bluuebunny/crossref_metadata_2025 huggingface dataset:
Association between dental health behaviours, mental/physical function and self‐feeding ability among the elderly: a cross‐sectional survey
Authors: Arai Koji , Sumi Yasunori , Uematsu Hiroshi , Miura Hiroko
Published: January 2004

Objectives: The aim of this study was to determine the association between dental health behaviour, mental/physical function and self‐feeding ability among the elderly. Subjects: A total of 414 elderly dental patients aged 65 years and older participated in this study. Methods: A survey was carried out for three years and seven months starting in January 1998 at the Chubu National Hospital. The patients or their carers were examined/interviewed about the severity of senile dementia, dental health behaviour, ability to rinse their mouths, ability to manage dentures, and ability to sit at a table during meals. To assess the association with self‐feeding ability among the elderly, cut‐offs were given for these variables, and then the odds ratios were calculated. Results: The strongest association to self‐feeding ability was marked by inability to rinse their own mouth, followed by inability to manage dentures, inability to sit at a table during meals, severe senile dementia and less frequency of toothbrushing. Conclusion: Elderly who have lost the feeding ability often could not maintain their dental health by themselves. Carers must provide not only a feeding service with acknowledgement of aspiration but oral care to prevent dental disease and fatal pneumonia in the elderly.
Continuing dental care for Highlands elderly: Current practice and attitudes of dental practitioners and home supervisors
Authors: Hally J. , Clarkson J.E. , Newton J.P.
Published: January 2004

Objective: To investigate current practice and attitudes of Highland dentists and home supervisors to continued dental care of elderly residents. Methods: A cross‐sectional questionnaire was designed to survey the current practice and attitudes of Highland dentists and residential care supervisors in their provision of dental care for the elderly at home and in long stay accommodation. Results: The response rate was 94% of dentists and 79% of homes. Despite 86% of dentists providing domiciliary care and 93% of homes transport to a surgery, no more than a quarter of residents had had contact with a dentist in the previous year. The distribution of residents varied with dependant individuals living in nursing units and the least dependant in residential homes. Only 1% of all residents were totally bed bound. Domiciliary patients were less likely to receive continuing care compared with those seen in a surgery and 75% of homes had to initiate dental care. In terms of patient referral, the majority of GDPs would refer uncooperative patients, salaried dentists would refer those with complex medical histories and community dentists would refer those requiring complex treatments. A dental assessment was undertaken in 46% of homes and 81% of these kept a record of dental care. Conclusion: This study highlights the need for a co‐ordinated, seamless continuing dental care service, tailored to the actual needs of the elderly individuals it is designed to serve, particularly in a remote and rural area.
Clinical reversal of root caries using ozone, double‐blind, randomised, controlled 18‐month trial
Authors: Holmes J
Published: January 2004

Objective: To assess the effect of an ozone delivery system, combined with the daily use of a remineralising patient kit, on the clinical severity of non‐cavitated leathery primary root carious lesions (PRCL's), in an older population group. Design: Atotal of 89 subjects, (age range 60–82, mean ± SD, 70.8 ± 6 years), each with two leathery PRCL's, were recruited. The two lesions in each subject were randomly assigned for treatment with ozone or air, in a double‐blind design, in a general dental practice. Subjects were recalled at three, six, 12 and 18 months. Lesions were clinically recorded at each visit as soft, leathery or hard, scored with a validated root caries severity index. Results: There were no observed adverse events. After three months, in the ozone‐treated group, 61 PRCL's (69%) had become hard and none had deteriorated, whilst in the control group, four PRCL's (4%) had become worse (p<0.01). At the six‐month recall, in the ozone group, seven PRCL's (8%) remained leathery, the remaining 82 (92%) PRCL's had become hard, whilst in the control group, 10 PRCL's had become worse (11%) and one had become hard (p<0.01). At 12 and 18 months, 87 Subjects attended. In the ozone group at 12 months, two PRCL's remained leathery, compared to 85 (98%) that had hardened, whilst in the control group 21 (24%) of the PRCL's had progressed from leathery to soft, i.e. became worse, 65 PRCL's (75%) were still leathery, and one remained hard (p<0.01). At 18 months, 87 (100%) of ozone‐treated PRCL's had arrested, whilst in the control group, 32 lesions (37%) of the PRCL's had worsened from leathery to soft (p<0.01), 54 (62%) PRCL's remained leathery and only one of the control PRCL's had reversed (p<0.01). Conclusions: Leathery non‐cavitated primary root caries can be arrested non‐operatively with ozone and remineralising products. This treatment regime is an effective alternative to conventional “drilling and filling”.
In Vitro and In Vivo Efficacies of the New Triazole Albaconazole against
Cryptococcus neoformans
Authors: Miller J. L. , Schell W. A. , Wills E. A. , Toffaletti D. L. , Boyce M. , Benjamin D. K. , Bartroli J. , Perfect J. R.
Published: January 2004

The activity of albaconazole (UR-9825; J. Uriach & Cía. S.A., Barcelona, Spain) was compared to that of fluconazole against 12 isolates of
Cryptococcus neoformans
in vitro and against 1 isolate in vivo in a rabbit model of cryptococcal meningitis. Albaconazole was 100-fold more potent in vitro than fluconazole on a per-weight basis and was fungicidal at potentially relevant concentrations for two isolates. MICs ranged from ≤0.0012 to 1.25 μg/ml, with the MICs for most isolates being between 0.039 and 0.156 μg/ml. Isolates were from human immunodeficiency virus (HIV)-infected and non-HIV-infected patients and were of serotypes A, B, and C; and the fluconazole MICs for some of the isolates were elevated. Infected rabbits were treated with either fluconazole or albaconazole at dosages ranging from 5 to 80 mg/kg of body weight/day. The peak concentrations of albaconazole in serum and cerebrospinal fluid (CSF) averaged 4.14 and 0.62 μg/ml, respectively, in animals receiving 80 mg/kg/day. Comparison of the concentrations in serum and CSF suggested a level of CSF penetration of approximately 15%. Despite limited penetration into the subarachnoid space, at all three doses tested albaconazole was as effective as fluconazole for the treatment of cryptococcal meningitis in rabbits.
In Vitro and In Vivo Activities of the Novel Azole Antifungal Agent R126638
Authors: Odds F. , Ausma J. , Van Gerven F. , Woestenborghs F. , Meerpoel L. , Heeres J. , Vanden Bossche H. , Borgers M.
Published: January 2004

