bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiple Outbreaks of Nosocomial Salmonellosis in Russia and Belarus Caused by a Single Clone of
Salmonella enterica
Serovar Typhimurium Producing an Extended-Spectrum β-Lactamase
Authors: Edelstein M. , Pimkin M. , Dmitrachenko T. , Semenov V. , Kozlova N. , Gladin D. , Baraniak A. , Stratchounski L.
Published: July 2004

Thirty-four cefotaxime-resistant
Salmonella enterica
serovar Typhimurium isolates representative of the isolates that caused outbreaks of gastroenteritis in 10 hospitals in seven regions of Russia and Belarus from 1994 to 2003 were analyzed. All isolates produced the CTX-M-5-like extended-spectrum β-lactamase, which confers high-level resistance to cefotaxime and ceftriaxone and decreased susceptibility to ceftazidime. The
bla
CTX-M
genes were located on small (7.4- to 12-kb) non-self-transferable plasmids approximately 20 bp downstream of the IS
Ecp1
insertion sequences. Some isolates carried additional conjugative plasmids mediating resistance to penicillin-inhibitor combinations and various non-β-lactam agents, including tetracycline, chloramphenicol, gentamicin, tobramycin, and co-trimoxazole. Despite the minor differences in susceptibility patterns, all isolates were considered clonally related on the basis of arbitrarily primed PCR and pulsed-field gel electrophoresis analysis. The similarities of the restriction profiles of the CTX-M-coding plasmids further supported the clonal origin of these isolates.
Unraveling the Mode of Action of the Antimalarial Choline Analog G25 inPlasmodium falciparumandSaccharomyces cerevisiae
Authors: Roggero Rodolphe , Zufferey Rachel , Minca Mihaela , Richier Eric , Calas Michele , Vial Henri , Ben Mamoun Choukri
Published: July 2004

Pharmacological studies have indicated that the choline analog G25 is a potent inhibitor ofPlasmodium falciparumgrowth in vitro and in vivo. Although choline transport has been suggested to be the target of G25, the exact mode of action of this compound is not known. Here we show that, similar to its effects onP. falciparum, G25 prevents choline entry intoSaccharomyces cerevisiaecells and inhibitsS. cerevisiaegrowth. However, we show that the uptake of this compound is not mediated by the choline carrier Hnm1. Anhnm1Δyeast mutant, which lacks the only choline transporter geneHNM1, was not altered in the transport of a labeled analog of this compound. Eleven yeast mutants lacking genes involved in different steps of phospholipid biosynthesis were analyzed for their sensitivity to G25. Four mutants affected in the de novo cytidyldiphosphate-choline-dependent phosphatidylcholine biosynthetic pathway and, surprisingly, a mutant strain lacking the phosphatidylserine decarboxylase-encoding genePSD1(but notPSD2) were found to be highly resistant to this compound. Based on these data forS. cerevisiae, labeling studies inP. falciparumwere performed to examine the effect of G25 on the biosynthetic pathways of the major phospholipids phosphatidylcholine and phosphatidylethanolamine. Labeling studies inP. falciparumand in vitro studies with recombinantP. falciparumphosphatidylserine decarboxylase further supported the inhibition of both the de novo phosphatidylcholine metabolic pathway and the synthesis of phosphatidylethanolamine from phosphatidylserine. Together, our data indicate that G25 specifically targets the pathways for synthesis of the two major phospholipids, phosphatidylcholine and phosphatidylethanolamine, to exert its antimalarial activity.
Zidovudine, Lamivudine, and Abacavir Have Different Effects on Resting Cells Infected with Human Immunodeficiency Virus In Vitro
Authors: Saavedra-Lozano Jesús , McCoig Cynthia C. , Cao Yanying , Vitetta Ellen S. , Ramilo Octavio
Published: July 2004

We have previously described an in vitro model for the evaluation of the effects of different immunomodulatory agents and immunotoxins (ITs) on cells latently infected with human immunodeficiency virus (HIV). We demonstrated that latently infected, replication-competent cells can be generated in vitro after eliminating CD25+cells with an IT. Thus, by selectively killing the productively infected cells with an anti-CD25 IT we can generate a population of latently infected cells. CD25−cells generated in this manner were treated with nucleoside analog reverse transcriptase inhibitors and subsequently activated with phytohemagglutinin in the presence of the drugs. The antiviral activities of zidovudine (ZDV), lamivudine (3TC), and abacavir (ABC) were evaluated by using this model. 3TC and ABC demonstrated significant activity in decreasing HIV production from recently infected resting cells following their activation, whereas the effect of ZDV was more modest. These results suggest that the differences in antiviral activity of nucleoside analogs on resting cells should be considered when designing drug combinations for the treatment of HIV infection. The model presented here offers a convenient alternative for evaluating the mechanism of action of new antiretroviral agents (J. Saavedra, C. Johnson, J. Koester, M. St. Claire, E. Vitteta, O. Ramilo, 37th Intersci. Conf. Antimicrob. Agents Chemother., abstr. I-59, 1997).
Prediction of Mechanisms of Action of Antibacterial Compounds by Gene Expression Profiling
Authors: Hutter Bernd , Schaab Christoph , Albrecht Sebastian , Borgmann Matthias , Brunner Nina A. , Freiberg Christoph , Ziegelbauer Karl , Rock Charles O. , Ivanov Igor , Loferer Hannes
Published: July 2004

We have generated a database of expression profiles carrying the transcriptional responses of the model organismBacillus subtilisfollowing treatment with 37 well-characterized antibacterial compounds of different classes. The database was used to build a predictor for the assignment of the mechanisms of action (MoAs) of antibacterial compounds by the use of support vector machines. This predictor was able to correctly classify the MoA class for most compounds tested. Furthermore, we provide evidence that the in vivo MoA of hexachlorophene does not match the MoA predicted from in vitro data, a situation frequently faced in drug discovery. A database of this kind may facilitate the prioritization of novel antibacterial entities in drug discovery programs. Potential applications and limitations are discussed.
DNA Sequence Analysis of Regions SurroundingblaCMY-2from MultipleSalmonellaPlasmid Backbones
Authors: Giles W. P. , Benson A. K. , Olson M. E. , Hutkins R. W. , Whichard J. M. , Winokur P. L. , Fey P. D.
Published: July 2004

