bluuebunny/crossref_metadata_2025 huggingface dataset:
Pharmacokinetics of Gentamicin C
1
, C
1a
, and C
2
in Beagles after a Single Intravenous Dose
Authors: Isoherranen Nina , Lavy Eran , Soback Stefan
Published: July 2002

The pharmacokinetics of gentamicin C
1
, C
2
, and C
1a
were studied in six beagles after administration of gentamicin at 4 mg/kg of body weight as a single intravenous bolus dose. Plasma concentrations of the gentamicin components were analyzed with a novel high-performance liquid chromatography method capable of identifying and quantifying each of the components. The pharmacokinetic analysis of the plasma concentration-versus-time data was performed using the noncompartmental approach. The results indicated significant differences in the pharmacokinetic characteristics between the gentamicin components C
1
, C
1a
, and C
2
. The mean residence times of gentamicin C
1
, C
1a
, and C
2
were 81 ± 13, 84 ± 12, and 79 ± 13 min (mean ± standard deviation), respectively. The half-lives of the respective components were 64 ± 12, 66 ± 12 and 63 ± 12 min. Clearance (CL) of gentamicin C
1
, 4.62 ± 0.71 ml min
−1
kg
−1
, was significantly higher (
P
= 0.0156) than CL of gentamicin C
1a
, 1.81 ± 0.26 ml min
−1
kg
−1
, and C
2
, 1.82 ± 0.25 ml min
−1
kg
−1
. Similarly, the volume of distribution at steady state (
V
ss
) of gentamicin C
1
, 0.36 ± 0.04 liter kg
−1
, was significantly higher (
P
= 0.0156) than the
V
ss
of gentamicin C
1a
, 0.14 ± 0.01 liter kg
−1
, and C
2
, 0.15 ± 0.02 liter kg
−1
. Tissue binding was considered the most likely cause for the difference. The difference may have clinical and toxicological significance.
Use of Drug Effect Interaction Modeling with Monte Carlo Simulation To Examine the Impact of Dosing Interval on the Projected Antiviral Activity of the Combination of Abacavir and Amprenavir
Authors: Drusano G. L. , D'Argenio D. Z. , Preston S. L. , Barone C. , Symonds W. , LaFon S. , Rogers M. , Prince W. , Bye A. , Bilello J. A.
Published: July 2002

The delineation of optimal regimens for combinations of agents is a difficult problem, in part because, to address it, one needs to (i) have effect relationships between the pathogen in question and the drugs in the combination, (ii) have knowledge of how the drugs interact (synergy, antagonism, and additivity), and (iii) address the issue of true between-patient variability in pharmacokinetics for the drugs in the population. We have developed an approach which employs a fully parametric assessment of drug interaction using the equation of W. R. Greco, G. Bravo, and J. C. Parsons (Pharmacol. Rev. 47:331–385, 1995) to generate an estimate of effects for the two drugs and have linked this approach to a population simulator, using Monte Carlo methods, which produce concentration-time profiles for the drugs in combination. This software automatically integrates the effect over a steady-state dosing interval and produces an estimate of the mean effect over a steady-state interval for each simulated subject. In this way, doses and schedules can be easily evaluated. This software allows for a rational choice of dose and schedule for evaluation in clinical trials. We evaluated different schedules of administration for the combination of the nucleoside analogue abacavir plus the human immunodeficiency virus type 1 protease inhibitor amprenavir. Amprenavir was simulated as either 800 mg every 8 h (q8h) or 1,200 mg q12h, each along with 300 mg q12h of abacavir. Both regimens produced excellent effects over the simulated population of 500 subjects, with average percentages of maximal effect (as determined from the in vitro assays) of 90.9% ± 11.4% and 80.9% ± 18.6%, respectively. This difference is statistically significant (
P
≪ 0.001). In addition, 68.8 and 46.0% of the population had an average percentage of maximal effect which was greater than or equal to 90% for the two regimens. We can conclude that the combination of abacavir plus amprenavir is a potent combination when it is given on either schedule. However, the more fractionated schedule for the protease inhibitor produced significantly better effects in combination. Clinicians need to explicitly balance the improvement in antiviral effect seen with the more fractionated regimen against the loss of compliance attendant to the use of such a regimen. This approach may be helpful in the preclinical evaluation of multidrug anti-infective regimens.
Antibacterial Action of Structurally Diverse Cationic Peptides on Gram-Positive Bacteria
Authors: Friedrich Carol L. , Moyles Dianne , Beveridge Terry J. , Hancock Robert E. W.
Published: July 2002

Antimicrobial cationic peptides are ubiquitous in nature and are thought to be a component of the first line of defense against infectious agents. It is widely believed that the killing mechanism of these peptides on bacteria involves an interaction with the cytoplasmic membrane. Cationic peptides from different structural classes were used in experiments with
Staphylococcus aureus
and other medically important gram-positive bacteria to gain insight into the mechanism of action. The membrane potential-sensitive fluorophore dipropylthiacarbocyanine was used to assess the interactions of selected antimicrobial peptides with the cytoplasmic membrane of
S. aureus
. Study of the kinetics of killing and membrane depolarization showed that, at early time points, membrane depolarization was incomplete, even when 90% or more of the bacteria had been killed. CP26, a 26-amino-acid α-helical peptide with a high MIC against
S. aureus
, still had the ability to permeabilize the membrane. Cytoplasmic-membrane permeabilization was a widespread ability and an action that may be necessary for reaching an intracellular target but in itself did not appear to be the killing mechanism. Transmission electron microscopy of
S. aureus
and
Staphylococcus epidermidis
treated with CP29 (a 26-amino-acid α-helical peptide), CP11CN (a 13-amino-acid, proline- and tryptophan-rich peptide), and Bac2A-NH
2
(a linearized version of the 12-amino-acid loop peptide bactenecin) showed variability in effects on bacterial structure. Mesosome-like structures were seen to develop in
S. aureus
, whereas cell wall effects and mesosomes were seen with
S. epidermidis
. Nuclear condensation and abherrent septation were occasionally seen in
S. epidermidis
. Our experiments indicated that these peptides vary in their mechanisms of action and that the mechanism of action likely does not solely involve cytoplasmic-membrane permeabilization.
Mutations in 23S rRNA and Ribosomal Protein L4 Account for Resistance in Pneumococcal Strains Selected In Vitro by Macrolide Passage
Authors: Tait-Kamradt A. , Davies T. , Cronan M. , Jacobs M. R. , Appelbaum P. C. , Sutcliffe J.
Published: July 2002