R126638 is a new triazole agent with potent antifungal activity in vitro against various dermatophytes,
Candida
spp., and
Malassezia
spp. Its activity against
Malassezia
spp. in vitro was superior to that of ketoconazole, the agent currently used for the treatment of
Malassezia
-related infections. R126638 showed activity comparable to or lower than that of itraconazole against dermatophytes in vitro; however, in guinea pig models of dermatophyte infections, R126638 given orally consistently showed antifungal activity superior to that of itraconazole, with 50% effective doses (ED
50
s) three- to more than eightfold lower than those of itraconazole, depending on the time of initiation and the duration of treatment. The ED
50
of R126638 in a mouse dermatophytosis model was more than fivefold lower than that of itraconazole. These data indicate that if the effects of R126638 seen when it is used to treat animals can be extrapolated to humans, the novel compound would be expected to show effects at doses lower than those of existing drugs and, hence, present a lower risk for side effects.
Replacement of Broad-Spectrum Cephalosporins by Piperacillin-Tazobactam: Impact on Sustained High Rates of Bacterial Resistance
Authors: Bantar Carlos , Vesco Eduardo , Heft Claudia , Salamone Francisco , Krayeski Marcelo , Gomez Hernán , Coassolo María Alicia , Fiorillo Alejandro , Franco Diego , Arango Carina , Duret Fernando , Oliva María Eugenia
Published: January 2004

We have previously observed a significant reduction of ceftriaxone resistance in
Proteus mirabilis
associated with an increase in the use of cefepime, along with a decrease in the consumption of broad-spectrum cephalosporins (CEP). However, we did not observe such a reduction with
Klebsiella pneumoniae
. Therefore, we sought to determine whether replacement of CEP by piperacillin-tazobactam might be useful in reducing sustained high rates of CEP resistance by this organism. We used a 6-month “before and after model”; during the second (intervention) period, most prescriptions of CEP were changed to piperacillin-tazobactam at the pharmacy. No additional barrier precautions were undertaken. During intervention, consumption of ceftazidime decreased from 17.73 to 1.14 defined daily doses (DDD) per 1,000 patient-days (
P
< 0.0001), whereas that of piperacillin-tazobactam increased from 0 to 30.57 DDD per 1,000 patient-days (
P
< 0.0001). The levels of resistance to CEP by
K. pneumoniae
and
P. mirabilis
decreased from 68.4 and 57.9% to 37.5 and 29.4%, respectively (
P
< 0.05). We conclude that replacement of ceftazidime by piperacillin-tazobactam might be a suitable strategy to decrease endemic CEP resistance by
K. pneumoniae
and
P. mirabilis
, even where there are high bacterial resistance rates and irrespective of any additional precautions for controlling nosocomial infection.
Antibody Array-Generated Profiles of Cytokine Release from THP-1 Leukemic Monocytes Exposed to Different Amphotericin B Formulations
Authors: Turtinen Lloyd W. , Prall David N. , Bremer Lindsay A. , Nauss Rachel E. , Hartsel Scott C.
Published: January 2004

Cytokine antibody arrays were used to establish the profiles of cytokine release from THP-1 monocytes exposed to different amphotericin B (AMB) drug delivery systems. Fungizone (FZ) and Amphotec (ABCD) caused the release of significantly more inflammatory molecules and the release of inflammatory molecules at higher levels than either AmBisome (L-AMB) or Abelcet (ABLC) after 6 h of treatment. Specifically, tumor necrosis factor alpha (TNF-α), interleukin-8 (IL-8), GRO-(αβγ), monocyte chemoattractant protein-1 (MCP-1), RANTES, IL-10, and IL-6 were detected and semiquantified with a chemiluminscence imaging system. TNF-α, IL-8, and MCP-1 were the most predominant; however, little if any TNF-α was present in ABLC- or L-AMB-treated cultures. The TNF- α and IL-8 levels determined by quantitative enzyme-linked immunosorbent assay correlated with the relative cytokine levels measured by using the antibody arrays. Although the viabilities of THP-l monocytes in all AMB-treated cultures were similar by trypan blue exclusion, the amount of lactic dehydrogenase released was significantly larger in FZ- and ABCD-treated cultures than in L-AMB- and ABLC-treated cultures, indicating more membrane perturbations with those formulations. Membrane cation channel formation was also measured in model cholesterol-containing large unilamellar vesicles to directly assess the ion channel formation ability of the system. Only FZ and ABCD induced significant ion currents at concentrations less than 1.5 × 10
−5
M. These results may help provide rationales for the immediate cytokine-mediated side effects observed with FZ and ABCD and the reduced side effects observed with L-AMB and ABLC.
Oral Treatment of Cowpox and Vaccinia Virus Infections in Mice with Ether Lipid Esters of Cidofovir
Authors: Quenelle Debra C. , Collins Deborah J. , Wan W. Brad , Beadle James R. , Hostetler Karl Y. , Kern Earl R.
Published: January 2004

