bluuebunny/crossref_metadata_2025 huggingface dataset:
Trypanosoma cruziInactivation in Human Platelet Concentrates and Plasma by a Psoralen (Amotosalen HCl) and Long-Wavelength UV
Authors: Van Voorhis Wesley C. , Barrett Lynn K. , Eastman Richard T. , Alfonso Ryan , Dupuis Kent
Published: January 2003

Trypanosoma cruzi,the protozoan pathogen that causes Chagas' disease, can be found in the blood of infected individuals for their entire life span. This presents a serious challenge in safeguarding blood products. Transmission ofT. cruzifrom blood products is a frequent occurrence in Latin America, where Chagas' disease is endemic. This study was designed to determine whetherT. cruzicould be inactivated in human platelet concentrates and plasma by a photochemical treatment process with long-wavelength UV A light (UVA, 320 to 400 nm) plus the psoralen amotosalen HCl (Cerus Corporation). Units of platelet concentrates (300 ml) and plasma (300 ml) were intentionally contaminated with approximately 106T. cruzitrypomastigotes, theT. cruziform found in the bloodstream, per ml. The viability ofT. cruziafter photochemical inactivation was determined by their ability to replicate in 3T3 fibroblasts. Controls, including treatment with 150 μM amotosalen or 3 J/cm2UVA alone, did not lead to reduction of the viability ofT. cruziin plasma or platelet concentrates. However, treatment with 150 μM amotosalen plus 3 J/cm2UVA inactivatedT. cruzito undetectable levels in plasma and platelet concentrates. This represented a >5.4-log reduction ofT. cruziin platelet concentrates and >5.0-log reduction ofT. cruziin plasma. We conclude that the amotosalen plus UVA photochemical inactivation technology is effective in inactivating high levels of protozoan pathogens, such asT. cruzi,in platelet concentrates and plasma, as has been previously shown for numerous viruses and bacteria.
MC21-A, a Bactericidal Antibiotic Produced by a New Marine Bacterium,
Pseudoalteromonas phenolica
sp. nov. O-BC30
T
, against Methicillin-Resistant
Staphylococcus aureus
Authors: Isnansetyo Alim , Kamei Yuto
Published: January 2003

We previously reported a new marine bacterium,
Pseudoalteromonas phenolica
sp. nov. O-BC30
T
, which produced a bactericidal antibiotic against methicillin-resistant
Staphylococcus aureus
(MRSA). In the present study, we purified an anti-MRSA substance (MC21-A) from the methanol extract of the cells of
P. phenolica
O-BC30
T
and analyzed its chemical structure. MC21-A was determined to be 3,3′,5,5′-tetrabromo-2,2′-biphenyldiol by spectrometric analyses. Its anti-MRSA activity against 10 clinical isolates of MRSA was comparable to that of vancomycin (MC21-A MICs, 1 to 2 μg/ml; vancomycin MICs, <0.25 to 2 μg/ml). This substance was also high active against
Enterococcus serolicida
,
Enterococcus faecium
, and
Enterococcus faecalis
but was less active against
Streptococcus
spp. A time-kill study also demonstrated that MC21-A was bactericidal and that its killing rate was much higher than that of vancomycin. The postantibiotic effect (PAE) of MC21-A against a clinical MRSA isolate, strain E 31243, was also comparable to that of vancomycin (MC21-A PAEs, 1.46 to 1.65 h; vancomycin PAEs, 0.84 to 1.43 h). However, a lysis experiment demonstrated that this substance failed to lyse MRSA cells. This substance also did not lyse human erythrocytes. A SYTOX Green staining experiment implied that this substance permeabilized the cell membrane of MRSA as its mode of action. When its activities against a hypersensitive
Escherichia coli
mutant (KO 1489) and wild-type strains were tested, MC21-A exhibited higher levels of activity against the former. Furthermore, MC21-A was not cytotoxic to human normal fibroblast, rat pheochromocytoma, and Vero cells at concentrations up to 50 μg/ml. These results suggest that MC21-A might be useful as a lead compound in the development of new types of anti-MRSA substances with modes of action different from those of vancomycin and teicoplanin.
Macrolide-Resistant
Streptococcus pneumoniae
and
Streptococcus pyogenes
in the Pediatric Population in Germany during 2000-2001
Authors: Reinert Ralf René , Lütticken Rudolf , Bryskier André , Al-Lahham Adnan
Published: January 2003

In a nationwide study in Germany covering 13 clinical microbiology laboratories, a total of 307
Streptococcus pyogenes
(mainly pharyngitis) and 333
Streptococcus pneumoniae
(respiratory tract infections) strains were collected from outpatients less than 16 years of age. The MICs of penicillin G, amoxicillin, cefotaxime, erythromycin A, clindamycin, levofloxacin, and telithromycin were determined by the microdilution method. In
S. pyogenes
isolates, resistance rates were as follows: penicillin, 0%; erythromycin A, 13.7%; and levofloxacin, 0%. Telithromycin showed good activity against
S. pyogenes
isolates (MIC
90
= 0.25 μg/ml; MIC range, 0.016 to 16 μg/ml). Three strains were found to be telithromycin-resistant (MIC ≥ 4 μg/ml). Erythromycin-resistant strains were characterized for the underlying resistance genotype, with 40.5% having the efflux type
mef
(A), 38.1% having the
erm
(A), and 9.5% having the
erm
(B) genotypes.
emm
typing of macrolide-resistant
S. pyogenes
isolates showed
emm
types 4 (45.2%), 77 (26.2%), and 12 (11.9%) to be predominant. In
S. pneumoniae
, resistance rates were as follows: penicillin intermediate, 7.5%; penicillin resistant, 0%; erythromycin A, 17.4%; and levofloxacin, 0%. Telithromycin was highly active against pneumococcal isolates (MIC
90
≤ 0.016 μg/ml; range, 0.016 to 0.5 μg/ml). The overall resistance profile of streptococcal respiratory tract isolates is still favorable, but macrolide resistance is of growing concern in Germany.
NP-1, a Rabbit α-Defensin, Prevents the Entry and Intercellular Spread of Herpes Simplex Virus Type 2
Authors: Sinha Sara , Cheshenko Natalia , Lehrer Robert I. , Herold Betsy C.
Published: January 2003

