bluuebunny/crossref_metadata_2025 huggingface dataset:
Intracellular Delivery and Antibacterial Activity of Gentamicin Encapsulated in pH-Sensitive Liposomes
Authors: Lutwyche Peter , Cordeiro Carol , Wiseman David J. , St-Louis Maryse , Uh Mitchell , Hope Michael J. , Webb Murray S. , Finlay B. Brett
Published: October 2018

Cell membranes are relatively impermeable to the antibiotic gentamicin, a factor that, along with the toxicity of gentamicin, precludes its use against many important intracellular bacterial infections. Liposomal encapsulation of this drug was used in order to achieve intracellular antibiotic delivery and therefore increase the drug’s therapeutic activity against intracellular pathogens. Gentamicin encapsulation in several dipalmitoylphosphatidylcholine (DPPC) and pH-sensitive dioleoylphosphatidylethanolamine (DOPE)-based carrier systems was characterized. To systematically test the antibacterial efficacies of these formulations, a tissue culture assay system was developed wherein murine macrophage-like J774A.1 cells were infected with bacteria and were then treated with encapsulated drug. Of these formulations, DOPE–
N
-succinyl-DOPE and DOPE–
N
-glutaryl-DOPE (70:30;mol:mol) containing small amounts of polyethyleneglycol-ceramide showed appreciable antibacterial activities, killing greater than 75% of intracellular vacuole-resident wild-type
Salmonella typhimurium
compared to the level of killing of the control formulations. These formulations also efficiently eliminated intracellular infections caused by a recombinant hemolysin-expressing
S. typhimurium
strain and a
Listeria monocytogenes
strain, both of which escape the vacuole and reside in the cytoplasm. Control non-pH-sensitive liposomal formulations of gentamicin had poor antibacterial activities. A fluorescence resonance energy transfer assay indicated that the efficacious formulations undergo a pH-dependent lipid mixing and fusion event. Intracellular delivery of the fluorescent molecules encapsulated in these formulations was confirmed by confocal fluorescence microscopy and was shown to be dependent on endosomal acidification. This work shows that encapsulation of membrane-impermeative antibiotics in appropriately designed lipid-based delivery systems can enable their use in treating intracellular infections and details the development of a general assay for testing the intracellular delivery of encapsulated drug formulations.
Secretion of Sparfloxacin from the Human Intestinal Caco-2 Cell Line Is Altered by P-Glycoprotein Inhibitors
Authors: Cormet-Boyaka Estelle , Huneau Jean-François , Mordrelle Agnès , Boyaka Prosper N. , Carbon Claude , Rubinstein Ethan , Tomé Daniel
Published: October 2018

The mechanism of intestinal secretion of the difluorinated quinolone sparfloxacin was investigated with the epithelial cell line Caco-2 and was compared to that of the P-glycoprotein (P-gp) substrate vinblastine. The P-gp inhibitors verapamil and progesterone significantly increased the epithelial cell accumulation of both vinblastine and sparfloxacin. This increase is likely to result from an inhibition of drug secretion since both vinblastine uptake and sparfloxacin uptake are known to proceed through a passive transmembrane diffusion. The unidirectional fluxes across cell monlayers grown on permeable filters indicated that a net secretion of sparfloxacin and vinblastine occurred across Caco-2 cells. These secretions were significantly inhibited by the MDR-reversing agent verapamil. We conclude that the P-gp is likely to be involved in the intestinal elimination of the difluorinated quinolone sparfloxacin.
Inhibitory Activities of Gatifloxacin (AM-1155), a Newly Developed Fluoroquinolone, against Bacterial and Mammalian Type II Topoisomerases
Authors: Takei Masaya , Fukuda Hideyuki , Yasue Tokutaro , Hosaka Masaki , Oomori Yasuo
Published: October 2018