Four newly synthesized ether lipid esters of cidofovir (CDV), hexadecyloxypropyl-CDV (HDP-CDV), octadecyloxyethyl-CDV (ODE-CDV), oleyloxypropyl-CDV (OLP-CDV), and oleyloxyethyl-CDV (OLE-CDV), were found to have enhanced activities against vaccinia virus (VV) and cowpox virus (CV) in vitro compared to those of CDV. The compounds were administered orally and were evaluated for their efficacies against lethal CV or VV infections in mice. HDP-CDV, ODE-CDV, and OLE-CDV were effective at preventing mortality from CV infection when treatments were initiated 24 h after viral inoculation, but only HDP-CDV and ODE-CDV maintained efficacy when treatments were initiated as late as 72 h postinfection. Oral pretreatment with HDP-CDV and ODE-CDV were also effective when they were given 5, 3, or 1 day prior to inoculation with CV, even when each compound was administered as a single dose. Both HDP-CDV and ODE-CDV were also effective against VV infections when they were administered orally 24 or 48 h after infection. In animals treated with HDP-CDV or ODE-CDV, the titers of both CV and VV in the liver, spleen, and kidney were reduced 3 to 7 log
10
. In contrast, virus replication in the lungs was not significantly reduced. These data indicate that HDP-CDV or ODE-CDV given orally is as effective as CDV given parenterally for the treatment of experimental CV and VV infections and suggest that these compounds may be useful for the treatment of orthopoxvirus infections in humans.
Orally Active Fusion Inhibitor of Respiratory Syncytial Virus
Authors: Cianci Christopher , Yu Kuo-Long , Combrink Keith , Sin Ny , Pearce Bradley , Wang Alan , Civiello Rita , Voss Stacey , Luo Guangxiang , Kadow Kathy , Genovesi Eugene V. , Venables Brian , Gulgeze Hatice , Trehan Ashok , James Jennifer , Lamb Lucinda , Medina Ivette , Roach Julia , Yang Zheng , Zadjura Lisa , Colonno Richard , Clark Junius , Meanwell Nicholas , Krystal Mark
Published: January 2004

BMS-433771 was found to be a potent inhibitor of respiratory syncytial virus (RSV) replication in vitro. It exhibited excellent potency against multiple laboratory and clinical isolates of both group A and B viruses, with an average 50% effective concentration of 20 nM. Mechanism-of-action studies demonstrated that BMS-433771 inhibits the fusion of lipid membranes during both the early virus entry stage and late-stage syncytium formation. After isolation of resistant viruses, resistance was mapped to a series of single amino acid mutations in the F1 subunit of the fusion protein. Upon oral administration, BMS-433771 was able to reduce viral titers in the lungs of mice infected with RSV. This new class of orally active RSV fusion inhibitors offers potential for clinical development.
Treatment of Advanced Human Immunodeficiency Virus Type 1 Disease with the Viral Entry Inhibitor PRO 542
Authors: Jacobson Jeffrey M. , Israel Robert J. , Lowy Israel , Ostrow Nancy A. , Vassilatos Linda S. , Barish Melanie , Tran Diep N. H. , Sullivan Brian M. , Ketas Thomas J. , O'Neill Tobias J. , Nagashima Kirsten A. , Huang Wei , Petropoulos Christos J. , Moore John P. , Maddon Paul J. , Olson William C.
Published: January 2004

Viral entry inhibitors represent an emerging mode of therapy for human immunodeficiency virus type 1 (HIV-1) infection. PRO 542 (CD4-immunoglobulin G2) is a tetravalent CD4-immunoglobulin fusion protein that broadly neutralizes primary HIV-1 isolates. PRO 542 binds to the viral surface glycoprotein gp120 and blocks attachment and entry of virus into CD4
+
cells. Previously, PRO 542 demonstrated antiviral activity without significant toxicity when tested at single doses ranging to 10 mg/kg. In this study, 12 HIV-infected individuals were treated with 25-mg/kg single-dose PRO 542 and then monitored for safety, antiviral effects, and PRO 542 pharmacokinetics for 6 weeks. The study examined two treatment cohorts that differed in the extent of HIV-1 disease progression. PRO 542 at 25 mg/kg was well tolerated and demonstrated a serum half-life of 3 days. Statistically significant acute reductions in HIV-1 RNA levels were observed across all study patients, and greater antiviral effects were observed in the cohort of patients with more advanced HIV-1 disease. In advanced disease (HIV-1 RNA > 100,000 copies/ml; CD4 lymphocytes < 200 cells/mm
3
), PRO 542 mediated an 80% response rate and statistically significant ≈0.5 log
10
mean reductions in viral load for 4 to 6 weeks posttreatment. Similar findings were obtained in an analysis of all (
n
= 11) advanced disease patients treated to date with single doses of PRO 542 ranging from 1 to 25 mg/kg. In addition, a significant correlation was observed between antiviral effects observed in vivo and viral susceptibility to PRO 542 in vitro. The findings support continued development of PRO 542 for salvage therapy of advanced HIV-1 disease.
Pharmacokinetics of Saquinavir plus Low-Dose Ritonavir in Human Immunodeficiency Virus-Infected Pregnant Women
Authors: Acosta Edward P. , Bardeguez Arlene , Zorrilla Carmen D. , Van Dyke Russell , Hughes Michael D. , Huang Sharon , Pompeo Lisa , Stek Alice M. , Pitt Jane , Watts D. Heather , Smith Elizabeth , Jiménez Eleanor , Mofenson Lynne ,   the Pediatric AIDS Clinical Trials Group 386 Protocol Team
Published: January 2004