Rabbit neutrophil peptide-1 (NP-1), a prototypic α-defensin, protects cells in vitro from infection by clinical and laboratory isolates of herpes simplex virus type 2 (HSV-2). Incubation of concentrated virus stocks for 1 h with noncytotoxic concentrations of NP-1 reduces subsequent infection by >98%. Pretreating cells with NP-1 for 1 h prior to inoculation with untreated virus also prevents infection. NP-1, a cationic peptide, does not compete with viral envelope glycoproteins for binding to cellular heparan sulfate receptors, but it prevents viral entry. No VP16, a major viral tegument protein, is transported to the cell nucleus in the presence of NP-1. Infectious center assays demonstrate that NP-1 also inhibits cell-to-cell viral spread. Thus, NP-1 prevents virally mediated fusion events, entry, and cell-to-cell spread. This unique mechanism of anti-HSV activity, coupled with established antibacterial and possible anti-human immunodeficiency virus type 1 activities of defensins, render this family of compounds excellent candidates for further development as topical microbicides.
Toward the Development of a Virus-Cell-Based Assay for the Discovery of Novel Compounds against Human Immunodeficiency Virus Type 1
Authors: Adelson Martin E. , Pacchia Annmarie L. , Kaul Malvika , Rando Robert F. , Ron Yacov , Peltz Stuart W. , Dougherty Joseph P.
Published: January 2003

The emergence of human immunodeficiency virus type 1 (HIV-1) strains resistant to highly active antiretroviral therapy necessitates continued drug discovery for the treatment of HIV-1 infection. Most current drug discovery strategies focus upon a single aspect of HIV-1 replication. A virus-cell-based assay, which can be adapted to high-throughput screening, would allow the screening of multiple targets simultaneously. HIV-1-based vector systems mimic the HIV-1 life cycle without yielding replication-competent virus, making them potentially important tools for the development of safe, wide-ranging, rapid, and cost-effective assays amenable to high-throughput screening. Since replication of vector virus is typically restricted to a single cycle, a crucial question is whether such an assay provides the needed sensitivity to detect potential HIV-1 inhibitors. With a stable, inducible vector virus-producing cell line, the inhibitory effects of four reverse transcriptase inhibitors (zidovudine, stavudine, lamivudine, and didanosine) and one protease inhibitor (indinavir) were assessed. It was found that HIV-1 vector virus titer was inhibited in a single cycle of replication up to 300-fold without affecting cell viability, indicating that the assay provides the necessary sensitivity for identifying antiviral molecules. Thus, it seems likely that HIV-1-derived vector systems can be utilized in a novel fashion to facilitate the development of a safe, efficient method for screening compound libraries for anti-HIV-1 activity.
Two Mechanisms for Human Immunodeficiency Virus Type 1 Inhibition by N-Terminal Modifications of RANTES
Authors: Pastore Cristina , Picchio Gastón R. , Galimi Francesco , Fish Richard , Hartley Oliver , Offord Robin E. , Mosier Donald E.
Published: January 2003

C-C chemokine receptor 5 (CCR5) is the primary coreceptor for human immunodeficiency virus type 1 (HIV-1) infection. Native chemokines that bind to CCR5 inhibit HIV-1 infection, albeit weakly, but chemically modified chemokines inhibit infection more efficiently. We have investigated the inhibitory mechanism of three N-terminally modified RANTES variants (AOP-, NNY-, and PSC-RANTES) with the MT-2 human T-cell line stably expressing either native or mutated CCR5. The RANTES analogues showed the same rank order (PSC > NNY > AOP) in their capacity to induce prolonged CCR5 internalization, inhibit surface reexpression, and prevent HIV-1 infection on MT-2 cells expressing wild-type CCR5 or CCR5 with four C-terminal serine phosphorylation sites mutated to alanine. None of the RANTES analogues caused internalization of a C-terminal cytoplasmic domain deletion mutant of CCR5, and each derivative had equal potency in inhibiting HIV-1 infection of MT-2 cells expressing this mutant. We conclude that the C-terminal cytoplasmic residues of CCR5 are necessary for receptor sequestration by RANTES analogues but that the process and the relative activity of each derivative are not dependent upon phosphorylation of the C-terminal serine residues. Two mechanisms of antiviral activity are demonstrated: receptor blockade and receptor sequestration. Potency correlates with the ability to induce CCR5 sequestration but not with receptor binding, suggesting that sequestration may make the greater contribution to antiviral activity.
Suboptimal Antibiotic Dosage as a Risk Factor for Selection of Penicillin-Resistant
Streptococcus pneumoniae
: In Vitro Kinetic Model
Authors: Odenholt Inga , Gustafsson Ingegerd , Löwdin Elisabeth , Cars Otto
Published: January 2003