We determined the inhibitory activities of gatifloxacin againstStaphylococcus aureustopoisomerase IV,Escherichia coliDNA gyrase, and HeLa cell topoisomerase II and compared them with those of several quinolones. The inhibitory activities of quinolones against these type II topoisomerases significantly correlated with their antibacterial activities or cytotoxicities (correlation coefficient [r] = 0.926 forS. aureus,r= 0.972 forE. coli, andr= 0.648 for HeLa cells). Gatifloxacin possessed potent inhibitory activities against bacterial type II topoisomerases (50% inhibitory concentration [IC50] = 13.8 μg/ml forS. aureustopoisomerase IV; IC50= 0.109 μg/ml forE. coliDNA gyrase) but the lowest activity against HeLa cell topoisomerase II (IC50= 265 μg/ml) among the quinolones tested. There was also a significant correlation between the inhibitory activities of quinolones againstS. aureustopoisomerase IV and those againstE. coliDNA gyrase (r= 0.969). However, the inhibitory activity against HeLa cell topoisomerase II did not correlate with that against either bacterial enzyme. The IC50of gatifloxacin for HeLa cell topoisomerase II was 19 and was more than 2,400 times higher than that forS. aureustopoisomerase IV and that forE. coliDNA gyrase. These ratios were higher than those for other quinolones, indicating that gatifloxacin possesses a higher selectivity for bacterial type II topoisomerases.
Single-Dose Pharmacokinetics of a Pleconaril (VP63843) Oral Solution and Effect of Food
Authors: Abdel-Rahman Susan M. , Kearns Gregory L.
Published: October 2018

Pleconaril is an orally active broad-spectrum antipicornaviral agent which demonstrates excellent penetration into the central nervous system, liver, and nasal epithelium. We report the results of a randomized two-way crossover study designed to characterize the disposition of a single dose (200 mg) of pleconaril oral solution in fed and fasting humans. Twelve healthy adult subjects (18.7 to 39 years of age) participated in this study. Each subject received a single 200-mg dose of pleconaril oral solution, both coadministered with a standard English breakfast and following a 10-h predose fast. There was a minimum 7-day washout period between pleconaril doses. Repeated blood samples (
n
= 10) were obtained over 24 h postdose, and the pleconaril level in plasma was quantified by gas chromatography. Plasma concentration-versus-time data were curve fitted for each subject by using a nonlinear weighted least-squares algorithm, and pharmacokinetic parameters were determined from the polyexponential estimates. Pleconaril disposition was best characterized by a one-compartment open model with first-order absorption. The apparent bioavailability of pleconaril oral solution was significantly increased with the administration of food. The area under the concentration-time curve and maximum concentration of pleconaril in plasma achieved following the standard English breakfast (i.e., 9.08 ± 3.23 mg/liter · h and 1.14 ± 0.58 mg/liter, respectively) were 2.2- and 2.5-fold higher, respectively than those achieved in the fasting state (i.e., 4.08 ± 2.74 mg/liter · h and 0.46 ± 0.30 mg/liter, respectively). Mean plasma pleconaril concentrations 12 h after a single 200-mg oral dose (fed, 0.25 ± 0.2 mg/liter; fasting, 0.11 ± 0.10 mg/liter) in healthy adults remained greater than that required to inhibit more than 90% of the enteroviruses in cell culture (i.e., 0.07 mg/liter). To enhance the oral bioavailability of pleconaril, coadministration with a fat-containing meal is recommended.
Enhancement of Antimicrobial Activity of Neuropeptide Y by N-Terminal Truncation
Authors: Shimizu Mayumi , Shigeri Yasushi , Tatsu Yoshiro , Yoshikawa Susumu , Yumoto Noboru
Published: October 2018

