bluuebunny/crossref_metadata_2025 huggingface dataset:
High-Efficiency Generation of Antibiotic-Resistant Strains of
Streptococcus pneumoniae
by PCR and Transformation
Authors: Martín-Galiano Antonio J. , de la Campa Adela G.
Published: March 2003

We designed a method by which to generate antibiotic-resistant strains of
Streptococcus pneumoniae
at frequencies 4 orders of magnitude greater than the spontaneous mutation rate. The method is based on the natural ability of this organism to be genetically transformed with PCR products carrying sequences homologous to its chromosome. The genes encoding the targets of ciprofloxacin (
parC
, encoding the ParC subunit of DNA topoisomerase IV), rifampin (
rpoB
, encoding the β subunit of RNA polymerase), and streptomycin (
rpsL
, encoding the S12 ribosomal protein) from susceptible laboratory strain R6 were amplified by PCR and used to transform the same strain. Resistant mutants were obtained with a frequency of 10
−4
to 10
−5
, depending on the fidelity of the DNA polymerase used for PCR amplifications. Ciprofloxacin-resistant mutants, for which the MICs were four-to eightfold higher than that for R6, carried a single mutation of a residue in the quinolone resistance-determining region: S79 (change to A, F, or Y) or D83 (change to N or V). Rifampin-resistant strains, for which the MICs were at least 133-fold higher than that for R6, contained a single mutation within cluster I of
rpoB
: S482 (change to P), Q486 (change to L), D489 (change to V), or H499 (change to L or Y). Streptomycin-resistant mutants, for which the MICs were at least 64-fold higher than that for R6, carried a mutation at either K56 (change to I, R, or T) or K101 (change to E). PCR products obtained from the mutants were able to transform R6 to resistance with high efficiency (>10
4
). This method could be used to efficiently obtain resistant mutants for any drug whose target is known.
Vancomycin Treatment Failure Associated with Heterogeneous Vancomycin-Intermediate
Staphylococcus aureus
in a Patient with Endocarditis and in the Rabbit Model of Endocarditis
Authors: Moore Matthew R. , Perdreau-Remington Françoise , Chambers Henry F.
Published: March 2003

Heterogeneous resistance to vancomycin is thought to precede emergence of intermediate susceptibility to vancomycin in
Staphylococcus aureus
, but the clinical significance of heterogeneous resistance is unknown. Paired
S. aureus
isolates from a patient with endocarditis who relapsed after vancomycin treatment were tested for heterogeneous resistance to vancomycin. The pretreatment and the relapse clinical isolates (strains SF1 and SF2, respectively) were genotyped by pulsed-field gel electrophoresis. Susceptibility to vancomycin was assessed by the broth dilution method, population analysis, and time-kill studies and in the rabbit model of endocarditis. Strains SF1 and SF2 had similar genotypes, and the vancomycin MICs for the strains were ≤2 μg/ml. SF2 exhibited heterogeneous resistance to vancomycin. Vancomycin eradicated SF1 in the rabbit model of endocarditis, while SF2 persisted at pretreatment levels. Vancomycin treatment failure in this patient with endocarditis was attributable to heterogeneous resistance to vancomycin.
Isolation of Antibiotic Hypersusceptibility Mutants of
Acinetobacter
spp. by Selection for DNA Release
Authors: Lee Hyunwoo , Vázquez-Laslop Nora , Klyachko Katya A. , Neyfakh Alex A.
Published: March 2003

Isolation of bacterial mutants hypersusceptible to antibiotics can reveal novel targets for antibiotic potentiators. However, identification of such mutants is a difficult task which normally requires laborious replica plating of thousands of colonies. The technique proposed here allows for the positive selection of genetic knockout mutants leading to hypersusceptibility. This technique, designated SDR (selection for DNA release), involves introduction of random insertions of a marker gene into the chromosome of a highly transformable bacterial species, followed by treatment of the obtained library with an antibiotic at subinhibitory concentrations. DNA released by lysing bacteria is collected and used to transform fresh bacteria, selecting for insertion of the marker gene. These selection cycles are repeated until variants with a hypersusceptibility phenotype caused by insertion of the marker begin to dominate in the library. This approach allowed for isolation of a number of mutants of the gram-negative opportunistic pathogen
Acinetobacter
sp. susceptible to 4- to 16-times-lower concentrations of ampicillin than wild-type bacteria. The mutations affected proteins involved in peptidoglycan turnover and, surprisingly, proteins involved in exopolysaccharide production. A further modification of the SDR technique is described which allows for selecting mutants hypersensitive to agents that affect bacterial physiology but do not cause cell lysis, e.g., inhibitors of translation. This application of SDR is illustrated here by identification of several mutants of
Acinetobacter
sp. with increased susceptibility (two- to fivefold decrease in the MIC) to erythromycin. The same technique can be used to identify prospective targets for potentiators of many other antibacterial agents.
Flucytosine-Fluconazole Cross-Resistance in Purine-Cytosine Permease-Deficient
Candida lusitaniae
Clinical Isolates: Indirect Evidence of a Fluconazole Uptake Transporter
Authors: Noël Thierry , François Fabienne , Paumard Patrick , Chastin Christiane , Brèthes Daniel , Villard Jean
Published: March 2003