The physiologic changes that occur during pregnancy make it difficult to predict antiretroviral pharmacokinetics (PKs), but few data exist on the PKs of protease inhibitors in human immunodeficiency virus (HIV)-infected pregnant women. The objective of the present study was to determine the PKs of ritonavir (RTV)-enhanced saquinavir (SQV) in HIV-infected pregnant women by an area under the curve (AUC)-targeted approach. A phase I, formal PK evaluation was conducted with HIV-infected pregnant woman during gestation, during labor and delivery, and at 6 weeks postpartum. The SQV-RTV regimen was 800/100 mg twice a day (b.i.d.), and nucleoside analogs were administered concomitantly. The SQV exposure targeted was an AUC at 24 h of 10,000 ng · h/ml. Participants were evaluated for 12-h steady-state PKs at each time period. Thirteen subjects completed the PK evaluations during gestation, 7 completed the PK evaluations at labor and delivery, and 12 completed the PK evaluations postpartum. The mean baseline weight was 67.4 kg, and the median length of gestation was 23.3 weeks. All subjects achieved SQV exposures in excess of the target AUC. The SQV AUCs at 12 h (AUC
12
s) during gestation (29,373 ± 17,524 ng · h/ml [mean ± standard deviation]), during labor and delivery (26,189 ± 22,138 ng · h/ml), and during the postpartum period (35,376 ± 26,379 ng · h/ml) were not significantly different. The mean values of the PK parameters for RTV were lower during gestation than during the postpartum period: for AUC
12
, 7,811 and 13,127 ng · h/ml, respectively; for trough concentrations, 376 and 632 ng/ml, respectively; and for maximum concentrations, 1,256 and 2,252 ng/ml, respectively (
P
≤ 0.05 for all comparisons). This is the first formal PK evaluation of a dual protease inhibitor regimen with HIV-infected pregnant women. The level of SQV exposure was sufficient at each time of evaluation. These data demonstrate large variability in SQV and RTV concentrations and suggest that RTV concentrations are altered by pregnancy. These PK results suggest that SQV-RTV at 800/100 mg b.i.d. appears to be a reasonable treatment option for this population.
Natural Variation of Drug Susceptibility in Wild-Type Human Immunodeficiency Virus Type 1
Authors: Parkin N. T. , Hellmann N. S. , Whitcomb J. M. , Kiss L. , Chappey C. , Petropoulos C. J.
Published: January 2004

Wild-type viruses from the ViroLogic phenotype-genotype database were evaluated to determine the upper confidence limit of the drug susceptibility distributions, or “biological cutoffs,” for the PhenoSense HIV phenotypic drug susceptibility assay. Definition of the natural variation in drug susceptibility in wild-type human immunodeficiency virus (HIV) type 1 isolates is necessary to determine the prevalence of innate drug resistance and to assess the capability of the PhenoSense assay to reliably measure subtle reductions in drug susceptibility. The biological cutoffs for each drug, defined by the 99th percentile of the fold change in the 50% inhibitory concentration distributions or the mean fold change plus 2 standard deviations, were lower than those previously reported for other phenotypic assays and lower than the clinically relevant cutoffs previously defined for the PhenoSense assay. The 99th percentile fold change values ranged from 1.2 (tenofovir) to 1.8 (zidovudine) for nucleoside reverse transcriptase RT inhibitors (RTIs), from 3.0 (efavirenz) to 6.2 (delavirdine) for nonnucleoside RTIs, and from 1.6 (lopinavir) to 3.6 (nelfinavir) for protease inhibitors. To evaluate the potential role of intrinsic assay variability in the observed variations in the drug susceptibilities of wild-type isolates, 10 reference viruses with different drug susceptibility patterns were tested 8 to 30 times each. The median coefficients of variation in fold change for the reference viruses ranged from 12 to 18% for all drugs except zidovudine (32%), strongly suggesting that the observed differences in wild-type virus susceptibility to the different drugs is related to intrinsic virus variability rather than assay variability. The low biological cutoffs and assay variability suggest that the PhenoSense HIV assay may assist in defining clinically relevant susceptibility cutoffs for resistance to antiretroviral drugs.
Gag Non-Cleavage Site Mutations Contribute to Full Recovery of Viral Fitness in Protease Inhibitor-Resistant Human Immunodeficiency Virus Type 1
Authors: Myint Lay , Matsuda Masakazu , Matsuda Zene , Yokomaku Yoshiyuki , Chiba Tomoko , Okano Aiko , Yamada Kaneo , Sugiura Wataru
Published: January 2004

It is well documented that human immunodeficiency virus type 1 (HIV-1) Gag cleavage site mutations (CSMs) emerge in conjunction with various HIV-1 mutations for protease inhibitor (PI) resistance and improve viral replication capacity, which is reduced by acquisition of the resistance. However, CSMs are not the only mutations that emerge in Gag during treatment; many mutations other than CSMs (non-CSMs) have been found to accumulate in the Gag region. In the present study we demonstrate the important role of Gag non-CSMs with regard to viral fitness recovery. We selected three Gag-protease sequences with different PI resistance-associated mutations and CSMs from patients with antiretroviral treatment failure. To clarify the significance of CSMs and non-CSMs, four types of recombinant viruses with different patterns in each sequence were constructed. These were the GP type (patient-derived Gag and protease), the P type (HXB2 Gag and patient-derived protease), the GP
−c
type (CSMs removed from the GP type), and the P
+c
type (CSMs in the HXB2 Gag frame and patient-derived protease). By comparison of these four types of recombinant viruses in each patient-derived Gag-protease sequence, we found that non-CSMs, which had no systematic pattern, make a significant contribution to viral fitness recovery. Our findings demonstrate a delicate interaction between the in vivo evolution of Gag and protease to evade drug selective pressure and the importance of Gag in evaluating drug-resistant viruses.
In Vitro Selection and Characterization of Resistance to Macrolides and Related Antibiotics in
Mycoplasma pneumoniae
Authors: Pereyre S. , Guyot C. , Renaudin H. , Charron A. , Bébéar C. , Bébéar C. M.
Published: January 2004