The mechanisms responsible for macrolide resistance in
Streptococcus pneumoniae
mutants, selected from susceptible strains by serial passage in azithromycin, were investigated. These mutants were resistant to 14- and 15-membered macrolides, but resistance could not be explained by any clinically relevant resistance determinant [
mef
(A),
erm
(A),
erm
(B),
erm
(C),
erm
(TR),
msr
(A),
mph
(A),
mph
(B),
mph
(C),
ere
(A),
ere
(B)]. An investigation into the sequences of 23S rRNAs in the mutant and parental strains revealed individual changes of C2611A, C2611G, A2058G, and A2059G (
Escherichia coli
numbering) in four mutants. Mutations at these residues in domain V of 23S rRNA have been noted to confer erythromycin resistance in other species. Not all four 23S rRNA alleles have to contain the mutation to confer resistance. Some of the mutations also confer coresistance to streptogramin B (C2611A, C2611G, and A2058G), 16-membered macrolides (all changes), and clindamycin (A2058G and A2059G). Interestingly, none of these mutations confer high-level resistance to telithromycin (HMR-3647). Further, two of the mutants which had no changes in their 23S rRNA sequences had changes in a highly conserved stretch of amino acids (
63
KPWRQKGTGRAR
74
) in ribosomal protein L4. One mutant contained a single amino acid change (G69C), while the other mutant had a 6-base insert, resulting in two amino acids (S and Q) being inserted between amino acids Q67 and K68. To our knowledge, this is the first description of mutations in 23S rRNA genes or ribosomal proteins in macrolide-resistant
S. pneumoniae
strains.
Analysis of
rdxA
and Involvement of Additional Genes Encoding NAD(P)H Flavin Oxidoreductase (FrxA) and Ferredoxin-Like Protein (FdxB) in Metronidazole Resistance of
Helicobacter pylori
Authors: Kwon Dong-Hyeon , El-Zaatari Fouad A. K. , Kato Mototsugu , Osato Michael S. , Reddy Rita , Yamaoka Yoshio , Graham David Y.
Published: July 2002

Metronidazole (Mtz) is a critical ingredient of modern multidrug therapies for
Helicobacter pylori
infection. Mtz resistance reduces the effectiveness of these combinations. Although null mutations in a
rdxA
gene that encodes oxygen-insensitive NAD(P)H nitroreductase was reported in Mtz-resistant
H. pylori
, an intact
rdxA
gene has also been reported in Mtz-resistant
H. pylori
, suggesting that additional Mtz resistance mechanisms exist in
H. pylori
. We explored the nature of Mtz resistance among 544 clinical
H. pylori
isolates to clarify the role of
rdxA
inactivation in Mtz resistance and to identify another gene(s) responsible for Mtz resistance in
H. pylori
. Mtz resistance was present in 33% (181 of 544) of the clinical isolates. There was marked heterogeneity of resistance, with Mtz MICs ranging from 8 to ≥256 μg/ml.
rdxA
inactivation resulted in Mtz MICs of up to 32 μg/ml for 6 Mtz-sensitive
H. pylori
strains and 128 μg/ml for one Mtz-sensitive strain. Single or dual (with
rdxA
) inactivation of genes that encode ferredoxin-like protein (designated
fdxB
) and NAD(P)H flavin oxidoreductase (
frxA
) also increased the MICs of Mtz for sensitive and resistant strains with low to moderate levels of Mtz resistance.
fdxB
inactivation resulted in a lower level of resistance than that from
rdxA
inactivation, whereas
frxA
inactivation resulted in MICs similar to those seen with
rdxA
inactivation. Further evidence for involvement of the
frxA
gene in Mtz resistance included the finding of a naturally inactivated
frxA
but an intact
rdxA
in an Mtz-resistant strain, complementation of Mtz sensitivity from an Mtz-sensitive strain to an Mtz-resistant strain or vice versa by use of naturally inactivated or functional
frxA
genes, respectively, and transformation of an Mtz-resistant
Escherichia coli
strain to an Mtz sensitive strain by a naturally functional
frxA
gene but not an inactivated
frxA
gene. These results are consistent with the hypothesis that null mutations in
fdxB
,
frxA
, or
rdxA
may be involved in Mtz resistance.
Antimicrobial Evaluation of
N
-Alkyl Betaines and
N
-Alkyl-
N
,
N
-Dimethylamine Oxides with Variations in Chain Length
Authors: Birnie Christine R. , Malamud Daniel , Schnaare Roger L.
Published: July 2002

Alkyl betaines and alkyl dimethylamine oxides have been shown to have pronounced antimicrobial activity when used individually or in combination. Although several studies have been conducted with these compounds in combinations, only equimolar concentrations of the C
12
/C
12
and C
16
/C
14
chain lengths for the betaine and the amine oxide, respectively, have been investigated. This study investigates the antimicrobial activity of a wide range of chain lengths (C
8
to C
18
) for both the betaine and amine oxide and attempts to correlate their micelle-forming capabilities with their biological activity. A broth microdilution method was used to determine the MICs of these compounds singly and in various molar ratio combinations. Activity against both
Staphylococcus aureus
and
Escherichia coli
was investigated. Antimicrobial activity was found to increase with increasing chain length for both homologous series up to a point, exhibiting a cutoff effect at chain lengths of approximately 16 for betaine and 14 for amine oxide. Additionally, the C
18
oleyl derivative of both compounds exhibited activity in the same range as the peak alkyl compounds. Critical micelle concentrations were correlated with MICs, inferring that micellar activity may contribute to the cutoff effect in biological activity.
Identification and Characterization of Inhibitors of Multidrug Resistance Efflux Pumps in
Pseudomonas aeruginosa
: Novel Agents for Combination Therapy
Authors: Lomovskaya Olga , Warren Mark S. , Lee Angela , Galazzo Jorge , Fronko Richard , Lee May , Blais Johanne , Cho Deidre , Chamberland Suzanne , Renau Tom , Leger Roger , Hecker Scott , Watkins Will , Hoshino Kazuki , Ishida Hiroko , Lee Ving J.
Published: July 2002