The emergence in the United States of resistance to expanded-spectrum cephalosporin (e.g., ceftriaxone) within the salmonellae has been associated primarily with three large (>100-kb) plasmids (designated types A, B, and C) and one 10.1-kb plasmid (type D) that carry theblaCMY-2gene. In the present study, the distribution of these four knownblaCMY-2-carrying plasmids among 35 ceftriaxone-resistantSalmonellaisolates obtained from 1998 to 2001 was examined. Twenty-three of these isolates wereSalmonella entericaserotype Newport, 10 wereSalmonella entericaserotype Typhimurium, 1 wasSalmonella entericaserotype Agona, and 1 wasSalmonella entericaserotype Reading. All 23 serotype Newport isolates carried a type C plasmid, and 5, 4, and 1 serovar Typhimurium isolate carried type B, A, and C plasmids, respectively. Both the serotype Agona and serotype Reading isolates carried type A plasmids. None of the isolates carried a type D plasmid. Hybridization data suggested that plasmid types A and C were highly related replicons. DNA sequencing revealed that the region surroundingblaCMY-2was highly conserved in all three plasmid types analyzed (types B, C, and D) and was related to a region surroundingblaCMY-5from theKlebsiella oxytocaplasmid pTKH11. These findings are consistent with a model in whichblaCMY-2has been disseminated primarily through plasmid transfer, and not by mobilization of the gene itself, to multipleSalmonellachromosomal backbones.
Effects of an Antibiotic Cycling Program on Antibiotic Prescribing Practices in an Intensive Care Unit
Authors: Merz Liana R. , Warren David K. , Kollef Marin H. , Fraser Victoria J.
Published: July 2004

Various interventions have been proposed to combat the increase of antibiotic resistance and influence antibiotic prescribing practices. A prospective cohort study in a medical intensive care unit was conducted to determine the effect of an antibiotic cycling program on patterns of antibiotic use and to determine patient factors associated with cycling adherence. Four major classes of antibiotics for empirical therapy of suspected gram-negative bacterial infections were rotated at 3- and 4-month intervals. During the study, 1,003 patients received antibiotic therapy with at least one of the study drugs; of the 792 receiving cycle antibiotics during the cycling period, 598 (75.5%) received an on-cycle drug. Compared to the baseline, cycling recommendations increased the use of the target cycle agent: the use of cephalosporins increased during cycle 1 (56 to 64% of total antibiotic days,
P
< 0.001), fluoroquinolone use increased in cycle 2 (24 to 55%,
P
< 0.001), carbapenem use increased during cycle 3 (14 to 38%,
P
< 0.001), and use of extended-spectrum penicillins increased in cycle 4 (5 to 36%,
P
< 0.001). Overall, 48% of total cycle antibiotic days were compliant with the cycling protocol. On average, 8.8 days per patient were spent receiving on-cycle drugs (range, 1 to 109). Cycle periods that specified carbapenem and fluoroquinolone use had the highest number of off-cycle days (62 and 64%). Predictors of on-cycle antibiotic use were increased severity of illness, as measured by an acute physiology and chronic health evaluation II score, and greater length of intensive care unit stay. In conclusion, the successful implementation of this cycling protocol increased antibiotic heterogeneity over time in the study unit.
Impact of Cethromycin (ABT-773) Therapy on Microbiological, Histologic, Immunologic, and Respiratory Indices in a Murine Model ofMycoplasma pneumoniaeLower Respiratory Infection
Authors: Ríos Ana María , Mejías Asunción , Chávez-Bueno Susana , Fonseca-Aten Mónica , Katz Kathy , Hatfield Jeanine , Gómez Ana María , Jafri Hasan S. , McCracken George H. , Ramilo Octavio , Hardy Robert Doug
Published: July 2004

Mycoplasma pneumoniaeis a major etiologic agent of acute lower respiratory infections. We evaluated the antimicrobial and immunologic effects of cethromycin (ABT-773), a ketolide antibiotic, for the treatment ofM. pneumoniaepneumonia in a mouse model. Eight-week-old BALB/c mice were inoculated intranasally once with 106CFU ofM. pneumoniaeon day 0. Treatment was started 24 h after inoculation. Groups of mice were treated subcutaneously with cethromycin at 25 mg/kg of body weight or with placebo daily until sacrifice. Five to ten mice per group were evaluated at days 1, 4, 7, and 10 after inoculation. Outcome variables included bronchoalveolar lavage (BAL) forM. pneumoniaequantitative culture and cytokine and chemokine concentration determinations by enzyme-linked immunosorbent assay (tumor necrosis factor alpha [TNF-α], gamma interferon [IFN-γ], interleukin-1β [IL-1β], IL-2, IL-4, IL-12, granulocyte-macrophage colony-stimulating factor, IL-8, monocyte chemoattractant protein 1 [MCP-1], and macrophage inflammatory protein 1α [MIP-1α]), histopathologic score of the lungs (HPS), and pulmonary function tests (PFT) using whole-body, unrestrained plethysmography at the baseline and post-methacholine exposure as indicators of airway obstruction (AO) and airway hyperresponsiveness (AHR), respectively. The cethromycin-treated mice had a greater reduction inM. pneumoniaeculture titers than placebo-treated mice, reaching statistical significance on days 7 and 10 (P< 0.05). HPS was significantly reduced in cethromycin-treated mice compared with placebo-treated mice on days 4, 7, and 10 (P< 0.05). Cytokine concentrations in BAL samples were reduced in mice that received cethromycin, and the differences were statistically significant for 7 of the 10 cytokines measured (TNF-α, IFN-γ, IL-1β, IL-8, IL-12, MCP-1, and MIP-1α) on day 4 (P< 0.05). PFT values were improved in the cethromycin-treated mice, with AO and AHR significantly reduced on day 4 (P< 0.05). In this mouse model, treatment with cethromycin significantly reducedM. pneumoniaeculture titers in BAL samples, cytokine and chemokine concentrations in BAL samples, histologic inflammation in the lungs, and disease severity as defined by AO and AHR.
Molecular Characterization of a Cephamycin-Hydrolyzing and Inhibitor-Resistant Class A β-Lactamase, GES-4, Possessing a Single G170S Substitution in the Ω-Loop
Authors: Wachino Jun-ichi , Doi Yohei , Yamane Kunikazu , Shibata Naohiro , Yagi Tetsuya , Kubota Takako , Arakawa Yoshichika
Published: July 2004