Macrolide-resistant mutants of
Mycoplasma pneumoniae
were selected in vitro from the susceptible reference strain M129, by 23 to 50 serial passages in subinhibitory concentrations of macrolides and related antibiotics, erythromycin A, azithromycin, josamycin, clindamycin, quinupristin, quinupristin-dalfopristin, pristinamycin, and telithromycin. Mutants for which the MICs are increased could be selected with all antibiotics except the streptogramin B quinupristin. Portions of genes encoding 23S rRNA (domains II and V) and ribosomal proteins L4 and L22 of mutants were amplified by PCR, and their nucleotide sequences were compared to those of the susceptible strain M129. No mutation could be detected in domain II of 23S rRNA. Two point mutations in domain V of 23S rRNA, C2611A and A2062G, were selected in the presence of erythromycin A, azithromycin, josamycin, quinupristin-dalfopristin, and telithromycin. Mutants selected in the presence of clindamycin and telithromycin harbored a single amino acid change (H70R or H70L, respectively) in ribosomal protein L4, whereas insertions of one, two, or three adjacent glycines at position 60 (
M. pneumoniae
numbering) were selected in the presence of both streptogramin combinations. Telithromycin was the sole antibiotic that selected for substitutions (P112R and A114T) and deletions (
111
IPRA
114
) in ribosomal protein L22. Three sequential mutational events in 23S rRNA and in both ribosomal proteins were required to categorize the strain as resistant to the ketolide. Azithromycin and erythromycin A were the only selector antibiotics that remained active (MICs, 0.06 and 1 μg/ml, respectively) on their mutants selected after 50 passages.
Genotyping of
Plasmodium falciparum
Pyrimethamine Resistance by Matrix-Assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry
Authors: Marks Florian , Meyer Christian G. , Sievertsen Jürgen , Timmann Christian , Evans Jennifer , Horstmann Rolf D. , May Jürgen
Published: January 2004

Increasing resistance, recrudescences, and treatment failure have led to the replacement of chloroquine with the combination of pyrimethamine (PYR) and sulfadoxine (SDX) as the first-line antimalarial drugs for treatment of uncomplicated
Plasmodium falciparum
malaria in several areas where this disease is endemic. The development of resistance to PYR-SDX is favored by incomplete treatment courses or by subtherapeutic levels in plasma. PYR-SDX resistance has been associated with several single-nucleotide polymorphisms (SNPs) in the
P. falciparum
dihydrofolate reductase (
pfdhfr
) and the
P. falciparum
dihydropteroate synthetase (
pfdhps
) genes. We have established assays based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) that conveniently allow the identification of SNPs associated with PYR resistance. Variants occurring at codon positions 16, 51, 59, and 108 of the
pfdhfr
gene were analyzed by MALDI-TOF MS in synthetic oligonucleotides to determine the detection threshold. In addition, 63 blood samples from subjects with
P. falciparum
parasitemia of various degrees were analyzed. The results were compared to those obtained by DNA sequencing of the respective gene fragment. The results of MALDI-TOF MS and DNA sequencing were consistent in 40 samples. In 23 samples two or three
pfdhfr
variants were detected by MALDI-TOF assays, whereas DNA-sequencing revealed one variant only. Simultaneous detection of two different mutations by biplex assays was, in principle, feasible. As demonstrated by the example of PYR resistance, MALDI-TOF MS allows for rapid and automated high-throughput assessment of drug sensitivity in
P. falciparum
malaria.
TheybxIGene ofBacillus subtilis168 Encodes a Class D β-Lactamase of Low Activity
Authors: Colombo Maria-Luigi , Hanique Sophie , Baurin Stéphane L. , Bauvois Cédric , De Vriendt Kris , Van Beeumen Jozef J. , Frère Jean-Marie , Joris Bernard
Published: January 2004

TheybxIgene ofBacillus subtilis168 encodes a preprotein of 267 amino acid residues, including a putative signal peptide of 23 residues. The YbxI primary structure exhibits high similarity scores with two members of the superfamily of the serine penicillin-recognizing enzymes: the class D β-lactamases and the hydrophilic carboxy-terminal domains of the BlaR and MecR penicillin receptors. To determine the function and the activity of this putative penicillin-recognizing enzyme, we have subcloned theybxIgene in the pET-26b expression vector. Transformation ofEscherichia coliBL21(DE3) by the recombinant plasmid pCIP51 resulted in the export of the mature YbxI in the periplasm as a water-soluble protein. The recombinant protein was purified to 95% homogeneity. YbxI interacts with several β-lactam antibiotics and can hydrolyze some of them. YbxI is not inactivated by clavulanic acid. The YbxI function and its enzymatic activity inB. subtilisremain unknown. The acyl-enzyme obtained after incubation of YbxI with a fluorescent derivative of ampicillin can be detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, confirming that YbxI can be acylated by β-lactam antibiotics. YbxI does not hydrolyze some of the standard substrates ofd-alanyl-d-alanine peptidases, the targets of penicillin. YbxI belongs to the penicillin-recognizing enzyme family but has an activity intermediate between those of a penicillin-binding protein and a β-lactamase.
Plasmid-Mediated 16S rRNA Methylase in
Serratia marcescens
Conferring High-Level Resistance to Aminoglycosides
Authors: Doi Yohei , Yokoyama Keiko , Yamane Kunikazu , Wachino Jun-ichi , Shibata Naohiro , Yagi Tetsuya , Shibayama Keigo , Kato Haru , Arakawa Yoshichika
Published: January 2004