An unusual interaction between flucytosine and fluconazole was observed when a collection of 60
Candida lusitaniae
clinical isolates was screened for cross-resistance. Among eight isolates resistant to flucytosine (MIC ≥ 128 μg/ml) and susceptible to fluconazole (0.5 < MIC < 2 μg/ml), four became flucytosine-fluconazole cross resistant when both antifungals were used simultaneously. Fluconazole resistance occurred only in the presence of high flucytosine concentrations, and the higher the fluconazole concentration used, the greater the flucytosine concentration necessary to trigger the cross-resistance. When the flucytosine- and fluconazole-resistant cells were grown in the presence of fluconazole alone, the cells reversed to fluconazole susceptibility. Genetic analyses of the progeny from crosses between resistant and sensitive isolates showed that resistance to flucytosine was derived from a recessive mutation in a single gene, whereas cross-resistance to fluconazole seemed to vary like a quantitative trait. We further demonstrated that the four clinical isolates were susceptible to 5-fluorouracil and that cytosine deaminase activity was unaffected. Kinetic transport studies with [
14
C]flucytosine showed that flucytosine resistance was due to a defect in the purine-cytosine permease. Our hypothesis was that extracellular flucytosine would subsequently behave as a competitive inhibitor of fluconazole uptake transport. Finally, in vitro selection of spontaneous and induced mutants indicated that such a cross-resistance mechanism could also affect other
Candida
species, including
C. albicans
,
C. tropicalis
, and
C. glabrata
. This is the first report of a putative fluconazole uptake transporter in
Candida
species and of a possible resistance mechanism associated with a deficiency in the uptake of this drug.
Integron-Containing IncU R Plasmids pRAS1 and pAr-32 from the Fish Pathogen
Aeromonas salmonicida
Authors: Sørum Henning , L'Abée-Lund Trine M. , Solberg Asta , Wold Anette
Published: March 2003

A 45-kb R plasmid, pRAS1, that confers resistance to tetracyclines, trimethoprim, and sulfonamides was isolated in 1989 from an atypical strain of the fish pathogen
Aeromonas salmonicida
. This plasmid could be transferred by conjugation to
Escherichia coli
with a high degree of efficiency (frequency, 0.48). The following year pRAS1 was isolated from
A. salmonicida
subsp.
salmonicida
in the same area. Incompatibility group U plasmid pRAS1 contained a drug resistance-determining region of 12 kb consisting of a class 1 integron similar to In4 of Tn
1696
but with a
dfrA16
gene cassette inserted. Close to IS
6100
at the right end of Tn4 was a truncated Tn
1721
. Restriction enzyme analysis showed that R plasmid pAr-32, isolated from
A. salmonicida
in Japan in 1970, had the same backbone structure as pRAS1, while the drug resistance-determining region contained a complex class 1 integron with an
aadA2
cassette; the chloramphenicol resistance gene
catA2
, as in In6 of pSa; and a duplicate of the 3′ conserved segment of the integron.
Engineering Anthracycline Biosynthesis toward Angucyclines
Authors: Metsä-Ketelä Mikko , Palmu Kaisa , Kunnari Tero , Ylihonko Kristiina , Mäntsälä Pekka
Published: March 2003

The biosynthesis pathways of two anthracyclines, nogalamycin and aclacinomycin, were directed toward angucyclines by using an angucycline-specific cyclase,
pgaF
, isolated from a silent antibiotic biosynthesis gene cluster. Addition of
pgaF
to a gene cassette that harbored the early biosynthesis genes of nogalamycin resulted in the production of two known angucyclinone metabolites, rabelomycin and its precursor, UWM6. Substrate flexibility of
pgaF
was demonstrated by replacement of the nogalamycin minimal polyketide synthase genes in the gene cassette with the equivalent aclacinomycin genes together with
aknE2
and
aknF
, which specify the unusual propionate starter unit in aclacinomycin biosynthesis. This modification led to the production of a novel angucyclinone, MM2002, in which the expected ethyl side chain was incorporated into the fourth ring.
Imipenem Resistance in a
Salmonella
Clinical Strain Due to Plasmid-Mediated Class A Carbapenemase KPC-2
Authors: Miriagou Vivi , Tzouvelekis Leonidas S. , Rossiter Shannon , Tzelepi Eva , Angulo Frederick J. , Whichard Jean M.
Published: March 2003

A
Salmonella enterica
serotype Cubana isolate exhibiting resistance to most β-lactam antibiotics, including oxyimino-cephalosporins and imipenem, was isolated from a 4-year-old boy with gastroenteritis in Maryland. β-Lactam resistance was mediated by a conjugative plasmid that encoded KPC-2, a class A carbapenemase previously found in a
Klebsiella pneumoniae
isolate from the Maryland area as well. Sequence analysis of the flanking regions indicated a potential association of
bla
KPC-2
with mobile structures.
Use of Genetically Engineered Phage To Deliver Antimicrobial Agents to Bacteria: an Alternative Therapy for Treatment of Bacterial Infections
Authors: Westwater Caroline , Kasman Laura M. , Schofield David A. , Werner Phillip A. , Dolan Joseph W. , Schmidt Michael G. , Norris James S.
Published: March 2003

The emergence and increasing prevalence of multidrug-resistant bacterial pathogens emphasizes the need for new and innovative antimicrobial strategies. Lytic phages, which kill their host following amplification and release of progeny phage into the environment, may offer an alternative strategy for combating bacterial infections. In this study, however, we describe the use of a nonlytic phage to specifically target and deliver DNA encoding bactericidal proteins to bacteria. To test the concept of using phage as a lethal-agent delivery vehicle, we used the M13 phagemid system and the addiction toxins Gef and ChpBK. Phage delivery of lethal-agent phagemids reduced target bacterial numbers by several orders of magnitude in vitro and in a bacteremic mouse model of infection. Given the powerful genetic engineering tools available and the present knowledge in phage biology, this technology may have potential use in antimicrobial therapies and DNA vaccine development.
Bactericidal Activities of Methoxyfluoroquinolones Gatifloxacin and Moxifloxacin against Aerobic and Anaerobic Respiratory Pathogens in Serum
Authors: Stein Gary E. , Schooley Sharon , Tyrrell Kerin L. , Citron Diane M. , Goldstein Ellie J. C.
Published: March 2003