Optimizing pharmacokinetic/pharmacodynamic indices of antibiotics to obtain clinical and microbiological efficacy is essential, but dosing regimens must also be tailored to minimize the risk for emergence of resistance. The aim of the present study was to investigate whether certain concentrations of benzylpenicillin are critical for the selection of resistant subpopulations. A mixed culture of
Streptococcus pneumoniae
containing ca. 90% susceptible (MIC = 0.031 mg/liter), 9% intermediate (MIC = 0.25 mg/liter), and 1% resistant (MIC = 8 mg/liter) was studied in an in vitro kinetic model. The time that concentrations exceeded the MIC (T>MIC) for the three strains in the culture was varied by different initial concentrations of benzylpenicillin. Samples for viable counts were withdrawn at different times during 24 h and seeded on blood agar plates and on selective antibiotic-containing plates. The T>MIC varied from 46 to 100% for the susceptible strain, from 6 to 100% for the intermediate strain, and from 0 to 48% for the resistant strain. Our study, which may mimic the clinical situation with carriage of a mixed population of
S. pneumoniae
with different antibiotic susceptibilities, has shown that selection of resistant bacteria may easily occur if dosing regimens are only targeted toward fully susceptible strains.
Activities of Azithromycin and Amphotericin B against
Naegleria fowleri
In Vitro and in a Mouse Model of Primary Amebic Meningoencephalitis
Authors: Goswick Shannon M. , Brenner George M.
Published: January 2003

Inhalation of fresh water containing the free-living ameba
Naegleria fowleri
may lead to a potentially fatal infection known as primary amebic meningoencephalitis. Amphotericin B is the only agent with established clinical efficacy in the treatment of primary amebic meningoencephalitis in humans, but therapy with this drug is often associated with adverse effects on the kidneys and other organs, and not all persons treated with amphotericin B have survived. We investigated the in vitro activity and in vivo efficacy of newer therapeutic agents in an attempt to identify other useful agents for treating primary amebic meningoencephalitis. Azithromycin has shown in vitro activity against
Acanthamoeba
spp. and in vivo activity against experimental toxoplasmosis. In our study, the MIC of azithromycin against
N. fowleri
was 13.4 μM (10 μg/ml), which was 123 times greater than the MIC of amphotericin B, which was 0.108 μM (0.1 μg/ml). Azithromycin protected 100% of mice infected with
N. fowleri
at a dose of 75 mg/kg/day for 5 days, whereas amphotericin B protected only 50% of mice at a dose of 7.5 mg/kg/day for 5 days, and all control mice died during the 28-day observation period. We conclude that azithromycin has both in vitro and in vivo activity versus
N. fowleri
and may be a useful addition to therapy for primary amebic meningoencephalitis.
Activity of Tigecycline (GAR-936), a Novel Glycylcycline, against Enterococci in the Mouse Peritonitis Model
Authors: Nannini Esteban C. , Pai Suresh R. , Singh Kavindra V. , Murray Barbara E.
Published: January 2003

A novel glycylcycline agent, tigecycline (GAR-936), was evaluated in vivo in the mouse model of peritonitis against three
Enterococcus faecalis
and four
Enterococcus faecium
isolates with different susceptibilities to vancomycin and tetracyclines, all of which were inhibited by ≤0.125 μg of tigecycline/ml. Using a single subcutaneous dose, tigecycline displayed a protective effect (50% protective dose, ≤5.7 mg/kg of body weight) against all strains tested, including two with Tn
925
(from the Tn
916
family), which contains the Tet(M) tetracycline resistance determinant, as well as VanA and VanB strains. As expected, tetracycline and minocycline were ineffective against the isolates carrying Tn
925
.
Activities of Tigecycline (GAR-936) against
Legionella pneumophila
In Vitro and in Guinea Pigs with
L. pneumophila
Pneumonia
Authors: Edelstein Paul H. , Weiss William J. , Edelstein Martha A. C.
Published: January 2003

The activities of tigecycline (Wyeth Research) against extracellular and intracellular
Legionella pneumophila
and for the treatment of guinea pigs with
L. pneumophila
pneumonia were studied. The tigecycline MIC at which 50% of strains are inhibited for 101 different
Legionella
sp. strains was 4 μg/ml versus 0.125 and 0.25 μg/ml for azithromycin and erythromycin, respectively. Tigecycline was about as active as erythromycin (tested at 1 μg/ml) against the F889 strain of
L. pneumophila
grown in guinea pig alveolar macrophages and more active than erythromycin against the F2111 strain. Azithromycin (0.25 μg/ml) was more active than (F889) or as active as (F2111) tigecycline (1 μg/ml) in the macrophage model. When tigecycline was given (7.5 mg/kg of body weight subcutaneously once) to guinea pigs with
L. pneumophila
pneumonia, the mean peak serum and lung levels were 2.3 and 1.8 μg/ml (1.2 and 1.5 μg/g) at 1 and 2 h postinjection, respectively. The serum and lung areas under the concentration time curve from 0 to 24 h were 13.7 and 15.8 μg · h/ml, respectively. Thirteen of 16 guinea pigs with
L. pneumophila
pneumonia treated with tigecycline (7.5 mg/kg subcutaneously once daily for 5 days) survived for 7 days post-antimicrobial therapy, as did 11 of 12 guinea pigs treated with azithromycin (15 mg/kg intraperitoneally once daily for 2 days). None of 12 guinea pigs treated with saline survived. Tigecycline-treated guinea pigs had average end of therapy lung counts of 1 × 10
6
CFU/g (range, 2.5 × 10
4
to 3.2 × 10
6
CFU/g) versus <1 × 10
2
CFU/g for azithromycin (range, undetectable to 100 CFU/g). A second guinea pig study examined the ability of tigecycline to clear
L. pneumophila
from the lung after 5 to 9 days of therapy; bacterial concentrations 1 day posttherapy ranged from log
10
4.2 to log
10
5.5 CFU/g for four different dosing regimens. Tigecycline is about as effective as erythromycin against intracellular
L. pneumophila
, but tigecycline inactivation by the test media confounded the interpretation of susceptibility data. Tigecycline was effective at preventing death from pneumonia in an animal model of Legionnaires' disease, warranting human clinical trials of the drug for the disease.
Population Pharmacokinetic Analysis of Nonlinear Behavior of Piperacillin during Intermittent or Continuous Infusion in Patients with Cystic Fibrosis
Authors: Vinks Alexander A. , den Hollander Jan G. , Overbeek Shelley E. , Jelliffe Roger W. , Mouton Johan W.
Published: January 2003