The nosocomial spread of six genetically related
Klebsiella pneumoniae
strains producing GES-type β-lactamases was found in a neonatal intensive care unit, and we previously reported that one of the six strains, strain KG525, produced a new β-lactamase, GES-3. In the present study, the molecular mechanism of cephamycin resistance observed in strain KG502, one of the six strains described above, was investigated. This strain was found to produce a variant of GES-3, namely, GES-4, which was responsible for resistance to both cephamycins (cefoxitin MIC, >128 μg/ml) and β-lactamase inhibitors (50% inhibitory concentration of clavulanic acid, 15.2 ± 1.7 μM). The GES-4 enzyme had a single G170S substitution in the Ω-loop region compared with the GES-3 sequence. This single amino acid substitution was closely involved with the augmented hydrolysis of cephamycins and carbapenems and the decreased affinities of β-lactamase inhibitors to GES-4. A cloning experiment and sequencing analysis revealed that strain KG502 possesses duplicate
bla
GES-4
genes mediated by two distinct class 1 integrons with similar gene cassette configurations. Moreover, the genetic environments of the
bla
GES-4
genes found in strain KG502 were almost identical to that of
bla
GES-3
in strain KG525. From these findings, these two phenotypically different strains were suggested to belong to a clonal lineage. The
bla
GES-4
gene found in strain KG502 might well emerge from a point mutation in the
bla
GES-3
gene harbored by its ancestor strains, such as strain KG525, under heavy antibiotic stress in order to acquire extended properties of resistance to cephamycins and carbapenems.
Estimation of Serum-Free 50-Percent Inhibitory Concentrations for Human Immunodeficiency Virus Protease Inhibitors Lopinavir and Ritonavir
Authors: Hickman Dean , Vasavanonda Sudthida , Nequist George , Colletti Lynn , Kati Warren M. , Bertz Richard , Hsu Ann , Kempf Dale J.
Published: July 2004

Using measured free fraction and 50% inhibitory concentration (IC50) values for the human immunodeficiency virus protease inhibitors lopinavir (LPV) and ritonavir (RTV) in tissue culture media with various protein concentrations ranging from 5 to 50%, we estimated serum-free IC50values for each drug. The range of serum-free IC50values (0.64 to 0.77 ng/ml for LPV and 3.0 to 5.0 ng/ml for RTV) did not exhibit a trend with increasing protein concentrations, despite a 10-fold difference in the free fraction value (0.006 to 0.063) for LPV and a 5-fold difference in the free fraction value (0.013 to 0.057) for RTV. The mean serum-free IC50by the MTT-MT4 assay (0.69 ng/ml for LPV and 4.0 ng/ml for RTV) may be the most accurate parameter for the estimation of the inhibitory quotient (IQ), a relative measure of in vivo potency defined as the ratio of the minimal free drug concentration in plasma (Ctrough,free) for a specific patient population and the serum-free IC50. Using this approach, we calculated the average IQs for protease inhibitor-naïve patients for LPV and RTV to be 67 and 5.6, respectively.
Therapeutic Efficacy of “Nubiotics” against Burn Wound Infection by
Pseudomonas aeruginosa
Authors: Dale Roderic M. K. , Schnell Glen , Wong Jonathan P.
Published: July 2004

“Nubiotics” are a novel class of proprietary protonated nucleic acid-based drugs shown to have potent in vitro antibacterial activities against a number of gram-positive and gram-negative bacteria. These nubiotics are evaluated here for their in vivo therapeutic efficacy for the treatment of burn wound infection caused by
Pseudomonas aeruginosa
. To achieve this, a burn wound infection model was established in mice by using a highly pathogenic burn wound clinical isolate of
P. aeruginosa
. Lethal doses of the bacteria were determined for two routes of infection (subcutaneous and topical), representing systemic and local forms of infection, respectively. Using this infection model, treatment with nubiotics by various routes of drug administration was evaluated and optimized. A total of 12 nubiotics and their analogues were tested and of these, Nu-2, -3, -4, and -5 were found to be extremely efficacious in the postexposure treatment of burn wound infection (60 to 100% survival rates versus 0% for untreated control [
P
< 0.05]). These nubiotics were effective when given either systemically by intravenous and/or subcutaneous administration or given locally to the affected site in the skin by topical application. Treatment by these two routes resulted in almost 100% survival rates and complete eradication of the bacteria from infection sites in the livers, spleens, and blood. These nubiotics were found to be as effective as intravenously administered ciprofloxacin, a potent and broad-spectrum fluoroquinolone. These results suggest that nubiotics may be a promising and effective approach for the treatment of burn wound infection caused by
P. aeruginosa
.
Rapid Real-Time PCR Genotyping of Mutations Associated with Sulfadoxine-Pyrimethamine Resistance in
Plasmodium falciparum
Authors: Alker Alisa P. , Mwapasa Victor , Meshnick Steven R.
Published: July 2004