Gatifloxacin (Bristol-Myers Squibb) and moxifloxacin (Bayer) are new methoxyfluoroquinolones with broad-spectrum activity against aerobic and anaerobic pathogens of the respiratory tract. In this investigation, we analyzed the bactericidal activity in serum over time of these antimicrobials against three aerobic (
Streptococcus pneumoniae, Haemophilus influenzae,
and
Staphylococcus aureus
) and four anaerobic (
Peptostreptococcus micros, Peptostreptococcus magnus, Fusobacterium nucleatum,
and
Prevotella melaninogenica
) bacteria associated with respiratory tract infections. Serum samples were obtained from 11 healthy male subjects following a single 400-mg oral dose of gatifloxacin and moxifloxacin. These samples were collected prior to and at 2, 6, 12, and 24 h after the dose of each drug. Gatifloxacin exhibited bactericidal activity for a median of 12 h against
Streptococcus pneumoniae
(MIC = 0.5 μg/ml),
Peptostreptococcus micros
(MIC = 0.25 μg/ml), and
F. nucleatum
(MIC = 0.5 μg/ml) and 24 h against
H. influenzae
(MIC = 0.03 μg/ml),
Staphylococcus aureus
(MIC = 0.125 μg/ml),
Peptostreptococcus magnus
(MIC = 0.125 μg/ml), and
Prevotella melaninogenica
(MIC = 0.5 μg/ml). Moxifloxacin exhibited bactericidal activity for a median of 24 h against
Streptococcus pneumoniae
(MIC = 0.125 μg/ml),
H. influenzae
(MIC = 0.015 μg/ml),
Staphylococcus aureus
(MIC = 0.06 μg/ml),
F. nucleatum
(MIC = 0.5 μg/ml),
Prevotella melaninogenica
(MIC =0.5 μg/ml),
Peptostreptococcus magnus
(MIC = 0.125 μg/ml), and
Peptostreptococcus micros
(MIC = 0.25 μg/ml). The results from this pharmacodynamic study suggest that these fluoroquinolones would have prolonged killing activity against these organisms in vivo and may have clinical utility in the treatment of mixed aerobic-anaerobic respiratory tract infections.
Rifampin Followed by Ceftriaxone for Experimental Meningitis Decreases Lipoteichoic Acid Concentrations in Cerebrospinal Fluid and Reduces Neuronal Damage in Comparison to Ceftriaxone Alone
Authors: Gerber Joachim , Pohl Karin , Sander Valeska , Bunkowski Stephanie , Nau Roland
Published: March 2003

Rifampin (RIF) releases smaller quantities of lipoteichoic acids (LTAs) from
Streptococcus pneumoniae
than ceftriaxone (CRO). Due to the rapid development of resistance, RIF cannot be used as a single agent for therapy of bacterial meningitis. For this reason, we compared the effect of treatment with RIF followed by treatment with CRO (RIF-CRO) or the effect of treatment with clindamycin (CLI) followed by treatment with CRO (CLI-CRO) to that of CRO alone on the concentrations of LTAs and teichoic acids in vitro. The effects of RIF-CRO on LTA concentrations in cerebrospinal fluid (CSF) and on neuronal injury were investigated in a rabbit model of
S. pneumoniae
meningitis. In vitro, bacterial titers were effectively reduced by CRO, RIF-CRO, and CLI-CRO when each drug was used at 10 μg/ml. The levels of release of LTAs after the initiation of therapy were lower in RIF-CRO- and CLI-CRO-treated cultures than in cultures treated with CRO alone (
P
< 0.05 from 3 to 12 h after initiation of treatment). Similarly, in rabbits, the increase in the amount of LTAs in CSF was lower in RIF-CRO-treated animals than in CRO-treated animals (
P
= 0.02). The density of dentate apoptotic granular cells was lower after RIF-CRO therapy than after CRO therapy (medians, 58.4 and 145.6/mm
2
, respectively; 25th quartiles, 36.3 and 81.7/mm
2
, respectively; 75th quartiles, 100.7 and 152.3/mm
2
, respectively;
P
= 0.03). Therefore, initiation of therapy with a protein synthesis-inhibiting antibacterial and continuation of therapy with a combination that includes a β-lactam may be a strategy to decrease neuronal injury in bacterial meningitis.
Daptomycin Pharmacokinetics and Safety following Administration of Escalating Doses Once Daily to Healthy Subjects
Authors: Dvorchik Barry H. , Brazier David , DeBruin Michael F. , Arbeit Robert D.
Published: March 2003

The purpose of this paper is to establish the pharmacokinetics and safety of escalating, once-daily doses of daptomycin, a novel lipopeptide antibiotic active against gram-positive pathogens, including those resistant to methicillin and vancomycin. This phase 1, multiple-dose, double-blind study involved 24 healthy subjects in three dose cohorts (4, 6, and 8 mg/kg of body weight) who were randomized to receive daptomycin or the control at a 3:1 ratio and administered the study medication by a 30-min intravenous infusion every 24 h for 7 to 14 days. Daptomycin pharmacokinetics was assessed by blood and urine sampling. Safety and tolerability were evaluated by monitoring adverse events (AEs) and laboratory parameters. Daptomycin pharmacokinetics was linear through 6 mg/kg, with a slight (∼20%) nonlinearity in the area under the curve and trough concentration at the highest dose studied (8 mg/kg). The pharmacokinetic parameters measured on the median day of the study period, (day 7) were half-life (∼9 h), volume of distribution (∼0.1 liters/kg), systemic clearance (∼8.2 ml/h/kg), and percentage of the drug excreted intact in urine from 0 to 24 h (∼54%). Daptomycin protein binding (mean amount bound, 91.7%) was independent of the drug concentration. No gender effect was observed. All subjects who received daptomycin completed the study. The frequencies and distributions of treatment-emergent AEs were similar for the subjects who received daptomycin and the control subjects. There were no serious AEs and no pattern of dose-related events. The pharmacokinetics of once-daily administration of daptomycin was linear through 6 mg/kg. For all three doses, plasma daptomycin concentrations were consistent and predictable throughout the dosing interval. Daptomycin was well tolerated when it was administered once daily at a dose as high as 8 mg/kg for 14 days.
Activities of Atazanavir (BMS-232632) against a Large Panel of Human Immunodeficiency Virus Type 1 Clinical Isolates Resistant to One or More Approved Protease Inhibitors
Authors: Colonno Richard J. , Thiry Alexandra , Limoli Kay , Parkin Neil
Published: March 2003