The purpose of this study was to describe the nonlinear pharmacokinetics of piperacillin observed during intermittent infusion and continuous infusion by using a nonparametric population modeling approach. Data were 120 serum piperacillin concentration measurements from eight adult cystic fibrosis (CF) patients. Individual pharmacokinetic parameter estimates during intermittent infusion or continuous infusion were calculated by noncompartmental analysis and with a maximum iterative two-stage Bayesian estimator. To simultaneously describe concentration-time data during intermittent infusion and continuous infusion, nonlinear models were parameterized as two-compartment Michaelis-Menten models. Models were fit to the data with the nonparametric expectation maximization algorithm. The calculations were executed on a remote supercomputer. Nonlinear models were evaluated by log-likelihood estimates, residual plots, and
R
2
values, and predictive performance was based on bias (mean weighted error [MWE]) and precision (mean weighted square error [MWSE]). A linear pharmacokinetic model could not describe combined intermittent infusion and continuous infusion data well. A good population model fit to the intermittent infusion and continuous infusion data was obtained with the constructed nonlinear models. Maximum a posteriori probability (MAP) Bayesian
R
2
values for the nonlinear models were 0.96 to 0.97. Median parameter estimates for the best nonlinear model were as follows:

K
m

, 58 ± 75 mg/liter (mean and standard deviation);
V
max
, 1,904 ± 1,009 mg/h; volume of distribution of the central compartment, 14.1 ± 3.0 liters;
k
12
, 0.63 ± 0.41 h
−1
; and
k
21
, 0.37 ± 0.19 h
−1
. The median bias (MWE) and precision (MWSE) values for MAP Bayesian estimation with the Michaelis-Menten model were 0.05 and 4.6 mg/liters, respectively. The developed nonlinear pharmacokinetic models can be used to optimize piperacillin therapy administered via continuous infusion in patients with CF and have distinct advantages over conventional linear models.
Population Pharmacokinetics of Linezolid in Patients Treated in a Compassionate-Use Program
Authors: Meagher Alison K. , Forrest Alan , Rayner Craig R. , Birmingham Mary C. , Schentag Jerome J.
Published: January 2003

Data obtained from 318 adult patients treated under the linezolid compassionate-use protocol were used to develop a population model of the pharmacokinetics of intravenous and oral linezolid. All of the patients received 600 mg of linezolid every 12 h, intravenously and/or orally. Blood samples (2 to 10 per patient; median, 4) were obtained and assayed for linezolid by high-performance liquid chromatography. These data and patient covariates were modeled by iterative two-stage analysis, and model discrimination was done by Akaike's information criterion. Of the patient covariates considered (age, sex, ideal body weight, baseline serum albumin, hepatic or renal dysfunction, underlying malignancy, organ transplantation, surgical status, global severity of illness, site of infection, route of administration, and location of care [intensive-care unit, general floor, or outpatient]), only normalized creatinine clearance (CL
CR
) and body weight explained significant portions of the variance and were incorporated into the pharmacokinetic model. The final model included central and peripheral compartments with parallel capacity-limited (nonrenal) and first-order (renal [CL
R
]) clearances. Volumes and clearances were normalized to the ideal body weight, and CL
R
was modeled as proportional to CL
CR
. Compared to previously studied adult volunteers, intrinsic clearance was ∼60% higher and the maximum rate of metabolism was twice as high in these debilitated patients, resulting in lower area under the time-concentration curve (AUC) values (
P
< 0.001). The derived 24-h AUC, averaged over the first 7 days of treatment, ranged between 57 and 871 (median, 191) μg/ml · 24 h. Despite these variations, linezolid provided high rates of clinical cure, as well as microbiological success, in the patients treated in the compassionate-use program. The mechanism(s) of these pharmacokinetic differences is unknown and requires further mechanistic study.
Improved Pharmacokinetics and Reduced Antibody Reactivity of Lysostaphin Conjugated to Polyethylene Glycol
Authors: Walsh Scott , Shah Anjali , Mond James
Published: January 2003

Lysostaphin is a 27-kDa endopeptidase that enzymatically disrupts the cell wall ofStaphylococcus aureusand is a promising candidate for treatingS. aureusblood-borne infections. It would be extremely useful to define conditions that would both increase lysostaphin's in vivo half-life to allow for more effective tissue distribution and reduce its immunogenicity. Conjugation of polyethylene glycol (PEG) to lysostaphin (PEGylation) was investigated as a means to accomplish these goals. Rather than using linear forms of PEG, branched PEGs were chosen as the initial candidates because their large spatial volumes prevent entry of the polymer into the enzyme's active sites, which could potentially reduce enzymatic function. Enzymatic activity for most PEGylated lysostaphins was reduced, but these compounds were still considerably active compared to unconjugated lysostaphin, with conjugates that had lower degrees of PEG modification having greater activity than those with higher degrees. PEGylated lysostaphin injected intravenously had a serum drug half-life of up to 24 h and resulted in much higher plasma drug concentrations than an equal dose of unconjugated lysostaphin, which had a half-life of less than 1 h. Finally, reduced binding affinity was shown for PEGylated lysostaphin in an antilysostaphin capture enzyme-linked immunosorbent assay, with some PEG-lysostaphin conjugates having binding affinities that were reduced more than 10-fold compared to unconjugated lysostaphin. These findings demonstrate that PEGylation of lysostaphin, while diminishing itsS. aureuskilling activity, results in prolonged serum drug persistence and reduced antibody binding. These features should significantly enhance lysostaphin's therapeutic value as an intravenous “antibiotic” againstS. aureus.
Prevalence of Plasmid-Mediated Quinolone Resistance
Authors: Jacoby George A. , Chow Nancy , Waites Ken B.
Published: January 2003