The resistance of
Plasmodium falciparum
to sulfadoxine-pyrimethamine (SP) is an emerging public health threat. Resistance to these drugs is associated with point mutations in the genes encoding dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR). We describe here an assay using real-time PCR and sequence-specific probes that detects these mutations. Using DNA from plasmids, cultured strains, and clinical samples, real-time PCR could distinguish four DHPS polymorphisms (codons 437, 540, 581, and 613) and three DHFR polymorphisms (codons 51, 59, and 108). This assay is rapid and sensitive, with a detection limit of 10 copies in most cases. This assay is amenable to large-scale studies of drug resistance.
Isoniazid Pharmacokinetics-Pharmacodynamics in an Aerosol Infection Model of Tuberculosis
Authors: Jayaram Ramesh , Shandil Radha. K. , Gaonkar Sheshagiri , Kaur Parvinder , Suresh B. L. , Mahesh B. N. , Jayashree R. , Nandi Vrinda , Bharath Sowmya , Kantharaj E. , Balasubramanian V.
Published: July 2004

Limited data exist on the pharmacokinetic-pharmacodynamic (PK-PD) parameters of the bactericidal activities of the available antimycobacterial drugs. We report on the PK-PD relationships for isoniazid. Isoniazid exhibited concentration (
C
)-dependent killing of
Mycobacterium tuberculosis
H37Rv in vitro, with a maximum reduction of 4 log
10
CFU/ml. In these studies, 50% of the maximum effect was achieved at a
C
/MIC ratio of 0.5, and the maximum effect did not increase with exposure times of up to 21 days. Conversely, isoniazid produced less than a 0.5-log
10
CFU/ml reduction in two different intracellular infection models (J774A.1 murine macrophages and whole human blood). In a murine model of aerosol infection, isoniazid therapy for 6 days produced a reduction of 1.4 log
10
CFU/lung. Dose fractionation studies demonstrated that the 24-h area under the concentration-time curve/MIC (
r
2
= 0.83) correlated best with the bactericidal efficacy, followed by the maximum concentration of drug in serum/MIC (
r
2
= 0.73).
Disposition of Artesunate and Dihydroartemisinin after Administration of Artesunate Suppositories in Children from Papua New Guinea with Uncomplicated Malaria
Authors: Karunajeewa Harin A. , Ilett Kenneth F. , Dufall Kitiya , Kemiki Adedayo , Bockarie Moses , Alpers Michael P. , Barrett P. Hugh , Vicini Paolo , Davis Timothy M. E.
Published: July 2004

A detailed pharmacokinetic analysis was performed with 47 children from Papua New Guinea with uncomplicated falciparum or vivax malaria treated with artesunate (ARTS) suppositories (Rectocaps) given in two doses of approximately 13 mg/kg of body weight 12 h apart. Following an intensive sampling protocol, samples were assayed for ARTS and its primary active metabolite, dihydroartemisinin (DHA), by liquid chromatography-mass spectrometry. A population pharmacokinetic model was developed to describe the data. Following administration of the first dose, the mean maximal concentrations of ARTS and DHA were 1,085 nmol/liter at 0.9 h and 2,525 nmol/liter at 2.3 h, respectively. The absorption half-life for ARTS was 2.3 h, and the conversion half-life (ARTS to DHA) was 0.27 h, while the elimination half-life of DHA was 0.71 h. The mean common volumes of distribution for ARTS and DHA relative to bioavailability were 42.8 and 2.04 liters/kg, respectively, and the mean clearance values relative to bioavailability were 6 and 2.2 liters/h/kg for ARTS and DHA, respectively. Substantial interpatient variability was observed, and the bioavailability of the second dose relative to that of the first was estimated to be 0.72. The covariates age, sex, and α-thalassemia genotype were not influential in the pharmacokinetic model development; but the inclusion of weight as a covariate significantly improved the performance of the model. An ARTS suppositories dose of 10 of 20 mg/kg is appropriate for use in children with uncomplicated malaria.
Role of Positional Hydrophobicity in the Leishmanicidal Activity of Magainin 2
Authors: Guerrero Esther , Saugar José María , Matsuzaki Katsumi , Rivas Luis
Published: July 2004

The emergence of membrane-active antimicrobial peptides as new alternatives against pathogens with multiantibiotic resistance requires the design of better analogues. Among the different physicochemical parameters involved in the optimization of linear antimicrobial peptides, positional hydrophobicity has recently been incorporated. This takes into consideration the concept of the topological distribution of hydrophobic residues throughout the sequence rather than the classical concept of hydrophobicity as a global parameter of the peptide, calculated as the summation of the individual hydrophobicities of the residues. In order to assess the contribution of this parameter to the leishmanicidal mechanisms of magainin 2 analogues, the activities of two of these analogues, MG-H1 (GIKKFLHIIWKFIKAFVGEIMNS) and MG-H2 (IIKKFLHSIWKFGKAFVGEIMNI), which have similar charges, amino acid compositions, and hydrophobicities but different positional hydrophobicities, against
Leishmania donovani
promastigotes were assayed (T. Tachi, R. F. Epand, R. M. Epand, and K. Matsuzaki, Biochemistry 41:10723-10731, 2002). The activities were compared with that of the parental peptide, F5W-magainin 2 (GIGKWLHSAKKFGKAFVGEIMNS). The three peptides were active at micromolar concentrations, in the order MG-H2 > MG-H1 > F5W-magainin 2. These activities differ from their hemolytic and bactericidal activities. The results demonstrate that positional hydrophobicity, which reflects the presence of short stretches of sequences rich in hydrophobic amino acids, plays an important role in the activities of leishmanicidal peptides.
Pharmacodynamic Evaluation of the Neutralization of Endotoxin by PMX622 in Mice
Authors: Lake Philip , DeLeo Jeffrey , Cerasoli Franklin , Logdberg Lennart , Weetall Marla , Handley Dean
Published: July 2004