To evaluate the cross-resistance profile of the human immunodeficiency virus type 1 protease inhibitor (PI) atazanavir (BMS-232632), a panel of 551 clinical isolates exhibiting a wide array of PI resistance profiles and a variety of genotypic patterns were assayed for susceptibility to atazanavir and six other PIs: amprenavir, indinavir, lopinavir, nelfinavir, ritonavir, and saquinavir. In general, reductions in atazanavir susceptibility in vitro required several amino acid changes and were relatively modest in degree, and susceptibility was retained among isolates resistant to one or two of the currently approved PIs. There was a clear trend toward loss of susceptibility to atazanavir, as isolates exhibited increasing levels of cross-resistance to multiple PIs. Atazanavir appeared to have a distinct resistance profile relative to each of the other six PIs tested based on susceptibility comparisons against this panel of resistant isolates. Analysis of the genotypic profiles of 943 PI-susceptible and -resistant clinical isolates identified a strong correlation between the presence of amino acid changes at specific residues (10I/V/F, 20R/M/I, 24I, 33I/F/V, 36I/L/V, 46I/L, 48V, 54V/L, 63P, 71V/T/I, 73C/S/T/A, 82A/F/S/T, 84V, and 90M) and decreased susceptibility to atazanavir. While no single substitution or combination of substitutions was predictive of atazanavir resistance (change, >3.0-fold), the presence of at least five of these substitutions correlated strongly with loss of atazanavir susceptibility. Mutations associated with reduced susceptibility to each of the other six PIs were also determined.
Phase I Safety and Pharmacokinetic Trials of 1263W94, a Novel Oral Anti-Human Cytomegalovirus Agent, in Healthy and Human Immunodeficiency Virus-Infected Subjects
Authors: Wang Laurene H. , Peck Richard W. , Yin Yin , Allanson Jane , Wiggs Rebecca , Wire Mary Beth
Published: March 2003

1263W94 [maribavir; 5,6-dichloro-2-(isopropylamino)-1, β-
l
-ribofuranosyl-1-
H
-benzimidazole], a novel benzimidazole compound, has been demonstrated to potently and selectively inhibit human cytomegalovirus replication in vitro and to have favorable safety profiles in animal species. Two phase I trials evaluated the safety and pharmacokinetics of escalating single doses of 1263W94 in 13 healthy subjects (dose, 50 to 1,600 mg) and 17 human immunodeficiency virus (HIV)-infected subjects (dose, 100 to 1,600 mg). No severe safety concerns were observed in the evaluation of adverse events, vital signs, electrocardiograms, and clinical laboratory tests following administration of a single dose of 1263W94. The most frequently reported adverse events in both populations were taste disturbance (80%) and headache (53%). 1263W94 was rapidly absorbed following oral administration, with peak concentrations in plasma (
C
max
) occurring 1 to 3 h after dosing. The increases in the
C
max
of 1263W94 and the area under the concentration-time curve from time zero to infinity (AUC
0-∞
) for 1263W94 were dose dependent;
C
max
increased slightly less than proportionally to the dose, and AUC
0-∞
increased slightly more than proportionally to the dose. 1263W94 was rapidly eliminated, with a mean half-life in plasma of 3 to 5 h; the half-life was independent of the dose level. Less than 2% of the 1263W94 dose administered was eliminated unchanged in urine. The principal metabolite of 1263W94 was 4469W94 (which is derived by N-dealkylation of 1263W94 via CYP3A4), which accounted for 30 to 40% of the dose in urine. Greater than 98% of the 1263W94 in plasma is bound to proteins, and the extent of binding appears to be constant over the dose range of 200 to 1,600 mg. In the trial with HIV-infected subjects, consumption of a high-fat meal decreased the 1263W94 AUC
0-∞
and
C
max
in plasma by ∼30%.
Chromosomal
aadD2
Encodes an Aminoglycoside Nucleotidyltransferase in
Bacillus clausii
Authors: Bozdogan Bülent , Galopin Sébastien , Gerbaud Guy , Courvalin Patrice , Leclercq Roland
Published: March 2003

Bacillus clausii
SIN is one of the four strains of
B. clausii
composing a probiotic administered to humans for the prevention of gastrointestinal side effects due to oral antibiotic therapy. The strain is resistant to kanamycin, tobramycin, and amikacin. A gene conferring aminoglycoside resistance was cloned into
Escherichia coli
and sequenced. The gene, called
aadD2
, encoding a putative 246-amino acid protein, shared 47% identity with
ant(4
′
)-Ia
from
Staphylococcus aureus
, which encodes an aminoglycoside 4′-
O
-nucleotidyltransferase. Phosphocellulose paper-binding assays indicated that the gene product was responsible for nucleotidylation of kanamycin, tobramycin, and amikacin. The
aadD2
gene was detected by DNA-DNA hybridization in the three other strains of the probiotic mixture and in the reference strain
B. clausii
DSM8716, although it did not confer resistance in these strains. Mutations in the sequence of the putative promoter for
aadD2
from
B. clausii
SIN resulted in higher identity with consensus promoter sequences and may account for aminoglycoside resistance in that strain. The
aadD2
gene was chromosomally located in all strains and was not transferable by conjugation. These data indicate that chromosomal
aadD2
is specific to
B. clausii
.
Molecular Determination of Point Mutation Haplotypes in the Dihydrofolate Reductase and Dihydropteroate Synthase of
Plasmodium falciparum
in Three Districts of Northern Tanzania
Authors: Pearce Richard J. , Drakeley Chris , Chandramohan Daniel , Mosha Frank , Roper Cally
Published: March 2003