Quinolone resistance encoded by the
qnr
gene and mediated by plasmid pMG252 was discovered in a clinical strain of
Klebsiella pneumoniae
that was isolated in 1994 at the University of Alabama at Birmingham Medical Center. The gene codes for a protein that protects DNA gyrase from quinolone inhibition and that belongs to the pentapeptide repeat family of proteins. The prevalence of the gene has been investigated by using PCR with
qnr
-specific primers with a sample of more than 350 gram-negative strains that originated in 18 countries and 24 states in the United States and that included many strains with plasmid-mediated AmpC or extended spectrum β-lactamase enzymes.
qnr
was found in isolates from the University of Alabama at Birmingham only during 6 months in 1994, despite the persistence of the gene for FOX-5 β-lactamase, which is linked to
qnr
on pMG252. Isolates from other locations were negative for
qnr
. The prevalence of
mcbG
in the same sample was also examined.
mcbG
encodes another member of the pentapeptide repeat family and is involved in immunity to microcin B17, which, like quinolones, targets DNA gyrase. A single clinical isolate contained
mcbG
on a transmissible R plasmid. This plasmid and one carrying the complete microcin B17 operon slightly decreased sparfloxacin susceptibility but had a much less protective effect than pMG252. Plasmid-mediated quinolone resistance was thus rare in the sample examined.
In Vivo Acquisition of Ceftriaxone Resistance in
Salmonella enterica
Serotype Anatum
Authors: Su Lin-Hui , Chiu Cheng-Hsun , Chu Chishih , Wang Mei-Hui , Chia Ju-Hsin , Wu Tsu-Lan
Published: January 2003

The emergence of resistance to antimicrobial agents within the salmonellas is a worldwide and severe problem. A case of treatment failure due to the emergence of resistance to ceftriaxone in
Salmonella enterica
serotype Anatum was studied.
S. enterica
serotype Anatum and
Escherichia coli
, both of which are susceptible to ceftriaxone, were initially isolated from a diabetic patient hospitalized for the treatment of wound and urinary tract infections. Resistant
S. enterica
serotype Anatum and
E. coli
strains were isolated concomitantly 2 weeks after the initiation of ceftriaxone therapy. The patient eventually died of a sepsis caused by the ceftriaxone-resistant salmonella. PCR, nucleotide sequence analysis, and DNA-DNA hybridization identified a
bla
CTX-M-3
gene located on a 95.1-kb plasmid from the ceftriaxone-resistant isolates of
S. enterica
serotype Anatum and
E. coli
. The plasmid was proved to be conjugative. Molecular fingerprinting showed that the susceptible and resistant strains were genetically indistinguishable. The emergence of resistance to ceftriaxone in
S. enterica
serotype Anatum was due to the in vivo acquisition of a plasmid containing the
bla
CTX-M-3
gene and was the cause for treatment failure in this patient.
Morphological and Genetic Differences in Two Isogenic
Staphylococcus aureus
Strains with Decreased Susceptibilities to Vancomycin
Authors: Reipert Andrea , Ehlert Kerstin , Kast Thomas , Bierbaum Gabriele
Published: January 2003

Many VISA (vancomycin intermediately resistant
Staphylococcus aureus
) strains are characterized by increased cell wall biosynthesis and decreased cross-linking of the peptide side chains, leading to accumulation of free
d
-alanyl-
d
-alanine termini in the peptidoglycan, which act as false target sites for vancomycin. A spontaneous mutant of methicillin-resistant VISA strain SA137/93A (vancomycin MIC [E-test], 8 μg/ml), called SA137/93G, showed increased resistance to vancomycin (MIC [E-test], 12 μg/ml). Analysis of the resistance profile of the mutant revealed a loss of β-lactam resistance with a concomitant increase in resistance to glycopeptides. In both strains, cell wall thickness was 1.4-fold greater than that of control isolates. However, cross-linking of the cell wall was drastically lower in SA137/93A than in SA137/93G. The sensitivity of strain SA137/93G to β-lactams was due to loss of the β-lactamase plasmid and a deletion that comprises 32.5 kb of the methicillin resistance cassette SCC
mec
, as well as 65.4 kb of chromosomal DNA. A spontaneous mutant of SA137/93G with higher sensitivity to vancomycin displayed a cell wall profile similar, in some respects, to that of an
fmhB
mutant. Results described here and elsewhere show that the only feature common to all VISA strains is a thickened cell wall, which may play a central role in the vancomycin resistance mechanism.
Mutations in
Aspergillus fumigatus
Resulting in Reduced Susceptibility to Posaconazole Appear To Be Restricted to a Single Amino Acid in the Cytochrome P450 14α-Demethylase
Authors: Mann Paul A. , Parmegiani Raulo M. , Wei Shui-Qing , Mendrick Cara A. , Li Xin , Loebenberg David , DiDomenico Beth , Hare Roberta S. , Walker Scott S. , McNicholas Paul M.
Published: January 2003

To better understand the molecular basis of posaconazole (POS) resistance in
Aspergillus fumigatus
, resistant laboratory isolates were selected. Spontaneous mutants arose at a frequency of 1 in 10
8
and fell into two susceptibility groups, moderately resistant and highly resistant. Azole resistance in
A. fumigatus
was previously associated with decreased drug accumulation. We therefore analyzed the mutants for changes in levels of transcripts of genes encoding efflux pumps (
mdr1
and
mdr2
) and/or alterations in accumulation of [
14
C]POS. No changes in either pump expression or drug accumulation were detected. Similarly, there was no change in expression of
cyp51A
or
cyp51B
, which encode the presumed target site for POS, cytochrome P450 14α-demethylase. DNA sequencing revealed that each resistant isolate carried a single point mutation in residue 54 of
cyp51A
. Mutations at the same locus were identified in three clinical
A. fumigatus
isolates exhibiting reduced POS susceptibility but not in susceptible clinical strains. To verify that these mutations were responsible for the resistance phenotype, we introduced them into the chromosome of a POS-susceptible
A. fumigatus
strain under the control of the glyceraldehyde phosphate dehydrogenase promoter. The transformants exhibited reductions in susceptibility to POS comparable to those exhibited by the original mutants, confirming that point mutations in the
cyp51A
gene in
A. fumigatus
can confer reduced susceptibility to POS.
Biochemical Characterization of the Acquired Metallo-β-Lactamase SPM-1 fromPseudomonas aeruginosa
Authors: Murphy Tanya A. , Simm Alan M. , Toleman Mark A. , Jones Ronald N. , Walsh Timothy R.
Published: January 2003

