further evaluation.
Kappacin, a Novel Antibacterial Peptide from Bovine Milk
Authors: Malkoski Marina , Dashper Stuart G. , O'Brien-Simpson Neil M. , Talbo Gert H. , Macris Mary , Cross Keith J. , Reynolds Eric C. 
Published: July 2002

ABSTRACT

Caseinomacropeptide (CMP) is a heterogeneous C-terminal fragment (residues 106 to 169) of bovine milk κ-casein composed of glycosylated and phosphorylated forms of different genetic variants. We have demonstrated that CMP has growth-inhibitory activity against the oral opportunistic pathogens
Streptococcus mutans
and
Porphyromonas gingivalis
and against
Escherichia coli
. CMP was fractionated using reversed-phase high-performance liquid chromatography (RP-HPLC), and each fraction was tested for activity against
S. mutans
in a 96-well-plate broth assay. Fractions were characterized by N-terminal sequence analysis and mass spectrometry. The active form of CMP was shown to be the nonglycosylated, phosphorylated κ-casein (residues 106 to 169) [κ-casein(106–169)], which we have designated kappacin. Endoproteinase Glu-C was used to hydrolyze CMP, and the generated peptides were separated using RP-HPLC and gel filtration-HPLC and then tested for activity against
S. mutans
. The peptide Ser(
P
)
149
κ-casein-A(138–158) was the only peptide generated by endoproteinase Glu-C digestion that exhibited growth-inhibitory activity. Peptides corresponding to the sequences of the inhibitory peptide Ser(
P
)
149
κ-casein-A(138–158) and its nonphosphorylated counterpart κ-casein-A(138–158) were chemically synthesized and tested for antibacterial activity. The synthetic Ser(
P
)
149
κ-casein-A(138–158) displayed growth-inhibitory activity against
S. mutans
(MIC, 59 μg/ml [26 μM]). The nonphosphorylated peptide, however, did not inhibit growth at the concentrations tested, indicating that phosphorylation is essential for activity.
Antigen Clearance during Treatment of Disseminated Histoplasmosis with Itraconazole versus Fluconazole in Patients with AIDS
Authors: Wheat L. Joseph , Connolly Patricia , Haddad Nicholas , Le Monte Ann , Brizendine Edward , Hafner Richard 
Published: July 2002

ABSTRACT

Antifungal treatment reduces the concentration of
Histoplasma
antigen in blood and urine, supporting a hypothesis that antigen clearance could be used to compare the activity of new agents for treating histoplasmosis. In separate trials in patients with AIDS, clinical response was similar with itraconazole (85%) and fluconazole (74%). Fungal blood cultures at week 4, however, were negative in a significantly higher proportion of patients treated with itraconazole (92.3%) than in those treated with fluconazole (61.9%) (
P
= 0.017). Baseline antigen concentrations were similar in the two groups: serum,
P
= 0.7235; and urine,
P
= 0.1360. After 4 weeks of treatment, the decline in antigen from baseline in serum was similar in the two treatment groups (
P
= 0.5237), as it was in urine (
P
= 0.4679). At week 12, the decline in antigen from baseline in serum also was similar in the two groups (
P
= 0.4911) and in urine (
P
= 0.2786). More rapid clearance of fungemia suggests that itraconazole is more effective than fluconazole in treating histoplasmosis. This study demonstrates that clearance of fungemia is a better measure of antifungal effect than clearance of antigen.
Discrepancy between Uptake and Intracellular Activity of Moxifloxacin in a
Staphylococcus aureus
-Human THP-1 Monocytic Cell Model
Authors: Paillard Delphine , Grellet Jean , Dubois Véronique , Saux Marie-Claude , Quentin Claudine 
Published: July 2002

ABSTRACT

The correlation between uptake of moxifloxacin by THP-1, a continuous line of monocytic cells devoid of intrinsic bactericidal properties, and its activity against
Staphylococcus aureus
ATCC 25923, a susceptible reference strain (MIC and minimal bactericidal concentration of moxifloxacin, 0.1 mg/liter), was studied in a 5-h assay. The uptake of the drug, added to the culture medium at 0.2 to 32 mg/liter, was evaluated by high-performance liquid chromatography. The ratio of the cellular to extracellular concentration of moxifloxacin reached, at equilibrium, 4.36 ± 0.39 in uninfected cells and 6.25 ± 0.41 in infected cells. The intracellular activity of moxifloxacin, introduced into the extracellular fluid at 0.06 to 8 mg/liter, was determined by the enumeration of viable bacteria. At concentrations ≤0.2 mg/liter, the drug inhibited only the intracellular bacterial growth, while at concentrations ≥0.5 mg/liter, it decreased the bacterial inoculum by less than 1 log
10
unit, with a maximum effect at 3 to 4 h, followed by regrowth of surviving bacteria to 80 to 120% of the original level at 5 h. In contrast, when killing curves were determined by using Mueller-Hinton broth with a similar inoculum (10
7
CFU/ml), moxifloxacin at concentrations ≥0.2 mg/liter reduced the inoculum by at least 3 log
10
units at 3 to 4 h, leaving ≤0.1% survival at 24 h. Persisters exhibited a fluoroquinolone susceptibility identical to that of
S. aureus
ATCC 25923. Our data indicate that moxifloxacin at therapeutic extracellular concentrations accumulates approximately sixfold in infected THP-1 cells and remains active intracellularly, but significantly less active than under in vitro conditions.
Pharmacodynamics of Abacavir in an In Vitro Hollow-Fiber Model System
Authors: Drusano G. L. , Bilello P. A. , Symonds W. T. , Stein D. S. , McDowell J. , Bye A. , Bilello J. A. 
Published: July 2002