The antimalarial combination of sulfadoxine and pyrimethamine (SP) was introduced as first-line treatment for uncomplicated malaria in Tanzania during 2001 following 18 years of second-line use. The genetic determinants of in vitro resistance to the two drugs individually are shown to be point mutations at seven sites in the dihydrofolate reductase gene (
dhfr
) conferring resistance to pyrimethamine and five sites in the dihydropteroate synthase (
dhps
) gene conferring resistance to sulfadoxine. Different combinations of mutations within each gene confer differing degrees of insensitivity, but information about the frequency with which allelic haplotypes occur has been lacking because of the complicating effects of multiple infection. Here we used a novel high-throughput sequence-specific oligonucleotide probe-based approach to examine the present resistance status of three
Plasmodium falciparum
populations in northern Tanzania. By using surveys of asymptomatic infections and screening for the presence of all known point mutations in
dhfr
and
dhps
genes, we showed that just five
dhfr
and three
dhps
allelic haplotypes are present. High frequencies of both triple-mutant
dhfr
and double-mutant
dhps
mutant alleles were found in addition to significant interregional heterogeneity in allele frequency. In vivo studies have shown that the cooccurrence of three
dhfr
mutations and two
dhps
mutations in an infection prior to treatment is statistically predictive of treatment failure. We have combined data for both loci to determine the frequency of two-locus genotypes. The triple-
dhfr
/double-
dhps
genotype is present in all three regions with frequencies ranging between 30 and 63%, indicating that treatment failure rates are likely to be high.
Pharmacokinetics and Efficacy of Linezolid in a Gerbil Model of
Streptococcus pneumoniae
-Induced Acute Otitis Media
Authors: Humphrey William R. , Shattuck Mark H. , Zielinski Raymond J. , Kuo Ming-Shang T. , Biermacher John J. , Smith Donald P. , Jensen Jana L. , Schaadt Ronda D. , Zurenko Gary E. , Richards Ivan M.
Published: March 2003

The oxazolidinone linezolid represents a new antibacterial class of potential benefit in managing multidrug-resistant gram-positive infections, including those caused by
Streptococcus pneumoniae
. In a gerbil model of acute otitis media (AOM) induced by either penicillin-resistant
S. pneumoniae
(PRSP; amoxicillin MIC = 8 μg/ml, linezolid MIC = 1 μg/ml) or penicillin-susceptible
S. pneumoniae
(PSSP; amoxicillin MIC = 0.015 μg/ml, linezolid MIC = 1 μg/ml), we explored the plasma and ear fluid levels of linezolid required to demonstrate efficacy. Threshold pathogen doses required to induce bilateral AOM (1,500 CFU/ear with PRSP; 30 CFU/ear with PSSP) were administered to gerbils by intrabullar injection on day 0. At peak infection (∼10
6
to 10
7
CFU/ear flush; day 2 for PRSP-AOM and day 3 for PSSP-AOM), twice-a-day oral doses of linezolid, amoxicillin, or vehicle were administered over 4.5 days prior to collection and assay of middle ear effluents for
S. pneumoniae
content. Linezolid doses of ≥10 mg/kg of body weight induced significant cure rates of ≥72% versus both PRSP and PSSP infections, whereas amoxicillin at ≤100 mg/kg was consistently effective only versus PSSP-AOM. Plasma and ear fluid levels of linezolid necessary to elicit pneumococcal eradication from the middle ear were measured by high-performance liquid chromatography-tandem mass spectrometry and found to be similar both within and between each infection protocol. The plasma-ear fluid pharmacodynamic profile associated with linezolid efficacy was a
T
>MIC of ≥42%, a
C
max
/MIC ratio of ≥3.1, and a (24-h area under the curve)/MIC ratio of ≥30 h. Application of this model will be useful in defining preclinical pharmacodynamic relationships of novel antibiotics necessary to cure
S. pneumoniae
-induced AOM.
Stability of Colistin and Colistin Methanesulfonate in Aqueous Media and Plasma as Determined by High-Performance Liquid Chromatography
Authors: Li Jian , Milne Robert W. , Nation Roger L. , Turnidge John D. , Coulthard Kingsley
Published: March 2003

The stabilities of colistin and colistin methanesulfonate (CMS) in different aqueous media were studied by specific high-performance liquid chromatography (HPLC) methods. Colistin was stable in water at 4 and 37°C for up to 60 days and 120 h, respectively. However, degradation was observed when colistin was stored in isotonic phosphate buffer (0.067 M, pH 7.4) and human plasma at 37°C. The stability of CMS from three different sources in water was explored by strong-anion-exchange (SAX) HPLC for CMS and by measuring the concentrations of colistin formed from the hydrolysis of CMS. The peaks of CMS in SAX HPLC disappeared almost completely after 12 h at 37°C, but appeared to remain intact for up to 2 days at 4°C. Over the same period, there was no formation of colistin at 4°C. In water, phosphate buffer, and plasma, there was rapid formation of colistin within 24 to 48 h at 37°C from the three sources of CMS. The hydrolysis products were assumed to be a complex mixture of many different sulfomethyl derivatives, including colistin. The stability of a fourth source of CMS in Mueller-Hinton broth examined during 30 min at 37°C revealed no formation of colistin. Along with previous microbiological studies, this suggested that different sulfomethyl CMSs possess intrinsic antibacterial activity. These results will be helpful for understanding the pharmacokinetics and pharmacodynamics of colistin and CMS in humans and animals.
MexXY-OprM Efflux Pump Is Necessary for Adaptive Resistance of
Pseudomonas aeruginosa
to Aminoglycosides
Authors: Hocquet Didier , Vogne Christelle , El Garch Farid , Vejux Anne , Gotoh Naomasa , Lee Angela , Lomovskaya Olga , Plésiat Patrick
Published: March 2003

Exposure of
Pseudomonas aeruginosa
to aminoglycosides frequently selects for recalcitrant subpopulations exhibiting an unstable, “adaptive” resistance to these antibiotics. In this study, we investigated the implication in the phenomenon of MexXY-OprM, an active efflux system known to export aminoglycosides in
P. aeruginosa
. Immunoblotting experiments demonstrated that the transporter MexY, but not the outer membrane pore OprM, was overproduced during the post-drug exposure adaptation period in wild-type strain PAO1. Furthermore, MexY production was dependent upon the degree of bacterial exposure to gentamicin (drug concentration). In contrast to parental strain PAO1, mutants defective in MexXY or in OprM were unable to develop adaptive resistance. Altogether, these results indicate that the resistance process requires the rapid production of MexXY and the interaction of these proteins with the constitutively produced component OprM.
In Vitro Antifungal Activity of Micafungin (FK463) against Dimorphic Fungi: Comparison of Yeast-Like and Mycelial Forms
Authors: Nakai Toru , Uno Jun , Ikeda Fumiaki , Tawara Shuichi , Nishimura Kazuko , Miyaji Makoto
Published: March 2003