Polymyxin B (PMB) binds to and neutralizes endotoxin, but its systemic clinical utility is limited by neuro- and nephrotoxicity. PMX622 is a covalent conjugate of PMB and Dextran-70 designed to retain the ability of PMB to neutralize endotoxin and to retain the favorable colloidal, pharmacokinetic, and metabolic properties of Dextran-70. PMX622 has demonstrated efficacy in a number of animal models and effectively neutralized endotoxin in phase I clinical trials. Here, we systematically evaluated the pharmacodynamic properties of PMX622 in a murine model of endotoxin-induced lethality in galactosamine-sensitized mice. PMX622 completely and dose dependently inhibited lethality in this model. A stoichiometric relationship was found between the endotoxin challenge dose and the dose of PMX622 needed for protection. PMX622 neutralized endotoxin from four different genera of gram-negative bacteria but notNeisseria meningitidis. PMX622 was significantly less toxic than PMB in the mouse, suggesting that PMX622 has a better margin of safety than PMB. The timing of PMX622 administration relative to endotoxin was crucial. PMX622 was active for several hours prior to the endotoxin challenge; however, PMX622 did not protect mice if administered ≥15 min after endotoxin challenge. This suggests that PMX622 would best be clinically used prophylactically rather than therapeutically. These studies will be crucial in designing and interpreting human clinical trials assessing PMX622 efficacy.
Comparison of the Susceptibilities of
Burkholderia pseudomallei
to Meropenem and Ceftazidime by Conventional and Intracellular Methods
Authors: Inglis T. J. J. , Rodrigues F. , Rigby P. , Norton R. , Currie B. J.
Published: July 2004

The effect of the two antibiotics ceftazidime and meropenem on a collection of 46
Burkholderia pseudomallei
isolates representing clinical and environmental sources across northern Australia was investigated by using a series of in vitro test methods. The susceptibility testing methods used included Kirby-Bauer disk diffusion, Etest MIC, broth microdilution MIC, and a modification of the microdilution method in which
Acanthamoeba
cells were added to simulate the effect of a professional phagocytic cell on test outcome. In a semiquantitative validation coculture series, the majority of bacteria were intracellular up to a multiplicity of infection of 10 bacteria to one ameba. The optical density and bacterial count (log
10
CFU/ml) correlated across the range tested (
r
2
= 0.77;
P
< 0.0001). Susceptibility test results were compared against clinical outcomes. The MICs of ceftazidime were consistently higher than those of meropenem by all three methods. The MICs of both agents were significantly higher when
Acanthamoeba
trophozoites were added to the broth microdilution method. Conventional and intracellular MIC results were consistent for clinical isolates from the Western Australian outbreak cluster despite the wide variety of clinical outcomes. Further development of the intracellular MIC method is expected to help assess the efficacy of antimicrobial agents on this bacterial species in an intracellular setting.
Nitric Oxide Generated from Isoniazid Activation by KatG: Source of Nitric Oxide and Activity againstMycobacterium tuberculosis
Authors: Timmins Graham S. , Master Sharon , Rusnak Frank , Deretic Vojo
Published: July 2004

Isonicotinic acid hydrazide (INH) is a frontline antituberculosis agent. Once taken up byMycobacterium tuberculosis, INH requires activation by the catalase-peroxidase KatG, converting INH from its prodrug form into a range of bactericidal reactive species. Here we used15N-labeled INH together with electron paramagnetic resonance spin trapping techniques to demonstrate that nitric oxide (NO˙) is generated from oxidation at the hydrazide nitrogens during the activation of INH byM. tuberculosisKatG. We also observed that a specific scavenger of NO˙ provided protection against the antimycobacterial activity of INH in bacterial culture. No significant increases in mycobacterial protein nitration were detected, suggesting that NO˙ and not peroxynitrite, a nitrating metabolite of NO·, is involved in antimycobacterial action. In conclusion, INH-derived NO·has biological activity, which directly contributes to the antimycobacterial action of INH.
Impact of Specific
pbp5
Mutations on Expression of β-Lactam Resistance in
Enterococcus faecium
Authors: Rice Louis B. , Bellais Samuel , Carias Lenore L. , Hutton-Thomas Rebecca , Bonomo Robert A. , Caspers Patrick , Page Malcolm G. P. , Gutmann Laurent
Published: July 2004

We tested the impact of individual PBP 5 mutations on expression of ampicillin resistance in
Enterococcus faecium
using a shuttle plasmid designed to facilitate expression of cloned
pbp5
in ampicillin-susceptible
E. faecium
D344SRF. Substitutions that had been implicated in contributing to the resistance of clinical strains conferred only modest levels of resistance when they were present as single point mutations. The levels of resistance were amplified when some mutations were present in combination. In particular, a methionine-to-alanine change at position 485 (in close proximity to the active site) combined with the insertion of a serine at position 466 (located in a loop that forms the outer edge of the active site) was associated with the highest levels of resistance to all β-lactams. Affinity for penicillin generally correlated with β-lactam MICs for the mutants, but these associations were not strictly proportional.
Inhibitors of
Leishmania mexicana
CRK3 Cyclin-Dependent Kinase: Chemical Library Screen and Antileishmanial Activity
Authors: Grant Karen M. , Dunion Morag H. , Yardley Vanessa , Skaltsounis Alexios-Leandros , Marko Doris , Eisenbrand Gerhard , Croft Simon L. , Meijer Laurent , Mottram Jeremy C.
Published: July 2004