Whole-cell assays were implemented to search for efflux pump inhibitors (EPIs) of the three multidrug resistance efflux pumps (MexAB-OprM, MexCD-OprJ, MexEF-OprN) that contribute to fluoroquinolone resistance in clinical isolates of
Pseudomonas aeruginosa
. Secondary assays were developed to identify lead compounds with exquisite activities as inhibitors. A broad-spectrum EPI which is active against all three known Mex efflux pumps from
P. aeruginosa
and their close
Escherichia coli
efflux pump homolog (AcrAB-TolC) was discovered. When this compound, MC-207,110, was used, the intrinsic resistance of
P. aeruginosa
to fluoroquinolones was decreased significantly (eightfold for levofloxacin). Acquired resistance due to the overexpression of efflux pumps was also decreased (32- to 64-fold reduction in the MIC of levofloxacin). Similarly, 32- to 64-fold reductions in MICs in the presence of MC-207,110 were observed for strains with overexpressed efflux pumps and various target mutations that confer resistance to levofloxacin (e.g.,
gyrA
and
parC
). We also compared the frequencies of emergence of levofloxacin-resistant variants in the wild-type strain at four times the MIC of levofloxacin (1 μg/ml) when it was used either alone or in combination with EPI. In the case of levofloxacin alone, the frequency was ∼10
−7
CFU/ml. In contrast, with an EPI, the frequency was below the level of detection (<10
−11
). In summary, we have demonstrated that inhibition of efflux pumps (i) decreased the level of intrinsic resistance significantly, (ii) reversed acquired resistance, and (iii) resulted in a decreased frequency of emergence of
P. aeruginosa
strains that are highly resistant to fluoroquinolones.
In Vitro Antimicrobial Activities of Bakuchiol against Oral Microorganisms
Authors: Katsura Harumi , Tsukiyama Ryo-Ichi , Suzuki Akiko , Kobayashi Makio
Published: July 2002

Bakuchiol was isolated from the seeds of
Psoralea corylifolia
, a tree native to China with various uses in traditional medicine, followed by extraction with ether and column chromatography combined with silica gel and octyldecyl silane. In this study, the antimicrobial activities of bakuchiol against some oral microorganisms were evaluated in vitro. The cell growth of
Streptococcus mutans
was inhibited in a bakuchiol concentration-dependent manner, and growth of
S. mutans
was completely prevented by 20 μg of bakuchiol per ml. The bactericidal effect of bakuchiol on
S. mutans
was dependent on temperature and stable under the following conditions: sucrose, 0 to 10% (wt/vol); pH, 3.0 to 7.0; organic acids (3% [wt/vol] citric and malic acids). Bakuchiol showed bactericidal effects against all bacteria tested, including
S. mutans
,
Streptococcus sanguis
,
Streptococcus salivarius
,
Streptococcus sobrinus
,
Enterococcus faecalis
,
Enterococcus faecium
,
Lactobacillus acidophilus
,
Lactobacillus casei
,
Lactobacillus plantarum
,
Actinomyces viscosus
, and
Porphyromonas gingivalis
, with MICs ranging from 1 to 4 μg/ml and the sterilizing concentration for 15 min ranging from 5 to 20 μg/ml. Furthermore, bakuchiol was also effective against adherent cells of
S. mutans
in water-insoluble glucan in the presence of sucrose and inhibited the reduction of pH in the broth. Thus, bakuchiol would be a useful compound for development of antibacterial agents against oral pathogens and has great potential for use in food additives and mouthwash for preventing and treating dental caries.
Antimicrobial Susceptibilities of 1,730
Haemophilus influenzae
Respiratory Tract Isolates in Spain in 1998-1999
Authors: Marco Francesc , Garcı́a-de-Lomas Juan , Garcı́a-Rey César , Bouza Emilio , Aguilar Lorenzo , Fernández-Mazarrasa Carlos ,   The Spanish Surveillance Group for Respiratory Pathogens
Published: July 2002

A β-lactamase prevalence of 23% was found among 1,730
Haemophilus influenzae
isolates. Ampicillin susceptibility was 70%, and 12% of β-lactamase-negative strains presented diminished susceptibility to ampicillin (BLNAR phenotype). Susceptibility of 90% was found for cefaclor and clarithromycin, whereas it was nearly 100% for cefotaxime, cefixime, azithromycin, and cefuroxime. Ciprofloxacin-resistant (0.1%) and β-lactamase-positive amoxicillin/clavulanate-resistant (BLPACR) phenotypes (0.1%) are anecdotal so far.
Novel Tetracycline Resistance Gene,
tet
(32), in the
Clostridium
-Related Human Colonic Anaerobe K10 and Its Transmission In Vitro to the Rumen Anaerobe
Butyrivibrio fibrisolvens
Authors: Melville Claire M. , Scott Karen P. , Mercer Derry K. , Flint Harry J.
Published: July 2002

A novel tetracycline resistance gene, designated
tet
(32), which confers a high level of tetracycline resistance, was identified in the
Clostridium-
related human colonic anaerobe K10, which also carries
tet
(W).
tet
(32) was transmissible in vitro to the rumen anaerobe
Butyrivibrio fibrisolvens
2221
R
. The predicted gene product of
tet
(32) has 76% amino acid identity with Tet(O). PCR amplification indicated that
tet
(32) is widely distributed in the ovine rumen and in porcine feces.
Antimicrobial Susceptibilities of 1,684
Streptococcus pneumoniae
and 2,039
Streptococcus pyogenes
Isolates and Their Ecological Relationships: Results of a 1-Year (1998–1999) Multicenter Surveillance Study in Spain
Authors: Pérez-Trallero E. , Fernández-Mazarrasa C. , Garcı́a-Rey C. , Bouza E. , Aguilar L. , Garcı́a-de-Lomas J. , Baquero F. ,   The Spanish Surveillance Group for Respiratory Pathogens
Published: July 2002