The activity of neuropeptide Y (NPY) against
Candida albicans
, which was revealed to be fungicidal, was enhanced significantly by the truncation of amino acid residues at the N terminus. The most active peptides (MICs, approximately 1 μM) were about 10-fold more potent than the intact NPY (MIC, approximately 10 μM). The enhancement was weakened by the replacement of the N terminus by negatively charged residues and/or acylation of the α-amino group. These results suggest that only the α-helical region of NPY is necessary for the antimicrobial activity and that the net charge of the peptide is important for the activity.
Pharmacokinetic Interaction between Ritonavir and Indinavir in Healthy Volunteers
Authors: Hsu Ann , Granneman G. Richard , Cao Guoliang , Carothers Lori , Japour Anthony , El-Shourbagy Tawakol , Dennis Suzana , Berg Jeanne , Erdman Keith , Leonard John M. , Sun Eugene
Published: October 2018

The pharmacokinetic interaction between indinavir and ritonavir was evaluated in five groups of healthy adult volunteers to explore the potential for twice-daily (b.i.d.) dosing of this combination. All subjects received 800 mg of indinavir every 8 h (q8h) on day 2. In addition, subjects in group I received one dose of 800 mg of indinavir on day 1 and 800 mg of indinavir q8h on day 17. Subjects in Groups II and IV each received one dose of 600 mg of indinavir on days 1 and 17, and subjects in groups III and V each received one dose of 400 mg of indinavir on days 1 and 17. During days 3 to 17, ritonavir placebo or ritonavir at 200, 300, 300, or 400 mg q12h was given to groups I, II, III, IV, and V, respectively. Ritonavir at steady state probably inhibited the cytochrome P-450 3A metabolism of indinavir and substantially increased plasma indinavir concentrations, with the area under the plasma concentration-time curve (AUC) increasing up to 475% and the peak concentration in serum (
C
max
) increasing up to 110%. The
C
max
/trough concentration ratio decreased from 50 in standard q8h regimens to less than 14 when indinavir was administered with ritonavir. For a constant indinavir dose, an increase in the ritonavir dose yielded similar indinavir AUCs,
C
max
s, and concentrations at 12 h (
C
12
s). For a constant ritonavir dose, an increase in the indinavir dose resulted in approximately proportional increases in the indinavir AUC, less than proportional increases in
C
max
, and slightly more than proportional increases in
C
12
. Ritonavir reduced between-subject variability in the indinavir AUC and trough concentrations and did not affect indinavir renal clearance. With the altered pharmacokinetic profile, indinavir likely could be given as a b.i.d. combination regimen with ritonavir. This could potentially improve patient compliance and thereby reduce treatment failures.
In Vitro Selection of Resistance to Four β-Lactams and Azithromycin in
Streptococcus pneumoniae
Authors: Pankuch Glenn A. , Jueneman Shane A. , Davies Todd A. , Jacobs Michael R. , Appelbaum Peter C.
Published: October 2018

Selection of resistance to amoxicillin (with or without clavulanate), cefaclor, cefuroxime, and azithromycin among six penicillin G- and azithromycin-susceptible pneumococcal strains and among four strains with intermediate penicillin sensitivities (azithromycin MICs, 0.125 to 4 μg/ml) was studied by performing 50 sequential subcultures in medium with sub-MICs of these antimicrobial agents. For only one of the six penicillin-susceptible strains did subculturing in medium with amoxicillin (with or without clavulanate) lead to an increased MIC, with the MIC rising from 0.008 to 0.125 μg/ml. Five of the six penicillin-susceptible strains showed increased azithromycin MICs (0.5 to >256.0 μg/ml) after 17 to 45 subcultures. Subculturing in medium with cefaclor did not affect the cefaclor MICs of three strains but and led to increased cefaclor MICs (from 0.5 to 2.0 to 4.0 μg/ml) for three of the six strains, with MICs of other β-lactams rising 1 to 3 twofold dilutions. Subculturing in cefuroxime led to increased cefuroxime MICs (from 0.03 to 0.06 μg/ml to 0.125 to 0.5 μg/ml) for all six strains without significantly altering the MICs of other β-lactams, except for one strain, which developed an increased cefaclor MIC. Subculturing in azithromycin did not affect β-lactam MICs. Subculturing of the four strains with decreased penicillin susceptibility in amoxicillin (with or without clavulanate) or cefuroxime did not select for β-lactam resistance. Subculturing of one strain in cefaclor led to an increase in MIC from 0.5 to 2.0 μg/ml after 19 passages. In contrast to strains that were initially azithromycin susceptible, which required >10 subcultures for resistance selection, three of four strains with azithromycin MICs of 0.125 to 4.0 μg/ml showed increased MICs after 7 to 13 passages, with the MICs increasing to 16 to 32 μg/ml. All azithromycin-resistant strains were clarithromycin resistant. With the exception of strains that contained
mefE
at the onset, no strains that developed resistance to azithromycin contained
ermB
or
mefE
, genes that have been found in macrolide-resistant pneumococci obtained from clinic patients.
Fluoroquinolone Action against Mycobacteria: Effects of C-8 Substituents on Growth, Survival, and Resistance
Authors: Dong Yuzhi , Xu Chen , Zhao Xilin , Domagala John , Drlica Karl
Published: October 2018