SPM-1 is a new metallo-β-lactamase recently identified inPseudomonas aeruginosastrain 48-1997A, isolated in Sao Paulo, Brazil. Kinetic analysis demonstrated that SPM-1 has a broad hydrolytic profile across a wide range of β-lactam antibiotics. Considerable variation was observed within the penicillin, cephalosporin, and carbapenem subfamilies; however, on the whole, SPM-1 appears to preferentially hydrolyze cephalosporins. The highestkcat/Kmratios (in micromolar per second) overall were observed for this subgroup. The hydrolytic profile of SPM-1 bears the most similarity to that of the metallo-β-lactamase IMP-1, yet for the most part, SPM-1 haskcat/Kmvalues higher than those of IMP-1. Zinc chelator studies established that progressive inhibition of SPM-1 by EDTA, dipicolinic acid, and 1-10-o-phenanthroline demonstrated a biexponential pattern in which none of the chelators completely inhibited SPM-1. A homology model of SPM-1 was developed on the basis of the IMP-1 crystal structure, which showed the protein folding and active-site structure characteristic of metallo-β-lactamases and which provides an explanation for the kinetic profiles observed.
The Antifungal Protein from
Aspergillus giganteus
Causes Membrane Permeabilization
Authors: Theis T. , Wedde M. , Meyer V. , Stahl U.
Published: January 2003

We investigated the inhibitory effects of the antifungal protein (AFP) from
Aspergillus giganteus
on the growth of several filamentous fungi. For this purpose, the MICs of AFP were determined and ranged from 0.1 μg/ml for
Fusarium oxysporum
to 200 μg/ml for
Aspergillus nidulans
. The antifungal activity of AFP was diminished in the presence of cations. We were able to show that incubation of AFP-sensitive fungi with the protein resulted in membrane permeabilization using an assay based on the uptake of the fluorescent dye SYTOX Green. No permeabilization by AFP could be detected at concentrations below the species-specific MIC. Furthermore, AFP-induced permeabilization could readily be detected after 5 min of incubation. Localization experiments with fluorescein isothiocyanate-labeled AFP and immunofluorescence staining with an AFP-specific antibody supported the observation that the protein interacts with membranes. After treatment of AFP-sensitive fungi with AFP, the protein was localized at the plasma membrane, whereas it was mainly detected inside the cells of AFP-resistant fungi. We conclude from these data that the growth-inhibitory effect of AFP is caused by permeabilization of the fungal membranes.
Genotypic Inhibitory Quotient as Predictor of Virological Response to Ritonavir-Amprenavir in Human Immunodeficiency Virus Type 1 Protease Inhibitor-Experienced Patients
Authors: Marcelin Anne-Geneviève , Lamotte Claire , Delaugerre Constance , Ktorza Nadine , Ait Mohand Hocine , Cacace Raquel , Bonmarchand Manuela , Wirden Marc , Simon Anne , Bossi Philippe , Bricaire François , Costagliola Dominique , Katlama Christine , Peytavin Gilles , Calvez Vincent
Published: January 2003

Forty-nine protease inhibitor (PI)-experienced but amprenavir (APV)-naïve patients experiencing virological failure were treated with ritonavir (RTV) (100 mg twice a day [b.i.d.]) plus APV (600 mg b.i.d.). Patients responded to therapy with a median viral load decrease of −1.32 log10by week 12. The addition of low-dose RTV enhanced the minimal APV concentration in plasma (APVCmin) up to 10-fold compared with that obtained with APV (1,200 mg b.i.d.) without RTV. Baseline PI resistance mutations (L10F/I/V, K20M/R, E35D, R41K, I54V, L63P, V82A/F/T/S, I84V) identified by univariate analysis and included in a genotypic score and APVCminat week 8 were predictive of the virological response at week 12. The response to APV plus RTV was significantly reduced in patients with six or more of the resistance mutations among the ones defined above. The genotypic inhibitory quotient, calculated as the ratio of the APVCminto the number of human immunodeficiency virus type 1 protease mutations, was a better predictor than the virological or pharmacological variables used alone. This genotypic inhibitory quotient could be used in therapeutic drug monitoring to define the concentrations in plasma needed to control replication of viruses with different levels of PI resistance, as measured by the number of PI resistance mutations.
Efficacy of CS-758, a Novel Triazole, against Experimental Fluconazole-Resistant Oropharyngeal Candidiasis in Mice
Authors: Kamai Yasuki , Kubota Mikie , Fukuoka Takashi , Kamai Yoko , Maeda Naoyuki , Hosokawa Tsunemichi , Shibayama Takahiro , Uchida Katsuhisa , Yamaguchi Hideyo , Kuwahara Shogo
Published: January 2003