A nationwide multicenter susceptibility surveillance study which included 1,684
Streptococcus pneumoniae
and 2,039
S. pyogenes
isolates was carried out over 1 year in order to assess the current resistance patterns for the two most important gram-positive microorganisms responsible for community-acquired infections in Spain. Susceptibility testing was done by a broth microdilution method according to National Committee for Clinical Laboratory Standards M100-S10 interpretative criteria. For
S. pneumoniae
, the prevalences of highly resistant strains were 5% for amoxicillin and amoxicillin-clavulanic acid; 7% for cefotaxime; 22% for penicillin; 31% for cefuroxime; 35% for erythromycin, clarithromycin, and azithromycin; and 42% for cefaclor. For
S. pyogenes
, the prevalence of erythromycin resistance was 20%. Efflux was encountered in 90% of
S. pyogenes
and 5% of
S. pneumoniae
isolates that exhibited erythromycin resistance. Erythromycin resistance was associated with clarithromycin and azithromycin in both species, regardless of phenotype. Despite the different nature of the mechanisms of resistance, a positive correlation (
r
= 0.612) between the two species in the prevalence of erythromycin resistance was found in site-by-site comparisons, suggesting some kind of link with antibiotic consumption. Regarding ciprofloxacin, the MIC was ≥4 μg/ml for 7% of
S. pneumoniae
and 3.5% of
S. pyogenes
isolates. Ciprofloxacin resistance (MIC, ≥4 μg/ml) was significantly (
P
< 0.05) associated with macrolide resistance in both
S. pyogenes
and
S. pneumoniae
and with penicillin nonsusceptibility in
S. pneumoniae
.
Cefepime, Piperacillin-Tazobactam, and the Inoculum Effect in Tests with Extended-Spectrum β-Lactamase-Producing
Enterobacteriaceae
Authors: Thomson Kenneth S. , Moland Ellen Smith
Published: July 2002

There is little information about the clinical effectiveness of cefepime and piperacillin-tazobactam in the treatment of infections caused by extended-spectrum β-lactamase (ESBL)-producing pathogens. Some inferences have been drawn from laboratory studies, which have usually involved only one or a few strains of ESBL-producing
Klebsiella pneumoniae
or
Escherichia coli
that produced only a limited range of ESBLs. Such studies are indirect, sometimes conflicting, indicators of efficacy. To extend previous laboratory findings, a study was designed to investigate organism-drug interactions by determining the in vitro activities of eight parenteral β-lactam agents against 82 clinical and laboratory strains of
Klebsiella
,
Escherichia
,
Enterobacter
,
Citrobacter
,
Serratia
,
Morganella
, and
Proteus
species that produced 22 different ESBLs, alone or in combination with other β-lactamases. Activities were determined in broth microdilution MIC tests using standard and 100-fold-higher inocula. An inoculum effect, defined as an eightfold or greater MIC increase on testing with the higher inoculum, was most consistently detected with cefepime, cefotaxime, and ceftriaxone and least frequently detected with meropenem and cefoteten. Piperacillin-tazobactam was intermediate between these two groups of agents. Although the inoculum effect is an in vitro laboratory phenomenon, if it has any predictive value in identifying increased risk of therapeutic failure in serious infections, these results support suggestions that cefepime may be a less-than-reliable agent for therapy of infections caused by ESBL-producing strains.
Amino Acid Repetitions in the Dihydropteroate Synthase of
Streptococcus pneumoniae
Lead to Sulfonamide Resistance with Limited Effects on Substrate

K
m
Authors: Haasum Ylva , Ström Katrin , Wehelie Rahma , Luna Vicki , Roberts Marilyn C. , Maskell Jeffrey P. , Hall Lucinda M. C. , Swedberg Göte
Published: July 2002

Sulfonamide resistance in
Streptococcus pneumoniae
is due to changes in the chromosomal
folP
(
sulA
) gene coding for dihydropteroate synthase (DHPS). The first reported laboratory-selected sulfonamide-resistant
S. pneumoniae
isolate had a 6-bp repetition, the
sul-d
mutation, leading to a repetition of the amino acids Ile
66
and Glu
67
in the gene product DHPS. More recently, clinical isolates showing this and other repetitions have been reported. WA-5, a clinical isolate from Washington State, contains a 6-bp repetition in the
folP
gene, identical to the
sul-d
mutation. The repetition was deleted by site-directed mutagenesis. Enzyme kinetic measurements showed that the deletion was associated with a 35-fold difference in

K
i

for sulfathiazole but changed the

K
m

for
p
-aminobenzoic acid only 2.5-fold and did not significantly change the

K
m

for 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine pyrophosphate. The enzyme characteristics of the deletion variant were identical to those of DHPS from a sulfonamide-susceptible strain. DHPS from clinical isolates with repetitions of Ser
61
had very similar enzyme characteristics to the DHPS from WA-5. The results confirm that the repetitions are sufficient for development of a resistant enzyme and suggest that the fitness cost to the organism of developing resistance may be very low.
Role of ATP-Binding-Cassette Transporter Genes in High-Frequency Acquisition of Resistance to Azole Antifungals in
Candida glabrata
Authors: Sanglard Dominique , Ischer Francoise , Bille Jacques
Published: July 2002