Serratia marcescens
S-95, which displayed an unusually high degree of resistance to aminoglycosides, including kanamycins and gentamicins, was isolated in 2002 from a patient in Japan. The resistance was mediated by a large plasmid which was nonconjugative but transferable to an
Escherichia coli
recipient by transformation. The gene responsible for the aminoglycoside resistance was cloned and sequenced. The deduced amino acid sequence of the resistance gene shared 82% identity with RmtA, which was recently identified as 16S rRNA methylase conferring high-level aminoglycoside resistance in
Pseudomonas aeruginosa
. Histidine-tagged recombinant protein showed methylation activity against
E. coli
16S rRNA. The novel aminoglycoside resistance gene was therefore designated
rmtB
. The genetic environment of
rmtB
was further investigated. The sequence immediately upstream of
rmtB
contained the right end of transposon Tn
3
, including
bla
TEM
, while an open reading frame possibly encoding a transposase was identified downstream of the gene. This is the first report describing 16S rRNA methylase production in
S. marcescens
. The aminoglycoside resistance mechanism mediated by production of 16S rRNA methylase and subsequent ribosomal protection used to be confined to aminoglycoside-producing actinomycetes. However, it is now identified among pathogenic bacteria, including
Enterobacteriaceae
and
P. aeruginosa
in Japan. This is a cause for concern since other treatment options are often limited in patients requiring highly potent aminoglycosides such as amikacin and tobramycin.
Effects of Interferon, Ribavirin, and Iminosugar Derivatives on Cells Persistently Infected with Noncytopathic Bovine Viral Diarrhea Virus
Authors: Durantel David , Carrouée-Durantel Sandra , Branza-Nichita Norica , Dwek Raymond A. , Zitzmann Nicole
Published: January 2004

Persistent infection with hepatitis C virus (HCV) is a major cause of chronic hepatitis in humans. In chronic carriers, the viral infection induces liver damage that predisposes the patient for cirrhosis and can lead to hepatocellular carcinoma. Current chemotherapies are limited to alpha interferon (IFN-α) used either alone or in combination with ribavirin (RBV). In addition to its limited efficacy, this treatment is frequently poorly tolerated because of its side effects. The urgently needed development of new drugs is made difficult by the lack of an in vitro or in vivo infectivity model, and no cell line has been found so far to reliably and reproducibly support HCV infection. For this reason, the closely related pestivirus bovine viral diarrhea virus (BVDV) has sometimes been used as a surrogate in vitro infectivity model. In this study we used an MDBK cell line persistently infected with noncytopathic BVDV to assess the antiviral effect of IFN-α and RBV, the two drugs currently in clinical use against HCV. The same system was then used to evaluate the potential of two classes of iminosugar derivates to clear noncytopathic BVDV infection from MDBK cells. We show that treatment with long-alkyl-chain deoxynojirimycin derivatives, which are inhibitors of the endoplasmic reticulum (ER)-resident α-glucosidases, can greatly reduce the amount of secreted enveloped viral RNA. Long-alkyl-chain deoxygalactonojirimycin derivatives, which do not inhibit ER α-glucosidases, were less potent but still more effective in this system than IFN-α or ribavirin.
Genetic Analysis and Complete Primary Structure of Microcin L
Authors: Pons Anne-Marie , Delalande François , Duarte Mariela , Benoit Stéphanie , Lanneluc Isabelle , Sablé Sophie , Van Dorsselaer Alain , Cottenceau Gilles
Published: January 2004

Escherichia coli
LR05, in addition to producing MccB17, J25, and D93, secretes microcin L, a newly discovered microcin that exhibits strong antibacterial activity against related
Enterobacteriaceae
, including
Salmonella enterica
serovars Typhimurium and Enteritidis. Microcin L was purified using a two-step procedure including solid-phase extraction and reverse-phase C
18
high-performance liquid chromatography. A 4,901-bp region of the DNA plasmid of
E. coli
LR05 was sequenced revealing that the microcin L cluster consists of four genes,
mclC
,
mclI
,
mclA
, and
mclB
. The structural gene
mclC
encoded a 105-amino-acid precursor with a 15-amino-acid N-terminal extension ending with a Gly-Ala motif upstream of the cleavage site. This motif is typical of the class II microcins and other gram-positive bacteriocins exported by ABC transporters. The
mclI
immunity gene was identified upstream of the
mclC
gene and encodes a 51-amino-acid protein with two potential transmembrane domains. Located on the reverse strand, two genes,
mclA
and
mclB
, encoded the proteins MclA and MclB, respectively. They bear strong relatedness with the ABC transporter proteins and accessory factors involved in the secretion of microcins H47, V, E492, and 24. The microcin L genetic system resembles the genetic organization of MccV. Furthermore the MccL primary structure has been determined. It is a 90-amino-acid peptide of 8,884 Da with two disulfide bridges. The N-terminal region has significant homologies with several gram-positive bacteriocins. The C-terminal 32-amino-acid sequence is 87.5% identical to that of MccV. Together, these results strongly indicate that microcin L is a gram-negative class II microcin.
Streptomyces clavuligerus
Has a Second Copy of the Proclavaminate Amidinohydrolase Gene
Authors: Jensen Susan E. , Wong Annie , Griffin Alison , Barton Barry
Published: January 2004

Past genetic studies have indicated that the genes encoding early enzymes of clavulanic acid biosynthesis may be duplicated in
Streptomyces clavuligerus
. We observed cross-hybridizing bands upon Southern analyses of proclavaminate amidinohydrolase (
pah
)-defective mutant strains of
S. clavuligerus
screened with a
pah
-specific probe. The DNA fragment responsible for this cross hybridization was cloned and sequenced and shown to encode a second copy of the
pah
gene. The new
pah
gene (
pah1
) was 1,056 bp in length, and its sequence was 72% identical to that of the original
pah
gene (
pah2
). Disruption mutants with defects in
pah1
showed no significant effects on production of clavulanic acid or any of the clavam metabolites with stereochemistries opposite that of clavulanic acid (
5S
clavams) produced by
S. clavuligerus
when they were grown on starch asparagine or soy medium. However, double mutants with defects in both
pah1
and
pah2
were defective in the production of both clavulanic acid and all of the
5S
clavam metabolites.
Pharmacokinetics of Total and Unbound Ertapenem in Healthy Elderly Subjects
Authors: Musson D. G. , Majumdar A. , Holland S. , Birk K. , Xi L. , Mistry G. , Sciberras D. , Muckow J. , Deutsch P. , Rogers J. D.
Published: January 2004