Fluoroquinolones trap gyrase on DNA as bacteriostatic complexes from which lethal DNA breaks are released. Substituents at the C-8 position increase activities of
N
-1-cyclopropyl fluoroquinolones against several bacterial species. In the present study, a C-8-methoxyl group improved bacteriostatic action against
gyrA
(gyrase-resistant) strains of
Mycobacterium tuberculosis
and
M. bovis
BCG. It also enhanced lethal action against gyrase mutants of
M. bovis
BCG. When cultures of
M. smegmatis
,
M. bovis
BCG, and
M. tuberculosis
were challenged with a C-8-methoxyl fluoroquinolone, no resistant mutant was recovered under conditions in which more than 1,000 mutants were obtained with a C-8-H control. A C-8-bromo substituent also increased bacteriostatic and lethal activities against a
gyrA
mutant of
M. bovis
BCG. When lethal activity was normalized to bacteriostatic activity, the C-8-methoxyl compound was more bactericidal than its C-8-H control, while the C-8-bromo fluoroquinolone was not. The C-8-methoxyl compound was also found to be more effective than the C-8-bromo fluoroquinolone at reducing selection of resistant mutants when each was compared to a C-8-H control over a broad concentration range. These data indicate that a C-8-methoxyl substituent, which facilitates attack of first-step gyrase mutants, may help make fluoroquinolones effective antituberculosis agents.
Intracellular Nucleotides of (−)-2′,3′-Deoxy-3′-Thiacytidine in Peripheral Blood Mononuclear Cells of a Patient Infected with Human Immunodeficiency Virus
Authors: Solas Caroline , Li Yu-Feng , Xie Meng-Yu , Sommadossi Jean-Pierre , Zhou Xiao-Jian
Published: October 2018

An analytical methodology was developed to quantitate the intracellular nucleotides including mono-, di-, and triphosphates and the diphosphocholine derivative of (−)-2′,3′-deoxy-3′-thiacytidine (3TC) in human peripheral blood mononuclear cells (PBMCs). The procedure includes the resolution of 3TC nucleotides by solid-phase extraction (SPE) on an anion-exchange cartridge, with subsequent enzyme digestion of the resulting phosphates to the parent drug that is ultimately quantitated by high-performance liquid chromatography with UV detection (HPLC-UV). Validation was performed with PBMCs from healthy donors exposed to [
3
H]3TC, leading to the formation of intracellular nucleotides that were quantitated by anion-exchange HPLC with radioactive detection (HPLC-RA). These nucleotide levels served as reference values and were used for cross-validation with data obtained by HPLC-UV. An excellent correlation was established between the results obtained by HPLC-RA and those obtained by HPLC-UV, with a slope of the regression lines close to unity and intercepts near nullity as well as a correlation coefficient close to unity for all 3TC phosphates. The assay was characterized by a limit of quantitation below 1 ng (amount on column) with a precision (percentage of coefficient of variation of repeated measurement) ranging from 0.8 to 18.1% and an accuracy (deviation of the amount determined by HPLC-UV from the nominal reference value) varying from −14.8 to 19.4%. This methodology was successfully applied to determine the quantity of 3TC nucleotides in PBMCs of a patient infected with human immunodeficiency virus after oral administration of 3TC and stavudine.
Penetration of Clindamycin and Its Metabolite N -Demethylclindamycin into Cerebrospinal Fluid following Intravenous Infusion of Clindamycin Phosphate in Patients with AIDS
Authors: Gatti Giorgio , Malena Marina , Casazza Rosetta , Borin Marie , Bassetti Matteo , Cruciani Mario
Published: October 2018