The CRK3 cyclin-dependent kinase of
Leishmania
has been shown by genetic manipulation of the parasite to be essential for proliferation. We present data which demonstrate that chemical inhibition of CRK3 impairs the parasite's viability within macrophages, thus further validating CRK3 as a potential drug target. A microtiter plate-based histone H1 kinase assay was developed to screen CRK3 against a chemical library enriched for protein kinase inhibitors. Twenty-seven potent CRK3 inhibitors were discovered and screened against
Leishmania donovani
amastigotes in vitro. Sixteen of the CRK3 inhibitors displayed antileishmanial activity, with a 50% effective dose (ED
50
) of less than 10 μM. These compounds fell into four chemical classes: the 2,6,9-trisubstituted purines, including the C-2-alkynylated purines; the indirubins; the paullones; and derivatives of the nonspecific kinase inhibitor staurosporine. The paullones and staurosporine derivatives were toxic to macrophages. The 2,6,9-trisubstituted purines inhibited CRK3 in vitro, with 50% inhibitory concentrations ranging from high nanomolar to low micromolar concentrations. The most potent inhibitors of CRK3 (compounds 98/516 and 97/344) belonged to the indirubin class; the 50% inhibitory concentrations for these inhibitors were 16 and 47 nM, respectively, and the ED
50
s for these inhibitors were 5.8 and 7.6 μM, respectively. In culture, the indirubins caused growth arrest, a change in DNA content, and aberrant cell types, all consistent with the intracellular inhibition of a cyclin-dependent kinase and disruption of cell cycle control. Thus, use of chemical inhibitors supports genetic studies to confirm CRK3 as a validated drug target in
Leishmania
and provides pharmacophores for further drug development.
Regulation of the Expression of Cell Wall Stress Stimulon Member GenemsrA1in Methicillin-Susceptible or -ResistantStaphylococcus aureus
Authors: Pechous Roger , Ledala Nagender , Wilkinson Brian J. , Jayaswal Radheshyam K.
Published: July 2004

Genome-wide transcriptional profiling studies of the response ofStaphylococcus aureusto cell wall-active antibiotics have led to the discovery of a cell wall stress stimulon of genes induced by these agents.msrA1, encoding methionine sulfoxide reductase, is a highly induced member gene of this stimulon. In the present study we show thatmsrA1induction by oxacillin is common to all methicillin-susceptible strains studied but did not occur in two homogeneous and two heterogeneous methicillin-resistant strains. However,msrA1was induced by vancomycin and/ord-cycloserine in methicillin-resistant strains. Lysozyme and lysostaphin treatment did not inducemsrA1expression. Oxacillin-inducedmsrA1expression was enhanced by ca. 30% in a SigB+derivative (SH1000) of the SigB-defective RN450 (NCTC 8325-4) strain.msrA1expression was not affected in mutants in the global regulatory systemsagrandsar. Glycerol monolaurate, an inhibitor of signal transduction, inhibited the oxacillin-induced transcription ofmsrA1and other cell wall stress stimulon member genes,vraSanddnaK. These observations suggest that the cell wall stress stimulon is induced by inhibition of the process of peptidoglycan biosynthesis, and the inhibitory effects of glycerol monolaurate indicate that gene expression is dependent on a signal transduction pathway.
Comparison of Gene Expression Profiles of
Candida albicans
Azole-Resistant Clinical Isolates and Laboratory Strains Exposed to Drugs Inducing Multidrug Transporters
Authors: Karababa Mahir , Coste Alix T. , Rognon Bénédicte , Bille Jacques , Sanglard Dominique
Published: July 2004

Azole resistance in
Candida albicans
can be due to upregulation of multidrug transporters belonging to ABC (ATP-binding cassette) transporters (
CDR1
and
CDR2
) or major facilitators (
CaMDR1
). Upregulation of these genes can also be achieved by exposure to fluphenazine, resulting in specific upregulation of
CDR1
and
CDR2
and by exposure to benomyl, resulting in specific
CaMDR1
upregulation. In this study, these two different states of gene upregulation were used to determine coregulated genes that often share similar functions or similar regulatory regions. The transcript profiles of a laboratory strain exposed to these drugs were therefore determined and compared with those of two matched pairs of azole-susceptible and -resistant strains expressing
CDR1
and
CDR2
(CDR strains) or
CaMDR1
(MDR isolates). The results obtained revealed that, among 42 commonly regulated genes (8.6% of all regulated genes) between fluphenazine-exposed cells and CDR isolates, the most upregulated were
CDR1
and
CDR2
as expected, but also
IFU5
,
RTA3
(which encodes putative membrane proteins),
HSP12
(which encodes heat shock protein), and
IPF4065
(which is potentially involved in stress response). Interestingly, all but
HSP12
and
IPF4065
contain a putative
cis
-acting drug responsive element in their promoters. Among the 57 genes (11.5% of all regulated genes) commonly regulated between benomyl-exposed cells and MDR isolates, the most upregulated were
CaMDR1
as expected but also genes with oxido-reductive functions such as
IFD
genes,
IPF5987
,
GRP2
(all belonging to the aldo-keto reductase family),
IPF7817
[NAD(P)H oxido-reductase], and
IPF17186
. Taken together, these results show that in vitro drug-induced gene expression only partially mimics expression profiles observed in azole-resistant clinical strains. Upregulated genes in both drug-exposed conditions and clinical strains are drug resistance genes but also genes that could be activated under cell damage conditions.
Novel Polymorphisms in
mec
Genes and a New
mec
Complex Type in Methicillin-Resistant
Staphylococcus aureus
Isolates Obtained in Rural Wisconsin
Authors: Shukla Sanjay K. , Ramaswamy Srinivas V. , Conradt Jennifer , Stemper Mary E. , Reich Robert , Reed Kurt D. , Graviss Edward A.
Published: July 2004