The therapeutic efficacy of CS-758, a novel triazole, was evaluated against experimental murine oropharyngeal candidiasis induced by
Candida albicans
with various susceptibilities to fluconazole. Against infections induced by strains with various susceptibilities to fluconazole, the efficacy of fluconazole was strongly correlated with the MIC of fluconazole, as measured by the NCCLS method, and agreed with the NCCLS interpretive breakpoints, suggesting that the efficacies of new drugs could be predicted by using this model. The results of the fungal burden study corresponded with the results of the histopathological study. CS-758 exhibited potent in vitro activity (MICs, 0.004 to 0.06 μg/ml) against the strains used in this murine model including fluconazole-susceptible dose-dependent and fluconazole-resistant strains (fluconazole MICs, 16 to 64 μg/ml). CS-758 exhibited excellent efficacy against the infections induced by all the strains including a fluconazole-resistant strain, and the reductions in viable cell counts were significant at 10 and 50 mg/kg of body weight/dose. Fluconazole was not effective even at 50 mg/kg/dose against infections induced by a fluconazole-resistant strain (fluconazole MIC, 64 μg/ml). These results suggest that CS-758 is a promising compound for the treatment of oropharyngeal candidiasis including fluconazole-refractory infections.
NK-Lysin and Its Shortened Analog NK-2 Exhibit Potent Activities againstTrypanosoma cruzi
Authors: Jacobs Thomas , Bruhn Heike , Gaworski Iris , Fleischer Bernhard , Leippe Matthias
Published: January 2003

Antimicrobial peptides are widespread in nature and have been evolutionarily conserved as essential tools for combating a variety of pathogens. Among the plethora of natural peptides and synthetic analogs thereof studied in recent years for their antimicrobial activities, only a very few are known to be effective against protozoan parasites. In the present study we investigated the activity of NK-lysin, a broad-spectrum effector polypeptide of mammalian cytotoxic lymphocytes, against trypomastigotes of the human pathogenTrypanosoma cruziin vitro. Moreover, the activity of a synthetic peptide named NK-2 that corresponds to the cationic core region of NK-lysin was tested in parallel against this parasite.T. cruziwas found to be highly susceptible to both peptides, as evidenced by inhibition of the mobility of trypomastigotes. The peptides rapidly permeabilized the plasma membrane of the parasite since micromolar concentrations resulted in the release of cytosolic enzymes within minutes. NK-lysin and NK-2 were even found to kill trypanosomes residing inside the human glioblastoma cell line 86HG39, but only NK-2 left the host cells apparently unharmed.
Culture and Antibiotic Susceptibility of
Bartonella quintana
in Human Erythrocytes
Authors: Rolain Jean-Marc , Maurin Max , Mallet Marie-Noëlle , Parzy Daniel , Raoult Didier
Published: January 2003

Bartonella quintana
, the agent of trench fever, has recently been implicated in various diseases, in particular, bacteremia and endocarditis in homeless people. The host cell of
Bartonella
spp. is believed to be the erythrocyte, and in the present study we demonstrate that
B. quintana
can be cultured in vitro in human erythrocytes. The bacteria were found to be intraerythrocytic by laser confocal microscopy with
Bartonella
species-specific monoclonal antibodies. Infections with
B. quintana
decreased the life span of erythrocytes in culture from 8.6 to 4.8 days. In the culture system we found that most of the antibiotics that we tested (doxycycline, fluoroquinolone compounds, and beta-lactams) were not bactericidal. Gentamicin was bactericidal at 4 μg/ml, as was rifampin, but to a lesser extent. At this concentration, gentamicin has been shown to enter erythrocytes slowly and to reach a peak level of 0.26 μg/ml after 24 h. At 0.26 μg/ml, however, we found that gentamicin was not able to kill extracellular
B. quintana
, even after 96 h of incubation. We hypothesize that erythrocytes may be a reservoir for
B. quintana
and that the bactericidal activity of gentamicin that we observed occurs mainly when the bacteria emerge from the erythrocytes and are found extracellularly. It would appear that gentamicin should be administered for at least 5 days to cure patients infected with
B. quintana
.
Effect of Amphotericin B on Larval Growth of
Echinococcus multilocularis
Authors: Reuter Stefan , Merkle Marion , Brehm Klaus , Kern Peter , Manfras Burkhard
Published: January 2003

Alveolar echinococcosis is caused by the parasitic cestode
Echinococcus multilocularis
. Benzimidazoles, namely, mebendazole and albendazole, are the only drugs available for the treatment of inoperable alveolar echinococcosis. At present, no therapeutic alternative is available for patients with progressive disease under treatment or for patients who are unable to tolerate the side effects of the benzimidazoles. In addition, benzimidazoles are only parasitostatic for
E. multilocularis
. Thus, new therapeutic options are of paramount importance. In the present study we examined the in vitro effect of amphotericin B on
E. multilocularis
larvae.
E. multilocularis
metacestodes grown in the peritoneal cavities of Mongolian gerbils were transferred into a culture system. Vesicles budded from the tissue blocks and increased in number and size during the first 5 weeks. After 6 weeks drugs were added and deleterious effects on the vesicles were observed macroscopically and microscopically. By use of this in vitro tissue culture model we demonstrated that amphotericin B effectively inhibits the growth of
E. multilocularis
metacestodes. This destructive effect was significantly more rapid with amphotericin B than with the benzimidazoles. Cyclic treatment was effective in suppressing parasite growth. However, amphotericin B appears to be parasitostatic for
E. multilocularis
larvae, and regrowth occurs even after extended periods. In summary, amphotericin B constitutes the first promising alternative for the treatment of alveolar echinococcosis in cases of intolerance or resistance to benzimidazoles. It holds promise as an effective treatment option for otherwise fatal courses of disease.
Pharmacokinetics (PK), Pharmacodynamics (PD), and PK-PD Integration of Danofloxacin in Sheep Biological Fluids
Authors: Aliabadi F. Shojaee , Landoni M. F. , Lees P.
Published: January 2003