Clindamycin, which is usually used in combination with pyrimethamine, has been proven effective in the treatment of cerebral toxoplasmosis in human immunodeficiency virus-infected patients. However, it is not known if clindamycin achieves inhibitory concentrations at the site of infection. Also, it has been hypothesized that the activity of clindamycin against
Toxoplasma gondii
may be due, at least in part, to a metabolite. We evaluated the penetration of clindamycin and its major metabolite,
N
-demethylclindamycin (NDC), into cerebrospinal fluid (CSF) of AIDS patients undergoing lumbar puncture for diagnostic purposes. A single, 1,200-mg dose of clindamycin was administered as a 45-min intravenous infusion beginning at 1.5 or 2.5 h before CSF sampling. The concentrations of clindamycin in CSF ranged from 0.091 to 0.429 mg/liter at 1.5 h and from 0.120 to 0.283 mg/liter at 2.5 h following the beginning of the infusion. The concentrations of clindamycin in CSF were well above the 50% inhibitory concentration of 0.001 mg/liter and the parasiticidal concentration of 0.006 mg/liter. NDC was undetectable both in plasma and in CSF. Our study provides a pharmacokinetic rationale for the clinical efficacy of clindamycin in the treatment of cerebral toxoplasmosis.
Coresistance to Zidovudine and Foscarnet Is Associated with Multiple Mutations in the Human Immunodeficiency Virus Type 1 Reverse Transcriptase
Authors: Tachedjian Gilda , French Martyn , Mills John
Published: October 2018

Human immunodeficiency virus type 1 (HIV-1) isolates obtained from a patient with AIDS were assessed for coresistance to foscarnet and zidovudine. An HIV-1 strain (AP20) coresistant to foscarnet and zidovudine was isolated after 20 months of continuous combination therapy. The reverse transcriptase (RT) gene of AP20 had 41 substitutions which were different from the HXB2-D sequence and 9 that were different from the sequence of its foscarnet-sensitive, zidovudine-resistant progenitor virus (AP6). Six of these mutations were nonpolymorphic (T39A, V108I, K166R, K219R, K223Q, and L228R). Both strains had the conventional mutations mediating zidovudine resistance. In vivo selection may result in HIV-1 strains that are coresistant to foscarnet and zidovudine, but coresistance appears to require a complex evolutionary path and multiple RT mutations.
Antibiotic-Induced Release of Lipoteichoic Acid and Peptidoglycan from
Staphylococcus aureus
: Quantitative Measurements and Biological Reactivities
Authors: van Langevelde P. , van Dissel J. T. , Ravensbergen E. , Appelmelk B. J. , Schrijver I. A. , Groeneveld P. H. P.
Published: October 2018