Candida glabrata
has been often isolated from AIDS patients with oropharyngeal candidiasis treated with azole antifungal agents, especially fluconazole. We recently showed that the ATP-binding-cassette (ABC) transporter gene
CgCDR1
was upregulated in
C. glabrata
clinical isolates resistant to azole antifungal agents (D. Sanglard, F. Ischer, D. Calabrese, P. A. Majcherczyk, and J. Bille, Antimicrob. Agents Chemother. 43:2753–2765, 1999). Deletion of
CgCDR1
in
C. glabrata
rendered the null mutant hypersusceptible to azole derivatives and showed the importance of this gene in mediating azole resistance. We observed that wild-type
C. glabrata
exposed to fluconazole in a medium containing the drug at 50 μg/ml developed resistance to this agent and other azoles at a surprisingly high frequency (2 × 10
−4
to 4 × 10
−4
). We show here that this high-frequency azole resistance (HFAR) acquired in vitro was due, at least in part, to the upregulation of
CgCDR1
. The
CgCDR1
deletion mutant DSY1041 could still develop HFAR but in a medium containing fluconazole at 5 μg/ml. In the HFAR strain derived from DSY1041, a distinct ABC transporter gene similar to
CgCDR1
, called
CgCDR2
, was upregulated. This gene was slightly expressed in clinical isolates but was upregulated in strains with the HFAR phenotype. Deletion of both
CgCDR1
and
CgCDR2
suppressed the development of HFAR in a medium containing fluconazole at 5 μg/ml, showing that both genes are important mediators of resistance to azole derivatives in
C. glabrata
. We also show here that the HFAR phenomenon was linked to the loss of mitochondria in
C. glabrata
. Mitochondrial loss could be obtained by treatment with ethidium bromide and resulted in acquisition of resistance to azole derivatives without previous exposure to these agents. Azole resistance obtained in vitro by HFAR or by agents stimulating mitochondrial loss was at least linked to the upregulation of both
CgCDR1
and
CgCDR2
.
In Vitro Generation of Novel Pyrimethamine Resistance Mutations in theToxoplasma gondiiDihydrofolate Reductase
Authors: Reynolds Mary G. , Oh Jung , Roos David S.
Published: July 2002

Pyrimethamine is a potent inhibitor of dihydrofolate reductase and is widely used in the treatment of opportunistic infections caused by the protozoan parasiteToxoplasma gondii. In order to assess the potential role ofdhfrsequence polymorphisms in drug treatment failures, we examined thedhfr-tsgenes of representative isolates forT. gondiivirulence types I, II, and III. These strains exhibit differences in their sensitivities to pyrimethamine but no differences in predicteddhfr-tsprotein sequences. To assess the potential for pyrimethamine-resistantdhfrmutants to emerge, three drug-sensitive variants of theT. gondii dhfr-tsgene (the wild-typeT. gondiisequence and two mutants engineered to reflect polymorphisms observed in drug-sensitivePlasmodium falciparum) were subjected to random mutagenesis and transfected into either wild-typeT. gondiiparasites ordhfr-deficientSaccharomyces cerevisiaeunder pyrimethamine selection. Three resistance mutations were identified, at amino acid residues 25 (Trp→Arg), 98 (Leu→Ser), and 134 (Leu→His).
Bactericidal Activities of Milk Lipids
Authors: Sprong R. Corinne , Hulstein Marco F. E. , Van der Meer Roelof
Published: July 2002

The bactericidal capacity of digestion products of bovine milk triglycerides and membrane lipids was tested in vitro usingEscherichia coliO157:H7,Salmonella enteritidis, Campylobacter jejuni, Listeria monocytogenes, andClostridium perfringens. C10:0and C12:0fatty acids and digestion products of sphingolipids appeared to be effective bactericidal agents, whereas digestion products of phosphoglycerides were moderately bactericidal. Thus, milk fat sphingolipids and triglycerides, particularly those containing C10:0and C12:0fatty acids, may protect against food-borne gastroenteritis.
Multidrug-Resistant Urinary Tract Isolates of
Escherichia coli:
Prevalence and Patient Demographics in the United States in 2000
Authors: Sahm Daniel F. , Thornsberry Clyde , Mayfield David C. , Jones Mark E. , Karlowsky James A.
Published: July 2002

Concurrent resistance to antimicrobials of different structural classes has arisen in a multitude of bacterial species and may complicate the therapeutic management of infections, including those of the urinary tract. To assess the current breadth of multidrug resistance among urinary isolates of
Escherichia coli,
the most prevalent pathogen contributing to these infections, all pertinent results in The Surveillance Network Database—USA from 1 January to 30 September 2000 were analyzed. Results were available for 38,835 urinary isolates of
E. coli
that had been tested against ampicillin, cephalothin, ciprofloxacin, nitrofurantoin, and trimethoprim-sulfamethoxazole. Of these isolates, 7.1% (2,763 of 38,835) were resistant to three or more agents and considered multidrug resistant. Among the multidrug-resistant isolates, 97.8% were resistant to ampicillin, 92.8% were resistant to trimethoprim-sulfamethoxazole, 86.6% were resistant to cephalothin, 38.8% were resistant to ciprofloxacin, and 7.7% were resistant to nitrofurantoin. The predominant phenotype among multidrug-resistant isolates (57.9%; 1,600 of 2,793) included resistance to ampicillin, cephalothin, and trimethoprim-sulfamethoxazole. This was the most common phenotype regardless of patient age, gender, or inpatient-outpatient status and in eight of the nine U.S. Bureau of the Census regions. Rates of multidrug resistance were demonstrated to be higher among males (10.4%) than females (6.6%), among patients >65 years of age (8.7%) than patients ≤17 (6.8%) and 18 to 65 (6.1%) years of age, and among inpatients (7.6%) than outpatients (6.9%). Regionally, the rates ranged from 4.3% in the West North Central region to 9.2% in the West South Central region. Given the current prevalence of multidrug resistance among urinary tract isolates of
E. coli
in the United States (7.1%), continued local, regional, and national surveillance is warranted.
Azithromycin Inhibits Quorum Sensing in
Pseudomonas aeruginosa
Authors: Tateda Kazuhiro , Comte Rachel , Pechere Jean-Claude , Köhler Thilo , Yamaguchi Keizo , Van Delden Christian
Published: July 2002