We determined allelic polymorphisms in the
mec
complexes of 524 methicillin-resistant
Staphylococcus aureus
isolates by partial or complete sequencing of three
mec
genes,
mecA, mecI
, and
mecR1
. The isolates had been collected over a 10-year period from patients living in rural Wisconsin, where the use of antibiotics was expected to be lower than in the bigger cities. Of the 18 mutation types identified, 16 had not been described previously. The five most common mutations were a mutation 7 nucleotides (nt) upstream from the start site (G→T) in the
mecA
promoter (34.7%), an E246G change encoded by
mecA
(2.2%), a cytosine insertion at codon 257 in
mecA
(13.2%), an N121K change encoded by
mecI
(7.8%), and a major
mecI
-
mecR1
deletion designated as a class B1
mec
complex deletion type (25.4%). There was a high degree of conservation of the amino acid sequence of MecR1. Strains with the same mutations had variable resistance to oxacillin, and the median MIC for isolates that harbored the 7-nt-upstream mutation was lower than that for strains harboring other mutations. Our data suggest that the
mecA
promoter mutation plays a more important role in determining methicillin resistance than mutations in
mecI
and
mecA
do. Eighty-five percent of the tested isolates (
n
= 148) with the
mecA
promoter mutation and the class B1
mec
complex deletion belonged to the same major clonal group, identified as MCG-2, and harbored the type IV staphylococcal cassette chromosome
mec
DNA. There was also a wide range of oxacillin MICs for strains with wild-type
mecA
,
mecI
, and
mecR1
sequences, suggesting that the genetic backgrounds of clinical strains are significant in determining susceptibility to methicillin.
1,2-Dithiole-3-Ones as Potent Inhibitors of the Bacterial 3-Ketoacyl Acyl Carrier Protein Synthase III (FabH)
Authors: He Xin , Reeve Anne McElwee , Desai Umesh R. , Kellogg Glen E. , Reynolds Kevin A.
Published: July 2004

The enzyme FabH catalyzes the initial step of fatty acid biosynthesis via a type II dissociated fatty acid synthase. The pivotal role of this essential enzyme, combined with its unique structural features and ubiquitous occurrence in bacteria, has made it an attractive new target for the development of antibacterial and antiparasitic compounds. We have searched the National Cancer Institute database for compounds bearing structural similarities to thiolactomycin, a natural product which exhibits a weak activity against FabH. This search has yielded several substituted 1,2-dithiole-3-ones that are potent inhibitors of FabH from bothEscherichia coli(ecFabH) andStaphylococcus aureus(saFabH). The most potent inhibitor was 4,5-dichloro-1,2-dithiole-3-one, which had 50% inhibitory concentration (IC50) values of 2 μM (ecFabH) and 0.16 μM (saFabH). The corresponding 3-thione analog exhibited comparable activities. Analogs in which the 4-chloro substituent was replaced with a phenyl group were also potent inhibitors, albeit somewhat less effectively (IC50values of 5.7 and 0.98 μM for ecFabH and saFabH, respectively). All of the 5-chlorinated inhibitors were most effective when they were preincubated with FabH in the absence of substrates. The resulting enzyme-inhibitor complex did not readily regain activity after excess inhibitor was removed, suggesting that a slow dissociation occurs. In stark contrast, a series of inhibitors in which the 5-chloro substituent was replaced with the isosteric and isoelectronic trifluoromethyl group were poorer inhibitors (IC50values typically ranging from 25 to >100 μM for both ecFabH and saFabH), did not require a preincubation period for maximal activity, and generated an enzyme-inhibitor complex which readily dissociated. Possible modes of binding of 5-chloro-1,2-dithiole-3-ones and 5-chloro-1,2-dithiole-3-thiones with FabH which account for the role of the 5-chloro substituent were considered.
Activities of Doripenem (S-4661) against Drug-Resistant Clinical Pathogens
Authors: Jones Ronald N. , Huynh Holly K. , Biedenbach Douglas J.
Published: July 2004

Doripenem (formerly S-4661), a new 1-β-methyl carbapenem, was challenged with a worldwide collection of 394 drug-refractory isolates. For endemic extended-spectrum β-lactamase- and stably derepressed AmpC-producing enteric bacilli, the doripenem MICs at which 90% of the isolates were inhibited (MIC
90
s) were 0.03 to 0.5 μg/ml, generally lower than those of comparator carbapenems. A greater proportion of strains among carbapenem-resistant nonfermentative gram-negative bacilli were inhibited by doripenem at ≤4 μg/ml, and doripenem was the most active carbapenem (MIC
90
, 1 to 4 μg/ml) against penicillin-resistant streptococci.
Lack of Evidence that DNA in Antibiotic Preparations Is a Source of Antibiotic Resistance Genes in Bacteria from Animal or Human Sources
Authors: Lau Susanna K. P. , Woo Patrick C. Y. , To Amanda P. C. , Lau Alexson T. K. , Yuen Kwok-yung
Published: July 2004

Although DNA encoding antibiotic resistance has been discovered in antibiotic preparations, its significance for the development of antibiotic resistance in bacteria is unknown. No phylogenetic evidence was obtained for recent horizontal transfer of antibiotic resistance genes from antibiotic-producing organisms to bacteria from human or animal sources.
Effect of pH on the In Vitro Activities of Amphotericin B, Itraconazole, and Flucytosine against
Aspergillus
Isolates
Authors: Te Dorsthorst D. T. A. , Mouton J. W. , van den Beukel C. J. P. , van der Lee H. A. L. , Meis J. F. G. M. , Verweij P. E.
Published: July 2004

The in vitro susceptibilities of 21
Aspergillus
isolates were tested against three antifungal agents in RPMI 1640 and yeast nitrogen base at pH 5.0 and 7.0 by a broth microdilution format of the NCCLS method. The MICs of amphotericin B and itraconazole were higher, while those of flucytosine were lower, at pH 5.0 than at pH 7.0. The poor correlation between in vitro results and clinical outcome could be due to a difference in pH between the in vitro susceptibility test and at the site of infection.
In Vitro Activities of the Newer Quinolones Garenoxacin, Gatifloxacin, and Gemifloxacin against Human Mycoplasmas
Authors: Pereyre S. , Renaudin H. , Bébéar C. , Bébéar C. M.
Published: July 2004

The activities of garenoxacin, gatifloxacin, and gemifloxacin were compared with those of four fluoroquinolones against human mycoplasmas and ureaplasmas, including fluoroquinolone-resistant genetically characterized strains. Garenoxacin exhibited the highest activity, followed by gemifloxacin, moxifloxacin, and gatifloxacin. The minimal bactericidal activities of these three compounds were lower than those of the four fluoroquinolones.
Rv2686c-Rv2687c-Rv2688c, an ABC Fluoroquinolone Efflux Pump in
Mycobacterium tuberculosis
Authors: Pasca Maria Rosalia , Guglierame Paola , Arcesi Fabio , Bellinzoni Marco , De Rossi Edda , Riccardi Giovanna
Published: July 2004