The characteristics of in vitro micafungin (FK463) antifungal activity against six species of dimorphic fungi were investigated in accordance with the NCCLS M27-A microdilution methods. MICs of micafungin, amphotericin B, itraconazole, and fluconazole for
Histoplasma capsulatum
var.
capsulatum
,
Blastomyces dermatitidis
,
Paracoccidioides brasiliensis
,
Penicillium marneffei
, and
Sporothrix schenckii
were determined both for the yeast-like form and mycelial form.
Coccidioides immitis
was tested only in its mycelial form. We have clearly demonstrated that the in vitro activity of micafungin depends considerably on the growth form of dimorphic fungi. Micafungin exhibited potent activity against the mycelial forms of
H. capsulatum
,
B. dermatitidis
, and
C. immitis
(MIC range, 0.0078 to 0.0625 μg/ml), while it was very weakly active against their yeast-like forms (MIC range, 32 to >64 μg/ml). Micafungin was also more active against the mycelial forms than the yeast-like forms of
Paracoccidioides brasiliensis
,
Penicillium marneffei
, and
S. schenckii
. The MICs of amphotericin B were 2 to 5 dilutions lower for the mycelial forms than for the yeast-like forms of
B. dermatitidis
and
Paracoccidioides brasiliensis
. There was no apparent difference in the activity of itraconazole between the two forms. The MICs of fluconazole for the yeast-like forms were generally lower than those for the mycelial forms, and considerably so for
B. dermatitidis
. These results suggest that the growth form employed in antifungal susceptibility testing of dimorphic fungi can considerably influence the interpretation of results. At present, it cannot be judged whether micafungin has clinical usefulness for dimorphic fungus infections, since for most fungi it remains uncertain which growth form correlates better with therapeutic outcome. However, the results of this study warrant further investigations of micafungin as a therapeutic agent for infections caused by dimorphic fungi.
Epidemiology of Rifampin ADP-Ribosyltransferase (
arr-2
) and Metallo-β-Lactamase (
bla
IMP-4
) Gene Cassettes in Class 1 Integrons in
Acinetobacter
Strains Isolated from Blood Cultures in 1997 to 2000
Authors: Houang Elizabeth T. S. , Chu Yiu-Wai , Lo Wai-Sing , Chu Ka-Yi , Cheng Augustine F. B.
Published: March 2003

We characterized two new gene cassettes in an
Acinetobacter
isolate: one harbored the metallo-β-lactamase (IMP-4) gene
bla
IMP-4
, the other harbored the rifampin ADP-ribosyltransferase (ARR-2) gene
arr-2
, and both arrayed with the aminoglycoside acetyltransferase [AAC(6′)-Ib
7
] gene cassette
aacA4
in two separate class 1 integrons. The epidemiology of these gene cassettes in isolates from blood cultures obtained from 1997 to 2000 was studied. Isolates bearing either the
bla
IMP-4
or the
arr-2
gene cassette or both represented 17.5% (10 of 57) of isolates in 1997, 16.1% (10 of 62) in 1998, 2.5% (1 of 40) in 1999, and 0% (0 of 58) in 2000. These two gene cassettes, probably borne on two separate integrons, were found in at least three genomic DNA groups, with evidence of clonal dissemination in the intensive care unit during 1997 to 1998. Seventeen of the 52
Acinetobacter baumannii
(genomic DNA group 2) isolates from 1997 to 2000 harbored
intI1
, but only one was positive for these gene cassettes, whereas 20 of the 21
intI1
-positive isolates of all other genomic DNA groups were positive for either or both of them. Reduced susceptibility to imipenem and rifampin was seen only in isolates harboring the
bla
IMP-4
and
arr-2
cassettes, respectively. The aminoglycoside phosphotransferase [APH(3′)-VIa] gene
aph(3′)-VIa
was detected in all 21 isolates for which the MIC of amikacin was ≥8 μg/ml, with or without
aacA4
, whereas
aacA4
alone was found in isolates for which the MIC of amikacin was 0.5 to 2 μg/ml. Significant differences between the 17
intI1
-positive and 47
intI1
-negative isolates belonging to genomic DNA group 3 from 1997 to 1998 in the MICs of amikacin, gentamicin, imipenem, sulfamethoxazole, and ceftazidime were observed (Mann-Whitney test,
P
< 0.001 to 0.01).
In Vitro Activities of Piperaquine and Other 4-Aminoquinolines against Clinical Isolates ofPlasmodium falciparumin Cameroon
Authors: Basco Leonardo K. , Ringwald Pascal
Published: March 2003

The spread of chloroquine-resistantPlasmodium falciparumcalls for a constant search for new drugs. The in vitro activity of piperaquine, a new Chinese synthetic drug belonging to the bisquinolines, was evaluated in 103 fresh clinical isolates ofP. falciparumin Cameroon, Central Africa, and compared with that of other 4-aminoquinoline and Mannich base derivatives and dihydroartemisinin. Piperaquine was highly active (geometric mean 50% inhibitory concentration, 38.9 nmol/liter; range, 7.76 to 78.3 nmol/liter) and equally active (P> 0.05) against the chloroquine-sensitive and the chloroquine-resistant isolates. There was a significant but low correlation of response between chloroquine and piperaquine (r= 0.257,P< 0.05). These results suggest that further development of piperaquine, in combination with dihydroartemisinin, holds promise for use in chloroquine-resistant regions of endemicity.
Pharmacokinetics and Metabolism of [14C]Ribavirin in Rats and Cynomolgus Monkeys
Authors: Lin Chin-Chung , Yeh Li-Tain , Luu Trong , Lourenco David , Lau Johnson Y. N.
Published: March 2003