We report that 2 μg of azithromycin/ml inhibits the quorum-sensing circuitry of
Pseudomonas aeruginosa
strain PAO1. Addition of synthetic autoinducers partially restored the expression of the trancriptional activator-encoding genes
lasR
and
rhlR
but not that of the autoinducer synthase-encoding gene
lasI
. We propose that azithromycin interferes with the synthesis of autoinducers, by an unknown mechanism, leading to a reduction of virulence factor production.
Continuous versus Intermittent Infusion of Vancomycin in Severe Staphylococcal Infections: Prospective Multicenter Randomized Study
Authors: Wysocki Marc , Delatour Frederique , Faurisson François , Rauss Alain , Pean Yves , Misset Benoit , Thomas Frank , Timsit Jean-François , Similowski Thomas , Mentec Herve , Mier Laurence , Dreyfuss Didier ,   The Study Group
Published: July 2002

A continuous infusion of vancomycin (CIV) may provide an alternative mode of infusion in severe hospital-acquired methicillin-resistant staphylococcal (MRS) infections. A multicenter, prospective, randomized study was designed to compare CIV (targeted plateau drug serum concentrations of 20 to 25 mg/liter) and intermittent infusions of vancomycin (IIV; targeted trough drug serum concentrations of 10 to 15 mg/liter) in 119 critically ill patients with MRS infections (bacteremic infections, 35%; pneumonia, 45%). Microbiological and clinical outcomes, safety, pharmacokinetics, ease of treatment adjustment, and cost were compared. Microbiological and clinical outcomes and safety were similar. CIV patients reached the targeted concentrations faster (36 ± 31 versus 51 ± 39 h,
P
= 0.029) and fewer samples were required for treatment monitoring than with IIV patients (7.7 ± 2.2 versus 11.8 ± 3.9 per treatment,
P
< 0.0001). The variability between patients in both the area under the serum concentration-time curve (AUC
24h
) and the daily dose given over 10 days of treatment was lower with CIV than with IIV (variances, 14,621 versus 53,975 mg
2
/liter
2
/h
2
[
P
= 0.026] and 414 versus 818 g
2
[
P
= 0.057], respectively). The 10-day treatment cost per patient was $454 ± 137 in the IIV group and was 23% lower in the CIV group ($321 ± 81:
P
< 0.0001). In summary, for comparable efficacy and tolerance, CIV may be a cost-effective alternative to IIV.
Integrons and Gene Cassettes in the
Enterobacteriaceae
Authors: White Peter A. , McIver Christopher J. , Rawlinson William D.
Published: July 2002

Integrons were detected in 59 of 120 (49%) urinary isolates of
Enterobacteriaceae
by PCR using degenerate primers targeted to conserved regions of class 1, 2, and 3 integrase genes. PCR sequencing analysis of the cassette arrays revealed a predominance of cassettes that confer resistance to the aminoglycosides and trimethoprim.
Phosphorylation of β-d-Ribosylbenzimidazoles Is Not Required for Activity against Human Cytomegalovirus
Authors: Krosky Paula M. , Borysko Katherine Z. , Nassiri M. Reza , Devivar Rodrigo V. , Ptak Roger G. , Davis Michelle G. , Biron Karen K. , Townsend Leroy B. , Drach John C.
Published: July 2002

We have previously reported that 2,5,6-trichloro-1-(β-d-ribofuranosyl)benzimidazole (TCRB) and its 2-bromo analog (2-bromo-5,6-dichloro-1-(β-d-ribofuranosy)benzimidazole [BDCRB]) are potent and selective inhibitors of human cytomegalovirus (HCMV) replication that block viral DNA maturation via HCMV gene products UL89 and UL56. To determine if phosphorylation is required for antiviral activity, the in vitro metabolism of BDCRB was examined and the antiviral activities of nonphosphorylatable 5′-deoxy analogs were determined. Reverse-phase high-performance liquid chromatography (HPLC) analysis of extracts from uninfected and HCMV-infected cells incubated with [3H]BDCRB revealed two major metabolites. Both were less polar than naturally occurring nucleoside monophosphates, but one peak coeluted with a BDCRB-5′-monophosphate (BDCRB-5′-MP) standard. Further analysis revealed, however, that neither metabolite partitioned with BDCRB-5′-MP on anion-exchange HPLC. Their retention patterns were not affected by incubation with alkaline phosphatase, thereby establishing that the compounds were not nucleoside 5′-monophosphates. Both compounds were detected in uninfected and HCMV-infected cells and in mouse live extracts, but neither has been identified. Like TCRB and BDCRB, the nonphosphorylatable 5′-deoxy analogs were potent and selective inhibitors of HCMV replication. The 5′-deoxy analogs maintained inhibition of HCMV replication upon removal of BDCRB, whereas an inhibitor of DNA synthesis did not. Similar to TCRB, its 5′-deoxy analog (5′-dTCRB) did not affect viral DNA synthesis, but 5′-dTCRB did inhibit viral DNA maturation to genome-length units. Additionally, virus isolates resistant to TCRB were also resistant to 5′-dTCRB and the 5′-deoxy analog of BDCRB. Taken together, these results confirm that TCRB, BDCRB, and their 5′-deoxy analogs have common mechanisms of action and establish that these benzimidazole ribonucleosides, unlike other antiviral nucleosides, do not require phosphorylation at the 5′ position for antiviral activity.
Antimicrobial Resistance among Clinical Isolates of
Streptococcus pneumoniae
in Canada during 2000
Authors: Low Donald E. , de Azavedo Joyce , Weiss Karl , Mazzulli Tony , Kuhn Magdalena , Church Deirdre , Forward Kevin , Zhanel George , Simor Andrew , McGeer A.
Published: July 2002