The fluoroquinolone antimicrobial drug danofloxacin was administered to sheep intravenously (i.v.) and intramuscularly (i.m.) at a dose of 1.25 mg/kg of body weight in a two-period crossover study. The pharmacokinetic properties of danofloxacin in serum, inflamed tissue cage fluid (exudate), and noninflamed tissue cage fluid (transudate) were established by using a tissue cage model. The in vitro and ex vivo activities of danofloxacin in serum, exudate, and transudate against a pathogenic strain ofMannheimia haemolyticawere established. Integration of in vivo pharmacokinetic data with the in vitro MIC provided mean values for the area under the curve (AUC)/MIC for serum, exudate, and transudate of 60.5, 85.6, and 45.7 h, respectively, after i.v. dosing and 55.9, 77.9, and 49.1 h, respectively, after i.m. dosing. After i.m. dosing, the maximum concentration/MIC ratios for serum, exudate, and transudate were 10.8, 3.0, and 1.6, respectively. The ex vivo growth inhibition data after i.m. dosing were fitted to the inhibitory sigmoidEmaxequation to provide the values of AUC/MIC required to produce bacteriostasis, bactericidal activity, and elimination of bacteria. The respective values for serum were 17.8, 20.2, and 28.7 h, and slightly higher values were obtained for transudate and exudate. It is proposed that use of these data might provide a novel approach to the rational design of dosage schedules.
Methodologies and Cell Lines Used for Antimicrobial Susceptibility Testing of
Chlamydia
spp
Authors: Suchland R. J. , Geisler W. M. , Stamm Walter E.
Published: January 2003

In vitro susceptibility testing was performed on strains of
Chlamydia trachomatis
,
Chlamydia pneumoniae
, and
Chlamydia psittaci
under various conditions, including the cell line utilized, the time between infection and the addition of an antimicrobial, the concentration of inoculum, and the effect of multiple passage on the minimal chlamydicidal concentrations for the antibiotics doxycycline, azithromycin, erythromycin, ofloxacin, and tetracycline. With macrolides, the MIC varied depending upon the cell line utilized. With all antimicrobials, the MIC was related to the time at which the antimicrobial was added after infection. By an optimized cell culture passage method, all strains of chlamydia tested demonstrated survival after exposure to high levels (>100 times the MIC) of antimicrobials. Furthermore, upon retest, these surviving organisms did not demonstrate increased MICs. Thus, this phenomenon does not reflect selection of antimicrobial-resistant mutants but rather survival of some organisms in high antimicrobial concentrations (heterotypic survival). An additional 44 clinical isolates of
C. trachomatis
from patients with single-incident infections were tested against those from patients with recurrent or persistent infections, and heterotypic survival was seen in all isolates tested; hence, in vitro resistance did not correlate with the patient's apparent clinical outcome.
MUC7 20-Mer: Investigation of Antimicrobial Activity, Secondary Structure, and Possible Mechanism of Antifungal Action
Authors: Bobek Libuse A. , Situ Hongsa
Published: January 2003

This study was aimed at examining the spectrum of antimicrobial activity of MUC7 20-mer (N-LAHQKPFIRKSYKCLHKRCR-C; residues 32 to 51 of MUC7, the low-molecular-weight human salivary mucin, comprised of 357 residues) and comparing its antifungal properties to those of salivary histatin 5 (Hsn-5). We also examined the secondary structure of the 20-mer and the possible mechanism of its antifungal action. Our results showed that MUC7 20-mer displays potent killing activity against a variety of fungi and both gram-positive and gram-negative bacteria at micromolar concentrations. Time-dependent killing of
Candida albicans
and
Cryptococcus neoformans
by MUC7 20-mer and Hsn-5 indicated differences in killing rates between MUC7 20-mer and Hsn-5. The secondary structure prediction showed that MUC7 20-mer adopts an amphiphilic helix with distinguishable hydrophilic and hydrophobic faces (a characteristic that is associated with antimicrobial activity). In comparison to that of Hsn-5, the fungicidal activity of MUC7 20-mer against
C. albicans
seems to be independent of fungal cellular metabolic activity, as evidenced by its killing potency at a low temperature (4°C) and in the presence of inhibitors of oxidative phosphorylation in the mitochondrial system. Fluorescence microscopy showed the ability of MUC7 20-mer to cross the fungal cell membrane and to accumulate inside the cells. The internalization of MUC7 20-mer was inhibited by divalent cations. Confocal microscopy of cells doubly labeled with MUC7 20-mer and a mitochondrion-specific dye indicated that mitochondria are not the target of MUC7 20-mer for either
C. albicans
or
C. neoformans
.
Sterilizing Activities of Fluoroquinolones against Rifampin-Tolerant Populations of
Mycobacterium tuberculosis
Authors: Hu Yanmin , Coates Anthony R. M. , Mitchison Denis A.
Published: January 2003

The bactericidal activities of ciprofloxacin, ofloxacin, levofloxacin, moxifloxacin, and gatifloxacin were tested in three models of rifampin-tolerant
Mycobacterium tuberculosis
persisters. Model 1 was a 100-day-old, unshaken, anaerobically adapted culture in which serial dilutions of the quinolones were incubated for 5 days and CFU counts were then done In models 2 and 3, 100 mg of rifampin/liter was added to the 100-day culture for 5 or 7 days to produce tolerant organisms that did not grow on plates; the rifampin was then washed off, fresh medium was added to allow recovery of growth on plates, and the culture was incubated for 7 days before CFU counts. In model 2, the quinolones were added after rifampin had been washed off, whereas in model 3 the quinolones were added to the cultures containing rifampin. In models 1 and 2, ciprofloxacin had the least bactericidal activity, ofloxacin and levofloxacin had greater activities, and moxifloxacin and gatifloxacin had the greatest activities. In model 3, ofloxacin had no detectable activity whereas moxifloxacin killed about log
10
0.279 CFU of the persisters per ml at concentrations attainable in lesions; isoniazid had virtually no activity. These findings predict that ofloxacin will not be found to have effective sterilizing activity in clinical studies now planned whereas moxifloxacin will be able to shorten treatment.