Antibiotics with different mechanisms of action may vary with respect to their effects on the release and immunostimulatory activities of cell wall fragments from gram-positive bacteria. Therefore, after
Staphylococcus aureus
was cultured for 4 h in the absence of antibiotics (control) and in the presence of β-lactam antibiotics (imipenem, flucloxacillin, or cefamandole) and protein synthesis-inhibiting antibiotics (erythromycin, clindamycin, or gentamicin), the lipoteichoic acid (LTA) and peptidoglycan (PG) levels in the bacterial supernatants were measured. β-Lactam antibiotics greatly enhanced the release of LTA and PG (4- to 9-fold and 60- to 85-fold, respectively), whereas protein synthesis inhibitors did not affect PG release and even inhibited the release of LTA compared to the amount of LTA released in control cultures. The capacity of β-lactam supernatants to stimulate the production of tumor necrosis factor alpha and interleukin-10 in human whole blood was significantly higher than that of protein synthesis inhibitor or control supernatants; the amounts of these cytokines released were directly proportional to the concentrations of PG and LTA in the supernatants. Enzymatic degradation of PG in the supernatants indicated that PG was mainly responsible for the observed biological reactivity.
Photosensitivity Reactions Caused by Lomefloxacin Hydrochloride: A Multicenter Survey
Authors: Arata Jirô , Horio Takeshi , Soejima Rinzo , Ohara Kenji
Published: October 2018

The photosensitivity effect of lomefloxacin hydrochloride (LFLX) was investigated in terms of patient background factors (sex, age, underlying disease, complications, history, occupation, and residential condition, etc.), treatment factors (daily dosage of LFLX, duration of treatment, total dose, concomitant drugs, and previous medication, etc.), and correlations among them. In 100 institutions throughout Japan, 4,284 patients were enrolled over a period of 2 years, beginning in October 1991, avoiding the accumulation of patients in any specific season. Since 8 patients did not visit again after enrollment, the clinical records of 4,276 patients were analyzed. Photosensitivity in 44 patients was found (1.03%), but the symptoms in most patients were not severe and improved after discontinuation of LFLX treatment. The photosensitivity reaction was more prevalent in patients who were 60 years of age and older with concomitant diseases and complications, in patients treated with a total amount of 20 g and more of LFLX for 30 days or longer, and in patients with a history of previous treatment with quinolone drugs. Although the incidence and degree of the photosensitivity reaction vary significantly among new quinolone drugs, every quinolone drug is potentially phototoxic. In particular, long-term use of LFLX should be avoided, and patients taking LFLX should be advised to abstain from prolonged exposure to sunlight.
Rapid Emergence of High-Level Resistance to Quinolones in
Campylobacter jejuni
Associated with Mutational Changes in
gyrA
and
parC
Authors: Gibreel Amera , Sjögren Eva , Kaijser Bertil , Wretlind Bengt , Sköld Ola
Published: October 2018

Quinolone resistance in clinical isolates of
Campylobacter jejuni
in Sweden increased more than 20-fold at the beginning of the 1990s. Resistance to 125 μg of ciprofloxacin per ml in clinical isolates was associated with chromosomal mutations in
C. jejuni
leading to a Thr-86-Ile substitution in the
gyrA
product and a Arg-139-Gln substitution in the
parC
product.
Joint Tolerance to β-Lactam and Fluoroquinolone Antibiotics in
Escherichia coli
Results from Overexpression of
hipA
Authors: Falla Timothy J. , Chopra Ian
Published: October 2018

The basis of joint tolerance to β-lactam and fluoroquinolone antibiotics in
Escherichia coli
mediated by
hipA
was examined. An antibiotic tolerance phenotype was produced by overexpression of
hipA
under conditions that did not affect the growth rate of the organism. Overexpressing
hipA
probably decreases the period in which bacteria are susceptible to the antibiotics by temporarily affecting some aspect of chromosome replication or cell division.
Effects of Bicyclomycin on RNA- and ATP-Binding Activities of Transcription Termination Factor Rho
Authors: Carrano Lucia , Bucci Cecilia , De Pascalis Roberto , Lavitola Alfredo , Manna Filomena , Corti Emiliana , Bruno Bruni Carmelo , Alifano Pietro
Published: October 2018