Absorption, pharmacokinetics, distribution, metabolism, and excretion of [14C]ribavirin were studied in rats (30 mg/kg of body weight) and cynomolgus monkeys (10 mg/kg) after intravenous (i.v.) and oral administration. The oral absorption and bioavailability were 83 and 59%, respectively, in rats and 87 and 55%, respectively, in monkeys. After i.v. administration, the elimination half-life (t[1/2]) was 9.9 h in rats and 130 h in monkeys and the total body clearance was 2,600 ml/h/kg in rats and 224 ml/h/kg in monkeys. The apparent volume of distribution was 11.4 liter/kg in rats and 29.4 liter/kg in monkeys. There was extensive distribution of drug-derived radioactivity into red blood cells and extensive metabolism of ribavirin in rats and a lesser degree of metabolism in monkeys. Excretion of total radioactivity in urine from rats accounted for 84% of the i.v. dose and 83% of the oral dose, whereas that from monkeys accounted for 47% of the i.v. dose and 67% of the oral dose. Several metabolites were observed in plasma and urine from both species. The amount of unchanged ribavirin in urine from both species was quite small after either i.v. or oral administration.
Time-Kill Studies of the Antianaerobe Activity of Garenoxacin Compared with Those of Nine Other Agents
Authors: Credito Kim L. , Jacobs Michael R. , Appelbaum Peter C.
Published: March 2003

The activities of garenoxacin, ciprofloxacin, levofloxacin, moxifloxacin, trovafloxacin, amoxicillin-clavulanate, piperacillin-tazobactam, imipenem, clindamycin, and metronidazole against 20 anaerobes were tested. At two times the MIC, garenoxacin was bactericidal against 19 of 20 strains after 48 h and against 17 of 20 after 24 h. Other drugs, except clindamycin (which gave lower killing rates), gave killing rates similar to those for garenoxacin.
Activities of ABT-773 against
Listeria monocytogenes
and Coryneform Bacteria of Clinical Interest
Authors: Conejo María del Carmen , Martínez-Martínez Luis , Pascual Álvaro , Suárez Ana Isabel , Perea Evelio J.
Published: March 2003

The in vitro activities of ABT-773 were evaluated against 15
Listeria monocytogenes
strains and 196 coryneform bacteria isolated from clinical samples. One hundred percent of the
L. monocytogenes
strains were inhibited by ≤0.015 μg of ABT-773/ml. MICs of ABT-773 (μg/ml) at which 50% of the isolates tested were inhibited (MIC
50
s) and MIC
90
s for other organisms were 0.125 and 0.5 (
Corynebacterium amycolatum
), 1 and >32 (
Corynebacterium jeikeium
), 0.03 and >32 (
Corynebacterium minutissimum
), >32 and >32 (
Corynebacterium pseudodiphtheriticum
and
Corynebacterium urealyticum
), 0.125 and >32 (
Corynebacterium striatum
), and 0.03 and 0.5 (
Rhodococcus equi
), respectively.
In Vitro Metronidazole and Tinidazole Activities against Metronidazole- Resistant Strains of
Trichomonas vaginalis
Authors: Crowell Andrea L. , Sanders-Lewis Kolby A. , Secor W. Evan
Published: March 2003

The in vitro activities of tinidazole and metronidazole against
Trichomonas vaginalis
isolates clinically resistant to metronidazole were compared. Minimal lethal concentrations (MLCs) of tinidazole were significantly lower than MLCs of metronidazole. Increased metronidazole resistance correlated with increased tinidazole resistance. These data support a role for tinidazole in the treatment of trichomoniasis.
Activity of and Resistance to Moxifloxacin in
Staphylococcus aureus
Authors: Ince Dilek , Zhang Xiamei , Hooper David C.
Published: March 2003

Moxifloxacin has enhanced potency against
Staphylococcus aureus
, lower propensity to select for resistant mutants, and higher bactericidal activity against highly resistant strains than ciprofloxacin. Despite similar activity against purified
S. aureus
topoisomerase IV and DNA gyrase, it selects for topoisomerase IV mutants, making topoisomerase IV the preferred target in vivo.
In Vitro Synergy of Caspofungin and Itraconazole against
Aspergillus
spp.: MIC versus Minimal Effective Concentration End Points
Authors: Shalit Itamar , Shadkchan Yona , Samra Zmira , Osherov Nir
Published: March 2003

Caspofungin and itraconazole were studied alone and in combination against 31 clinical isolates of
Aspergillus
spp. according to NCCLS M38-P guidelines. MICs and microscopic minimal effective concentrations (MECs) were recorded, and synergy was calculated by using both end points. Synergy or synergy to additivity was found in 30 of 31 isolates by using MIC end points. With MEC end points no synergy was found and indifference was detected in 26 of 31 strains.
Genetic Characterization of Fluoroquinolone-Resistant
Streptococcus pneumoniae
Strains Isolated during Ciprofloxacin Therapy from a Patient with Bronchiectasis
Authors: de la Campa Adela G. , Ferrandiz María-José , Tubau Fe , Pallarés Román , Manresa Federico , Liñares Josefina
Published: March 2003

Five Spain
9V-3
Streptococcus pneumoniae
strains were isolated from a patient with bronchiectasis who had received long-term ciprofloxacin therapy. One ciprofloxacin-susceptible strain was isolated before treatment, and four ciprofloxacin-resistant strains were isolated during treatment. The resistant strains were derived from the susceptible strain either by a
parC
mutation (low-level resistance) or by
parC
and
gyrA
mutations (high-level resistance). This study shows that ciprofloxacin therapy in a patient colonized by susceptible
S. pneumoniae
may select fluoroquinolone-resistant mutants.
Multiplex PCR for Detection of Aminoglycoside Resistance Genes in Enterococci
Authors: Vakulenko Sergei B. , Donabedian Susan M. , Voskresenskiy Anatoliy M. , Zervos Marcus J. , Lerner Stephen A. , Chow Joseph W.
Published: March 2003

A multiplex PCR procedure for detecting the aminoglycoside resistance genes
aac(6′)-Ie-aph(2")-Ia
,
aph(2")-Ib
,
aph(2")-Ic
,
aph(2")-Id
,
aph(3′)-IIIa
, and
ant(4′)-Ia
was evaluated and found to determine accurately the presence of these genes in enterococci.
Integron-Associated Antibiotic Resistance in
Salmonella enterica
Serovar Typhi from Asia
Authors: Ploy Marie-Cécile , Chainier Delphine , Tran Thi Nhu Hoa , Poilane Isabelle , Cruaud Philippe , Denis François , Collignon Anne , Lambert Thierry
Published: March 2003