A total of 2,245 clinical isolates of
Streptococcus pneumoniae
were collected from 63 microbiology laboratories from across Canada during 2000 and characterized at a central laboratory. Of these isolates, 12.4% were not susceptible to penicillin (penicillin MIC, ≥0.12 μg/ml) and 5.8% were resistant (MIC, ≥2 μg/ml). Resistance rates among non-β-lactam agents were the following: macrolides, 11.1%; clindamycin, 5.7%; chloramphenicol, 2.2%; levofloxacin, 0.9%; gatifloxacin, 0.8%; moxifloxacin, 0.4%; and trimethoprim-sulfamethoxazole, 11.3%. The MICs at which 90% of the isolates were inhibited (MIC
90
s) of the fluoroquinolones were the following: gemifloxacin, 0.03 μg/ml; BMS-284756, 0.06 μg/ml; moxifloxacin, 0.12 μg/ml; gatifloxacin, 0.25 μg/ml; levofloxacin, 1 μg/ml; and ciprofloxacin, 1 μg/ml. Of 578 isolates from the lower respiratory tract, 21 (3.6%) were inhibited at ciprofloxacin MICs of ≥4 μg/ml. None of the 768 isolates from children were inhibited at ciprofloxacin MICs of ≥4 μg/ml, compared to 3 of 731 (0.6%) from those ages 15 to 64 (all of these >60 years old), and 27 of 707 (3.8%) from those over 65. The MIC
90
s for ABT-773 and telithromycin were 0.015 μg/ml for macrolide-susceptible isolates and 0.12 and 0.5 μg/ml, respectively, for macrolide-resistant isolates. The MIC of linezolid was ≤2 μg/ml for all isolates. Many of the new antimicrobial agents tested in this study appear to have potential for the treatment of multidrug-resistant strains of pneumococci.
Accessory Gene Regulator (
agr
) Locus in Geographically Diverse
Staphylococcus aureus
Isolates with Reduced Susceptibility to Vancomycin
Authors: Sakoulas George , Eliopoulos George M. , Moellering Robert C. , Wennersten Christine , Venkataraman Lata , Novick Richard P. , Gold Howard S.
Published: July 2002

The majority of infections with glycopeptide intermediate-level resistant
Staphylococcus aureus
(GISA) originate in biomedical devices, suggesting a possible increased ability of these strains to produce biofilm. Loss of function of the accessory gene regulator (
agr
) of
S. aureus
has been suggested to confer an enhanced ability to bind to polystyrene. We studied
agr
in GISA, hetero-GISA, and related glycopeptide-susceptible
S. aureus
isolates. All GISA strains from diverse geographic origins belong to
agr
group II. All GISA strains were defective in
agr
function, as demonstrated by their inability to produce delta-hemolysin. Hetero-GISA isolate A5940 demonstrated a nonsense mutation in
agrA
that was not present in a pulsed-field gel electrophoresis-indistinguishable vancomycin-susceptible isolate from the same patient. Various other
agr
point mutations were noted in several clinical GISA and hetero-GISA isolates. A laboratory-generated
agr
-null strain demonstrated a small but reproducible increase in vancomycin heteroresistance after growth in vitro in subinhibitory concentrations of vancomycin. This was not seen in the isogenic
agr
group II parent strain in which
agr
was intact. The in vitro bactericidal activity of vancomycin was attenuated in the
agr
-null strain compared to the parent strain. These findings imply that compromised
agr
function is advantageous to clinical isolates of
S. aureus
toward the development of vancomycin heteroresistance, perhaps through the development of vancomycin tolerance.
Long-Term Shifts in Patterns of Antibiotic Resistance in Enteric Bacteria
Authors: Houndt Tara , Ochman Howard
Published: July 2002

Several mechanisms are responsible for the ability of microorganisms to tolerate antibiotics, and the incidence of resistance to these compounds within bacterial species has increased since the commercial use of antibiotics became widespread. To establish the extent of and changes in the diversity of antibiotic resistance patterns in natural populations, we determined the MICs of five antibiotics for collections of enteric bacteria isolated from diverse hosts and geographic locations and during periods before and after commercial application of antibiotics began. All of the pre-antibiotic era strains were susceptible to high levels of these antibiotics, whereas 20% of strains from contemporary populations of
Escherichia coli
and
Salmonella enterica
displayed high-level resistance to at least one of the antibiotics. In addition to the increase in the frequency of high-level resistance, background levels, conferred by genes providing nonspecific low-level resistance to multiple antibiotics, were significantly higher among contemporary strains. Changes in the incidence and levels of antibiotic resistance are not confined to particular segments of the bacterial population and reflect responses to the increased exposure of bacteria to antimicrobial compounds over the past several decades.
Glycine Betaine, Carnitine, and Choline Enhance Salinity Tolerance and Prevent the Accumulation of Sodium to a Level Inhibiting Growth of
Tetragenococcus halophila
Authors: Robert Hervé , Le Marrec Claire , Blanco Carlos , Jebbar Mohamed
Published: July 2002

Natural-abundance
13
C-nuclear magnetic resonance was used to probe the intracellular organic solute content of the moderately halophilic bacterium
Tetragenococcus halophila
. When grown in complex growth media supplemented or not with NaCl,
T. halophila
accumulates glycine betaine and carnitine. Unlike other moderate halophiles,
T. halophila
was not able to produce potent osmoprotectants (such as ectoines and glycine betaine) through de novo synthesis when cultured in defined medium under hyperosmotic constraint. Addition of 2 mM carnitine, glycine betaine, or choline to defined medium improved growth parameters, not only at high salinity (up to 2.5 M NaCl) but also in media lacking NaCl. These compounds were taken up when available in the surrounding medium. The transport activity occurred at low and high salinities and seems to be constitutive. Glycine betaine and carnitine were accumulated by
T. halophila
in an unmodified form, while exogenously provided choline led to an intracellular accumulation of glycine betaine. This is the first evidence of the existence of a choline-glycine betaine pathway in a lactic acid bacterium. An assay showed that the compatible solutes strikingly repressed the accumulation of glutamate and slightly increased the intracellular potassium level only at high salinity. Interestingly, osmoprotectant-treated cells were able to maintain the intracellular sodium concentration at a relatively constant level (200 to 300 nmol/mg [dry weight]), independent of the NaCl concentration of the medium. In contrast, in the absence of osmoprotectant, the intracellular sodium content increased sharply from 200 to 2,060 nmol/mg (dry weight) when the salinity of the medium was raised from 1 to 2 M. Indeed, the imported compatible solutes play an actual role in regulating the intracellular Na
+
content and confer a much higher salt tolerance to
T. halophila.
Characterization and Role of the Branched-Chain Aminotransferase (BcaT) Isolated from
Lactococcus lactis
subsp.
cremoris
NCDO 763
Authors: Yvon Mireille , Chambellon Emilie , Bolotin Alexander , Roudot-Algaron Florence
Published: July 2002