Ertapenem is a new once-a-day parenteral carbapenem antimicrobial agent. The pharmacokinetics of unbound and total concentrations of ertapenem in plasma were investigated in elderly subjects and compared with historical data from young adults. In a single- and multiple-dose study, healthy elderly males and females (
n
= 14) 65 years old or older were given a 1-g intravenous (i.v.) dose once daily for 7 days. Plasma and urine samples collected for 24 h on days 1 and 7 following administration of the 1-g doses were analyzed by reversed-phase high-performance liquid chromatography. Areas under the concentration-time curve from 0 h to infinity (AUC
0-∞
) for elderly females and males were similar following administration of 1-g single i.v. doses, and thus, the genders were pooled in subsequent analyses. Concentrations in plasma and the half-life of ertapenem were generally higher and longer, respectively, in elderly subjects than in young adults. The mean AUC
0-∞
of total ertapenem in the elderly was 39% higher than that in young subjects following administration of a 1-g dose. The differences were slightly greater for the mean AUC
0-∞
of unbound ertapenem (71%). The unbound fraction of ertapenem in elderly subjects (∼5 to 11%) was generally greater than that in young adults (∼5 to 8%). As in young adults, ertapenem did not accumulate upon multiple dosing in the elderly. The pharmacokinetics of ertapenem in elderly subjects, while slightly different from those in young adults, do not require a dosage adjustment for elderly patients.
New Class of Bacterial Phenylalanyl-tRNA Synthetase Inhibitors with High Potency and Broad-Spectrum Activity
Authors: Beyer Dieter , Kroll Hein-Peter , Endermann Rainer , Schiffer Guido , Siegel Stephan , Bauser Marcus , Pohlmann Jens , Brands Michael , Ziegelbauer Karl , Haebich Dieter , Eymann Christine , Brötz-Oesterhelt Heike
Published: January 2004

Phenylalanyl (Phe)-tRNA synthetase (Phe-RS) is an essential enzyme which catalyzes the transfer of phenylalanine to the Phe-specific transfer RNA (tRNA
Phe
), a key step in protein biosynthesis. Phenyl-thiazolylurea-sulfonamides were identified as a novel class of potent inhibitors of bacterial Phe-RS by high-throughput screening and chemical variation of the screening hit. The compounds inhibit Phe-RS of
Escherichia coli
,
Haemophilus influenzae
,
Streptococcus pneumoniae
, and
Staphylococcus aureus
, with 50% inhibitory concentrations in the nanomolar range. Enzyme kinetic measurements demonstrated that the compounds bind competitively with respect to the natural substrate Phe. All derivatives are highly selective for the bacterial Phe-RS versus the corresponding mammalian cytoplasmic and human mitochondrial enzymes. Phenyl-thiazolylurea-sulfonamides displayed good in vitro activity against
Staphylococcus
,
Streptococcus
,
Haemophilus
, and
Moraxella
strains, reaching MICs below 1 μg/ml. The antibacterial activity was partly antagonized by increasing concentrations of Phe in the culture broth in accordance with the competitive binding mode. Further evidence that inhibition of tRNA
Phe
charging is the antibacterial principle of this compound class was obtained by proteome analysis of
Bacillus subtilis
. Here, the phenyl-thiazolylurea-sulfonamides induced a protein pattern indicative of the stringent response. In addition, an
E. coli
strain carrying a
relA
mutation and defective in stringent response was more susceptible than its isogenic
relA
+
parent strain. In vivo efficacy was investigated in a murine
S. aureus
sepsis model and a
S. pneumoniae
sepsis model in rats. Treatment with the phenyl-thiazolylurea-sulfonamides reduced the bacterial titer in various organs by up to 3 log units, supporting the potential value of Phe-RS as a target in antibacterial therapy.
Epidemiology of Conjugative Plasmid-Mediated AmpC β-Lactamases in the United States
Authors: Alvarez M. , Tran J. H. , Chow N. , Jacoby G. A.
Published: January 2004

A sample of 752 resistant
Klebsiella pneumoniae
,
Klebsiella oxytoca
, and
Escherichia coli
strains from 70 sites in 25 U.S. states and the District of Columbia was examined for transmissibility of resistance to ceftazidime and the nature of the plasmid-mediated β-lactamase involved. Fifty-nine percent of the
K. pneumoniae
, 24% of the
K. oxytoca
, and 44% of the
E. coli
isolates transferred resistance to ceftazidime. Plasmids encoding AmpC-type β-lactamase were found in 8.5% of the
K. pneumoniae
samples, 6.9% of the
K. oxytoca
samples, and 4% of the
E. coli
samples, at 20 of the 70 sites and in 10 of the 25 states. ACT-1 β-lactamase was found at eight sites, four of which were near New York City, where the ACT-1 enzyme was first discovered; ACT-1 β-lactamase was also found in Massachusetts, Pennsylvania, and Virginia. FOX-5 β-lactamase was also found at eight sites, mainly in southeastern states but also in New York. Two
E. coli
strains produced CMY-2, and one
K. pneumoniae
strain produced DHA-1 β-lactamase. Pulsed-field gel electrophoresis and plasmid analysis suggested that AmpC-mediated resistance spread both by strain and plasmid dissemination. All AmpC β-lactamase-containing isolates were resistant to cefoxitin, but so were 11% of strains containing transmissible SHV- and TEM-type extended-spectrum β-lactamases. A β-lactamase inhibitor test was helpful in distinguishing the two types of resistance but was not definitive since 24% of clinical isolates producing AmpC β-lactamase had a positive response to clavulanic acid. Coexistence of AmpC and extended-spectrum β-lactamases was the main reason for these discrepancies. Plasmid-mediated AmpC-type enzymes are thus responsible for an appreciable fraction of resistance in clinical isolates of
Klebsiella
spp. and
E. coli
, are disseminated around the United States, and are not so easily distinguished from other enzymes that mediate resistance to oxyimino-β-lactams.
Duplex Real-Time PCR Assay for Rapid Detection of Ampicillin-Resistant
Enterococcus faecium
Authors: Mohn Stein Christian , Ulvik Arve , Jureen Roland , Willems Rob J. L. , Top Janetta , Leavis Helen , Harthug Stig , Langeland Nina
Published: January 2004