Eighteen of 25 isolates of
Salmonella enterica
serovar Typhi were multidrug resistant and contained class 1 integrons with a single cassette,
dfrVII
or
aadA1
. The
dfrVII
-containing integron was likely borne on an IncHI1 plasmid.
Salmonella
serovar Typhi could become resistant to broad-spectrum cephalosporins by integrating cassettes, such as
veb-1
, a common cassette in Asia.
Novel Tetracycline Resistance Determinant from the Oral Metagenome
Authors: Diaz-Torres M. L. , McNab R. , Spratt D. A. , Villedieu A. , Hunt N. , Wilson M. , Mullany P.
Published: March 2003

A major drawback of most studies on how bacteria become resistant to antibiotics is that they concentrate mainly on bacteria that can be cultivated in the laboratory. In the present study, we cloned part of the oral metagenome and isolated a novel tetracycline resistance gene,
tet
(37), which inactivates tetracycline.
Molecular Characterization and Antimicrobial Susceptibility of Fluoroquinolone-Resistant or -Susceptible
Streptococcus pneumoniae
from Hong Kong
Authors: Morrissey Ian , Farrell David J. , Bakker Sarah , Buckridge Sylvie , Felmingham David
Published: March 2003

Fluoroquinolone resistance in
Streptococcus pneumoniae
isolated from Hong Kong as part of Prospective Resistant Organism Tracking and Epidemiology for the Ketolide Telithromycin 1999/2000 was found to be due to the spread of the Spain
23F
-1 clone (mainly a Spain
23F
-1-14 variant). All the isolates were multidrug resistant but were susceptible to quinupristin-dalfopristin, linezolid, and telithromycin. The Spain
23F
-1 clone also occurred among antimicrobial-susceptible isolates, which suggests that the primary source of this clone may have been Asia rather than Iberia.
Activity of Posaconazole against
Pseudallescheria boydii
: In Vitro and In Vivo Assays
Authors: González Gloria M. , Tijerina Rolando , Najvar Laura K. , Bocanegra Rosie , Rinaldi Michael G. , Loebenberg David , Graybill John R.
Published: March 2003

Thirty isolates of
Pseudallescheria boydii
were tested to compare the in vitro activity of posaconazole with those of fluconazole and itraconazole, using NCCLS methods. Posaconazole was evaluated in an immunosuppressed mouse model of disseminated pseudallescheriasis. Posaconazole was more effective than itraconazole and as effective as fluconazole in preventing death and significantly reducing the CFU of
P. boydii
from tissues.
Tissue Penetration by Ertapenem, a Parenteral Carbapenem Administered Once Daily, in Suction-Induced Skin Blister Fluid in Healthy Young Volunteers
Authors: Laethem T. , De Lepeleire I. , McCrea J. , Zhang J. , Majumdar A. , Musson D. , Rogers D. , Li S. , Guillaume M. , Parneix-Spake A. , Deutsch P.
Published: March 2003

The penetration of 1 g of intravenous ertapenem once daily for 3 days in suction-induced skin blisters was evaluated. Ten forearm blisters were formed (
n
= 12) 12 h prior to the last dose. Concentrations of ertapenem in blister fluid exceeded 4 μg/ml (the MIC at which 90% of the isolates tested are eliminated) for the entire dosing interval. The area under the concentration-time curve for 0 to 24 h ratio of blister fluid to plasma was 61% (90% confidence interval, 56, 65%) suggesting good blister penetration.
Antibiotic Resistance in Bacteria Isolated from Subgingival Plaque in a Norwegian Population with Refractory Marginal Periodontitis
Authors: Handal Trude , Caugant Dominique A. , Olsen Ingar
Published: March 2003

The prevalence of β-lactamase-producing bacteria in subgingival plaque from patients with refractory periodontitis in Norway was assessed by the chromogenic nitrocefin method. β-Lactamase activity was detected in 68% of the patients. Enzyme-producing strains belonged predominantly to the genus
Prevotella
; others were staphylococci, enteric gram-negative rods, and
Bacillus
spp.
In Vitro Activity of a New Antibiotic, NVP-PDF386 (VRC4887), against
Chlamydia pneumoniae
Authors: Roblin Patricia M. , Hammerschlag Margaret R.
Published: March 2003

The in vitro activity of NVP-PDF386 (VRC4887), a novel new peptide deformylase inhibitor, and those of levofloxacin and clarithromycin were tested against 21 isolates of
Chlamydia pneumoniae
. The MIC at which 90% of the isolates were inhibited and the minimal bactericidal concentration at which 90% of the isolates were killed by NVP-PDF386 for all isolates of
C. pneumoniae
were 0.008 μg/ml (range, 0.008 to 0.015 μg/ml) compared to 0.25 and 0.06 μg/ml for levofloxacin and clarithromycin, respectively.
Trypanocidal and Leishmanicidal Properties of Substitution-Containing Chalcones
Authors: Lunardi Fabiane , Guzela Michel , Rodrigues Andrea T. , Corrêa Rogério , Eger-Mangrich Iriane , Steindel Mário , Grisard Edmundo C. , Assreuy Jamil , Calixto João B. , Santos Adair R. S.
Published: March 2003

Ten chalcones were synthesized and tested as potential leishmanicidal and trypanocidal agents. All tested compounds caused concentration-dependent inhibition of the in vitro growth ofLeishmania braziliensisandTrypanosoma cruziwith no significant toxic effect towards host macrophages. Our results show that the positions of the substituents seem to be critical for their antiprotozoal activities.
Efficacy of Micafungin Alone or in Combination against Systemic Murine Aspergillosis
Authors: Luque Javier Capilla , Clemons Karl V. , Stevens David A.
Published: March 2003

We tested the efficacy of micafungin (FK) alone or in combination with other antifungals against systemic murine aspergillosis. FK alone at 10 mg/kg of body weight/dose prolonged survival (
P
= 0.01) and reduced CFU in the brain and kidney. Combination therapy that used suboptimal FK with amphotericin B or itraconazole prolonged survival. Although no survivors were free of infection, no antagonism was seen. Nikkomycin Z with FK showed significantly greater potency (
P
< 0.01) than either alone.