The
Mycobacterium tuberculosis
Rv2686c-Rv2687c-Rv2688c operon, encoding an ABC transporter, conferred resistance to ciprofloxacin and, to a lesser extent, norfloxacin, moxifloxacin, and sparfloxacin to
Mycobacterium smegmatis
. The resistance level decreased in the presence of the efflux pump inhibitors reserpine, carbonyl cyanide
m
-chlorophenylhydrazone, and verapamil. Energy-dependent efflux of ciprofloxacin from
M. smegmatis
cells containing the Rv2686c-Rv2687c-Rv2688c operon was observed.
Extended-Spectrum-Cephalosporin Resistance in
Salmonella enterica
Isolates of Animal Origin
Authors: Gray Jeffrey T. , Hungerford Laura L. , Fedorka-Cray Paula J. , Headrick Marcia L.
Published: July 2004

A total of 112 out of 5,709
Salmonella enterica
isolates from domestic animal species exhibited decreased susceptibilities to ceftiofur and ceftriaxone, and each possessed the
bla
CMY
gene. Ten
Salmonella
serotypes were significantly more likely to include resistant isolates. Isolates from turkeys, horses, cats, and dogs were significantly more likely to include resistant isolates.
High Prevalence of the
ermB
Gene among Erythromycin-Resistant
Streptococcus pneumoniae
Isolates in Germany during the Winter of 2000-2001 and In Vitro Activity of Telithromycin
Authors: Kresken Michael , Henrichfreise Beate , Bagel Simone , Brauers Johannes , Wiedemann Bernd
Published: July 2004

Of 595 isolates of
Streptococcus pneumoniae
from outpatients with respiratory tract infections, collected from 17 microbiology laboratories, 14.1% were resistant to erythromycin. Eighty-three erythromycin-resistant isolates were genetically analyzed, 83.1% of which harbored the
ermB
gene. Only four isolates (4.8%) harbored the
mefA
gene. Telithromycin exhibited potent activity against all isolates.
Role of β-Lactamases and Porins in Resistance to Ertapenem and Other β-Lactams in
Klebsiella pneumoniae
Authors: Jacoby George A. , Mills Debra M. , Chow Nancy
Published: July 2004

High-level resistance to ertapenem was produced by β-lactamases of groups 1, 2f, and 3 in a strain of
Klebsiella pneumoniae
deficient in Omp35 and Omp36. From a wild-type strain producing ACT-1 β-lactamase, ertapenem-resistant mutants for which the ertapenem MICs were up to 128 μg/ml and expression of outer membrane proteins was diminished could be selected.
α
1
-Antitrypsin Binds to and Interferes with Functionality of EspB from Atypical and Typical Enteropathogenic
Escherichia coli
Strains
Authors: Knappstein Sabine , Ide Tina , Schmidt M. Alexander , Heusipp Gerhard
Published: July 2004

Enteropathogenic
Escherichia coli
(EPEC), including diffusely adhering atypical
E. coli
, strains use a type III secretion system to deliver effector proteins into the membrane and cytoplasm of infected cells. The
E. coli
secreted proteins A, B, and D (EspA, EspB, and EspD) are required for the formation of the characteristic attaching and effacing (A/E) lesions. EspB and EspD are thought to form a translocation pore in the host cell membrane through which effector proteins are injected into the host cytosol. Besides its function in pore formation, EspB has been found in the cytosol and implicated to function as an effector protein. We screened for putative host cell proteins interacting with EspB of atypical EPEC strains and identified α
1
-antitrypsin (AAT) as a binding partner for EspB. AAT binds to EspB in pull-down and overlay experiments and also to EspD in overlay experiments. In agreement with the role of EspB and EspD in pore formation, EPEC-mediated hemolysis of red blood cells is strongly reduced by AAT in a concentration-dependent manner, indicating that AAT interferes with type III secretion by inhibiting the formation of the translocation pore. This is further supported by a decreased actin polymerization after infection of HeLa or CaCo-2 cells with EPEC in the presence of physiologically relevant concentrations of AAT. In this study, we identify AAT as a new binding partner for EspB and EspD, suggesting a previously unappreciated role for AAT in host cell defense against EPEC infections and potentially also against other bacterial pathogens.
Long-Term Multiepitopic Cytotoxic-T-Lymphocyte Responses Induced in Chimpanzees by Combinations ofPlasmodium falciparumLiver-Stage Peptides and Lipopeptides
Authors: BenMohamed Lbachir , Thomas Alan , Druilhe Pierre
Published: July 2004

Preclinical immunogenicity studies of 12 malaria peptides, selected from fourPlasmodium falciparumantigens (Ags), namely, LSA1, LSA3, SALSA, and STARP, that are expressed at the pre-erythrocytic (sporozoite and liver) stages of the human parasite were carried out in chimpanzees. To strengthen their immunogenicity, six of these synthetic peptides were modified by the C-terminal addition of a single palmitoyl chain (lipopeptides) and delivered without adjuvant, whereas the remaining six unmodified peptides were emulsified and delivered by using Montanide ISA51 adjuvant. We have previously reported that these peptides and lipopeptides induce high B-cell and CD4+-T-helper responses in chimpanzees. In this report, we show their ability to induce multiepitopic and long-lasting antigen-specific CD8+cytotoxic-T-lymphocyte (CTL) responses. The magnitude, consistency, and memory of CTL responses generated by LSA3 peptides point to the strong immunogenicity of this liver-stage Ag. These findings support the screening strategy used to select the fourP. falciparumpre-erythrocytic Ags and emphasize their valuable immunogenic properties. The successful immunization of nonhuman primates with combinations of corresponding peptides in a mineral oil emulsion (ISA51) and lipopeptides in saline provide a vaccine formulation that can be tested in humans.