Rapid and accurate identification of carriers of resistant microorganisms is an important aspect of efficient infection control in hospitals. Traditional identification methods of antibiotic-resistant bacteria usually take at least 3 to 4 days after sampling. A duplex real-time PCR assay was developed for rapid detection of ampicillin-resistant
Enterococcus faecium
(ARE). Primers and probes that are used in this assay specifically detected the
d-
Ala
-d-
Ala ligase gene of
E. faecium
and the modified penicillin-binding protein 5 gene (
pbp5
) carrying the Glu-to-Val substitution at position 629 (Val-629) in a set of 129 tested
E. faecium
strains with known
pbp5
sequence. Presence of the Val-629 in the strain set from 11 different countries was highly correlated with ampicillin resistance. In a screening of hospitalized patients, the real-time PCR assay yielded a sensitivity and a specificity for the detection of ARE colonization of 95% and 100%, respectively. The results were obtained 4 h after samples were harvested from overnight broth of rectal swab samples, identifying both species and the resistance marker mutation in
pbp5
. This novel assay reliably identifies ARE 2 to 3 days more quickly than traditional culture methods, thereby increasing laboratory throughput, making it useful for rectal screening of ARE. The assay demonstrates the advantages of real-time PCR for detection of nosocomial pathogens.
Differential Gene Expression in Auristatin PHE-Treated
Cryptococcus neoformans
Authors: Woyke Tanja , Berens Michael E. , Hoelzinger Dominique B. , Pettit George R. , Winkelmann Günther , Pettit Robin K.
Published: January 2004

The antifungal pentapeptide auristatin PHE was recently shown to interfere with microtubule dynamics and nuclear and cellular division in the opportunistic pathogen
Cryptococcus neoformans.
To gain a broader understanding of the cellular response of
C. neoformans
to auristatin PHE, mRNA differential display (DD) and reverse transcriptase PCR (RT-PCR) were applied. Examination of approximately 60% of the cell transcriptome from cells treated with 1.5 times the MIC (7.89 μM) of auristatin PHE for 90 min revealed 29 transcript expression differences between control and drug-treated populations. Differential expression of seven of the transcripts was confirmed by RT-PCR, as was drug-dependent modulation of an additional seven transcripts by RT-PCR only. Among genes found to be differentially expressed were those encoding proteins involved in transport, cell cycle regulation, signal transduction, cell stress, DNA repair, nucleotide metabolism, and capsule production. For example,
RHO1
and an open reading frame (ORF) encoding a protein with 91% similarity to the
Schizophyllum commune
14-3-3 protein, both involved in cell cycle regulation, were down-regulated, as was the gene encoding the multidrug efflux pump Afr1p. An ORF encoding a protein with 57% identity to the heat shock protein HSP104 in
Pleurotus sajor
-
caju
was up-regulated. Also, three transcripts of unknown function were responsive to auristatin PHE, which may eventually contribute to the elucidation of the function of their gene products. Further study of these differentially expressed genes and expression of their corresponding proteins are warranted to evaluate how they may be involved in the mechanism of action of auristatin PHE. This information may also contribute to an explanation of the selectivity of auristatin PHE for
C. neoformans
. This is the first report of drug action using DD in
C. neoformans.
Three-Dimensional Models of Wild-Type and Mutated Forms of Cytochrome P450 14α-Sterol Demethylases from
Aspergillus fumigatus
and
Candida albicans
Provide Insights into Posaconazole Binding
Authors: Xiao Li , Madison Vincent , Chau Andrew S. , Loebenberg David , Palermo Robert E. , McNicholas Paul M.
Published: January 2004

The cytochrome P450 sterol 14α-demethylase enzyme (CYP51) is the target of azole antifungals. Azoles block ergosterol synthesis, and thereby fungal growth, by binding in the active-site cavity of the enzyme and ligating the iron atom of the heme cofactor through a nitrogen atom of the azole. Mutations in and around the CYP51 active site have resulted in azole resistance. In this work, homology models of the CYP51 enzymes from
Aspergillus fumigatus
and
Candida albicans
were constructed based on the X-ray crystal structure of CYP51 from
Mycobacterium tuberculosis
. Using these models, binding modes for voriconazole (VOR), fluconazole (FLZ), itraconazole (ITZ), and posaconazole (POS) were predicted from docking calculations. Previous work had demonstrated that mutations in the vicinity of the heme cofactor had a greater impact on the binding of FLZ and VOR than on the binding of POS and ITZ. Our modeling data suggest that the long side chains of POS and ITZ occupy a specific channel within CYP51 and that this additional interaction, which is not available to VOR and FLZ, serves to stabilize the binding of these azoles to the mutated CYP51 proteins. The model also predicts that mutations that were previously shown to specifically impact POS susceptibility in
A. fumigatus
and
C. albicans
act by interfering with the binding of the long side chain.