ABSTRACT

Abacavir is a potent new carbocyclic nucleoside analogue. We employed our hollow-fiber pharmacodynamic modeling system to examine the antiretroviral effects of different abacavir exposures, as well as the impact of the schedule of drug administration on efficacy. Dose ranging of abacavir revealed that a concentration of four times the 50% effective concentration (EC
50
) (approximately the EC
95
) was required to inhibit the replication of human immunodeficiency virus type 1 (HIV-1) (strain MN) either in a continuous-infusion hollow-fiber experiment or in a classical tissue culture flask experiment. In contrast to earlier work with another drug class (HIV-1 protease inhibitors), addition of physiological amounts of the human drug binding proteins albumin and α
1
acid glycoprotein revealed that there was little impact on the antiviral effect of the drug. Comparison of equivalent exposures (an area under the concentration-time curve [AUC] developed by approximately 500 mg per day of orally administered abacavir), either in a continuous-infusion mode or as a single oral dose of abacavir, demonstrated no difference in the ability to suppress either strain III
B
or strain MN. Comparison of administration of 250 mg every 12 h (q12h) versus once-daily administration of 500 mg for strain MN again showed no significant difference in suppressive effect. These experiments were carried out over 8 to 15 days. Because of these promising initial results, we extended the experiment to 30 days and examined three different schedules of administration that generated the same AUC at 24 h (AUC
24
): 300 mg q12h, 600 mg q24h, and 1,200 mg q48h. The aim of the last of these regimens was to definitively demonstrate schedule failure. There was little difference between the 1,200-mg q48h treatment group and the untreated control at 30 days. Likewise, there was little difference between the 600-mg q24h and 300-mg q12h treatment groups. However, at circa day 18 of the experiment, there was a small increase in viral output of p24 in the once-daily dosing unit. Examination of virus from all groups demonstrated no phenotypic or genotypic differences. The small difference in hollow-fiber unit p24 in the once-daily dosing group was not due to emergence of resistance over the 30-day single-drug exposure. We conclude that the dose of abacavir currently being studied in clinical trials (300 mg orally q12h) will be efficacious for the majority of sensitive clinical isolates of HIV-1. These in vitro data also suggest that this drug may be able to be administered to patients on a once-daily basis at a dose of 600 mg.
Azole Cross-Resistance in
Aspergillus fumigatus
Authors: Mosquera J. , Denning D. W. 
Published: July 2002

ABSTRACT

We susceptibility tested 17 clinical isolates of
Aspergillus fumigatus
, for most of which MICs of itraconazole were elevated (MIC at which 50% of the isolates tested are inhibited, 16 μg/ml), against itraconazole, posaconazole, ravuconazole, and voriconazole. Posaconazole was the most active against itraconazole-susceptible isolates. A complex pattern of cross-resistance and hypersusceptibility was seen with voriconazole and ravuconazole, suggesting marked differences in activity and mechanisms of resistance.
Synergistic Activities of Macrolide Antibiotics against
Pseudomonas aeruginosa
,
Burkholderia cepacia
,
Stenotrophomonas maltophilia
, and
Alcaligenes xylosoxidans
Isolated from Patients with Cystic Fibrosis
Authors: Saiman Lisa , Chen Yunhua , Gabriel Pablo San , Knirsch Charles 
Published: July 2002

ABSTRACT

Azithromycin and clarithromycin were paired with other antibiotics to test synergistic activity against 300 multidrug-resistant pathogens isolated from cystic fibrosis (CF) patients. Clarithromycin-tobramycin was most active against
Pseudomonas aeruginosa
and inhibited 58% of strains. Azithromycin-trimethoprim-sulfamethoxazole, azithromycin-ceftazidime, and azithromycin-doxycycline or azithromycin-trimethoprim-sulfamethoxazole inhibited 40, 20, and 22% of
Stenotrophomonas maltophilia
,
Burkholderia cepacia
complex, and
Achromobacter
(
Alcaligenes
)
xylosoxidans
strains, respectively.