Bicyclomycin is a commercially important antibiotic that has been shown to be effective against many gram-negative bacteria. Genetic and biochemical evidence indicates that the antibiotic interferes with RNA metabolism in
Escherichia coli
by inhibiting the activity of transcription termination factor Rho. However, the precise mechanism of inhibition is not completely known. In this study we have used in vitro transcription assays to analyze the effects of bicyclomycin on the termination step of transcription. The Rho-dependent transcription termination region located within the
hisG
cistron of
Salmonella typhimurium
has been used as an experimental system. The possible interference of the antibiotic with the various functions of factor Rho, such as RNA binding at the primary site, ATP binding, and hexamer formation, has been investigated by RNA gel mobility shift, photochemical cross-linking, and gel filtration experiments. The results of these studies demonstrate that bicyclomycin does not interfere with the binding of Rho to the loading site on nascent RNA. Binding of the factor to ATP is not impeded, on the contrary, the antibiotic appears to decrease the apparent equilibrium dissociation constant for ATP in photochemical cross-linking experiments. The available evidence suggests that this decrease might be due to an interference with the correct positioning of ATP within the nucleotide-binding pocket leading b an inherent block of ATP hydrolysis. Possibly, as a consequence of this interference, the antibiotic also prevents ATP-dependent stabilization of Rho hexamers.
In Vitro Activity of BAY 12-8039, a New Fluoroquinolone, against Mycoplasmas
Authors: Bébéar C. M. , Renaudin H. , Boudjadja A. , Bébéar C.
Published: October 2018

The in vitro activity of the fluoroquinolone BAY 12-8039 against 66 strains of different mycoplasma species and 30 strains of
Ureaplasma urealyticum
was compared with those of three other antimicrobial agents. BAY 12-8039 at 0.5 μg/ml inhibited 100% of all the mycoplasmal and ureaplasmal strains tested. The minimal bactericidal concentrations of BAY 12-8039 increased only two- to eightfold compared to the MICs. Furthermore, they were comparable to those of sparfloxacin and lower than those of doxycycline and clarithromycin.
Antibiotic-Induced Lipopolysaccharide (LPS) Release fromSalmonella typhi: Delay between Killing by Ceftazidime and Imipenem and Release of LPS
Authors: van Langevelde Petra , Kwappenberg Kitty M. C. , Groeneveld Paul H. P. , Mattie Herman , van Dissel Jaap T.
Published: October 2018

It has been suggested that the antibiotic-induced release of lipopolysaccharide (LPS) is an important cause of the development of septic shock in patients treated for severe infections caused by gram-negative bacteria. β-Lactam antibiotics change the integrity of the bacterial cell envelope by binding to penicillin-binding proteins (PBP) in the membrane and thus may affect the amount of LPS that is released and the kinetics of that release. In this respect, ceftazidime at intermediate concentrations binds with a high affinity to PBP 3 and PBP 1a and thus can induce filament formation in addition to killing, whereas imipenem preferentially binds to PBP 2 and PBP 1b, leading to spheroplast formation and rapid cell lysis. We investigated the effects of these antibiotics on the killing and the release of the radioactively labelled LPS ofSalmonella typhiTy 21A. A mathematical model was developed to calculate the delay between bacterial killing and LPS release, designated the lag time. At antibiotic concentrations inducing equal killing, the amount of LPS released was the same for both antibiotics. Only after 6 h of incubation at antibiotic concentrations above 0.5 μg/ml, the amount of3H-LPS released was slightly higher (∼1.2-fold) in incubations with ceftazidime than in those with imipenem, and the maximum releases of the total label were 33.2% ± 0.89% and 27.1% ± 0.45%, respectively. Despite the clear concentration-dependent effect on the bacterial killing and subsequent LPS release, the lag time was independent of the antibiotic concentration. For ceftazidime as well as imipenem the lag time amounted to approximately 60 min. In conclusion, our findings imply that the mechanism of antibiotic-induced LPS release is independent of the PBP affinities for these β-lactam antibiotics. Furthermore, once the organism is killed by either imipenem or ceftazidime, the rate of LPS release fromS. typhidoes not differ according to the antibiotic with which the organism is killed, and there is little difference in the relative amount of LPS released.
Use of Pharmacodynamic Parameters To Predict Efficacy of Combination Therapy by Using Fractional Inhibitory Concentration Kinetics
Authors: den Hollander Jan G. , Mouton Johan W. , Verbrugh Henri A.
Published: October 2018