In
Lactococcus lactis
, which is widely used as a starter in the cheese industry, the first step of aromatic and branched-chain amino acid degradation is a transamination which is catalyzed by two major aminotransferases. We have previously purified and characterized biochemically and genetically the aromatic aminotransferase, AraT. In the present study, we purified and studied the second enzyme, the branched-chain aminotransferase, BcaT. We cloned and sequenced the corresponding gene and used a mutant, along with the luciferase gene as the reporter, to study the role of the enzyme in amino acid metabolism and to reveal the regulation of gene transcription. BcaT catalyzes transamination of the three branched-chain amino acids and methionine and belongs to class IV of the pyridoxal 5′-phosphate-dependent aminotransferases. In contrast to most of the previously described bacterial BcaTs, which are hexameric, this enzyme is homodimeric. It is responsible for 90% of the total isoleucine and valine aminotransferase activity of the cell and for 50 and 40% of the activity towards leucine and methionine, respectively. The original role of BcaT was probably biosynthetic since expression of its gene was repressed by free amino acids and especially by isoleucine. However, in dairy strains, which are auxotrophic for branched-chain amino acids, BcaT functions only as a catabolic enzyme that initiates the conversion of major aroma precursors. Since this enzyme is still active under cheese-ripening conditions, it certainly plays a major role in cheese flavor development.
Staphylococcal Surface Display of Metal-Binding Polyhistidyl Peptides
Authors: Samuelson Patrik , Wernérus Henrik , Svedberg Malin , Ståhl Stefan
Published: July 2002

Recombinant
Staphylococcus xylosus
and
Staphylococcus carnosus
strains were generated with surface-exposed chimeric proteins containing polyhistidyl peptides designed for binding to divalent metal ions. Surface accessibility of the chimeric surface proteins was demonstrated and the chimeric surface proteins were found to be functional in terms of metal binding, since the recombinant staphylococcal cells were shown to have gained Ni
2+
- and Cd
2+
-binding capacity, suggesting that such bacteria could find use in bioremediation of heavy metals. This is, to our knowledge, the first time that recombinant, surface-exposed metal-binding peptides have been expressed on gram-positive bacteria. Potential environmental or biosensor applications for such recombinant staphylococci as biosorbents are discussed.
Physiological Analysis of the Expression of the Styrene Degradation Gene Cluster in
Pseudomonas fluorescens
ST
Authors: Santos Pedro Miguel , Blatny Janet Martha , Di Bartolo Ilaria , Valla Svein , Zennaro Elisabetta
Published: July 2002

The effects of different carbon sources on expression of the styrene catabolism genes in
Pseudomonas fluorescens
ST were analyzed by using a promoter probe vector, pPR9TT, which contains transcription terminators upstream and downstream of the β-galactosidase reporter system. Expression of the promoter of the
stySR
operon, which codes for the styrene two-component regulatory system, was found to be constitutive and not subject to catabolite repression. This was confirmed by the results of an analysis of the
stySR
transcript in
P. fluorescens
ST cells grown on different carbon sources. The promoter of the operon of the upper pathway, designated P
styA
, was induced by styrene and repressed to different extents by organic acids or carbohydrates. In particular, cells grown on succinate or lactate in the presence of styrene started to exhibit β-galactosidase activity during the mid-exponential growth phase, before the preferred carbon sources were depleted, indicating that there is a threshold succinate and lactate concentration which allows induction of styrene catabolic genes. In contrast, cells grown on glucose, acetate, or glutamate and styrene exhibited a diauxic growth curve, and β-galactosidase activity was detected only after the end of the exponential growth phase. In each experiment the reliability of the reporter system constructed was verified by comparing the β-galactosidase activity and the activity of the styrene monooxygenase encoded by the first gene of the styrene catabolic operon.
The Chromosomal Arsenic Resistance Genes of
Thiobacillus ferrooxidans
Have an Unusual Arrangement and Confer Increased Arsenic and Antimony Resistance to
Escherichia coli
Authors: Butcher Bronwyn G. , Deane Shelly M. , Rawlings Douglas E.
Published: July 2002

The chromosomal arsenic resistance genes of the acidophilic, chemolithoautotrophic, biomining bacterium
Thiobacillus ferrooxidans
were cloned and sequenced. Homologues of four arsenic resistance genes,
arsB
,
arsC
,
arsH
, and a putative
arsR
gene, were identified. The
T. ferrooxidans arsB
(arsenite export) and
arsC
(arsenate reductase) gene products were functional when they were cloned in an
Escherichia coli ars
deletion mutant and conferred increased resistance to arsenite, arsenate, and antimony. Therefore, despite the fact that the
ars
genes originated from an obligately acidophilic bacterium, they were functional in
E. coli
. Although
T. ferrooxidans
is gram negative, its ArsC was more closely related to the ArsC molecules of gram-positive bacteria. Furthermore, a functional
trxA
(thioredoxin) gene was required for ArsC-mediated arsenate resistance in
E. coli
; this finding confirmed the gram-positive ArsC-like status of this resistance and indicated that the division of ArsC molecules based on Gram staining results is artificial. Although
arsH
was expressed in an
E. coli
-derived in vitro transcription-translation system, ArsH was not required for and did not enhance arsenic resistance in
E. coli
. The
T. ferrooxidans ars
genes were arranged in an unusual manner, and the putative
arsR
and
arsC
genes and the
arsBH
genes were translated in opposite directions. This divergent orientation was conserved in the four
T. ferrooxidans
strains investigated.