Combination therapy with antimicrobial agents can be used against bacteria that have reduced susceptibilities to single agents. We studied various tobramycin and ceftazidime dosing regimens against four resistant
Pseudomonas aeruginosa
strains in an in vitro pharmacokinetic model to determine the usability of combination therapy for the treatment of infections due to resistant bacterial strains. For the selection of an optimal dosing regimen it is necessary to determine which pharmacodynamic parameter best predicts efficacy during combination therapy and to find a simple method for susceptibility testing. An easy-to-use, previously described E-test method was evaluated as a test for susceptibility to combination therapy. That test resulted in a MIC
combi
, which is the MIC of, for example, tobramycin in the presence of ceftazidime. By dividing the tobramycin and ceftazidime concentration by the MIC
combi
at each time point during the dosing interval, fractional inhibitory concentration (FIC) curves were constructed, and from these curves new pharmacodynamic parameters for combination therapy were calculated (i.e., AUC
combi
,
C
max-combi
,
T
>MIC-combi
, and
T
>FICi
, where AUC
combi
,
C
max-combi
,
T
>MIC-combi
, and
T
>FICi
are the area under the FIC
combi
curve, the peak concentration of FIC
combi
, the time that the concentration of the combination is above the MIC
combi
, and the time above the FIC index, respectively). By stepwise multilinear regression analysis, the pharmacodynamic parameter
T
>FICi
proved to be the best predictor of therapeutic efficacy during combination therapy with tobramycin and ceftazidime (
R
2
= 0.6821;
P
< 0.01). We conclude that for combination therapy with tobramycin and ceftazidime the
T
>FICi
is the parameter best predictive of efficacy and that the E-test for susceptibility testing of combination therapy gives promising results. These new pharmacodynamic parameters for combination therapy promise to provide better insight into the rationale behind combination therapy.
Imiquimod, a Patient-Applied Immune-Response Modifier for Treatment of External Genital Warts
Authors: Beutner Karl R. , Tyring Stephen K. , Trofatter Kenneth F. , Douglas John M. , Spruance Spotswood , Owens Mary L. , Fox Terry L. , Hougham Andrina J. , Schmitt Kathy A.
Published: October 2018

Genital human papillomavirus infection is one of the most common sexually transmitted diseases. Imiquimod is a new agent, an immune-response modifier, that has been demonstrated to have potent in vivo antiviral and antitumor effects in animal models. The present prospective, multicenter, double-blind, randomized, vehicle-controlled trial evaluated the efficacy and safety of daily patient-applied imiquimod for up to 16 weeks for the treatment of external genital warts. Wart recurrence was investigated during a 12-week treatment-free follow-up period. In the intent-to-treat analysis, baseline warts cleared from 49 of 94 (52%) patients treated with 5% imiquimod cream, 13 of 90 (14%) patients treated with 1% imiquimod cream, and 3 of 95 (4%) vehicle-treated patients; the differences between the groups treated with vehicle and imiquimod were significant (P< 0.0001). For subjects who completed the follow-up period, recurrence rates after a complete response were 19% (9 of 48 patients) in the 5% imiquimod cream group, 17% (2 of 12) in the 1% imiquimod cream group, and 0% (0 of 3) in the vehicle-treated group. There were no systemic reactions, although local skin reactions (generally of mild or moderate severity) were common, particularly in the 5% imiquimod cream group. Local reactions caused two patients to discontinue treatment. The most frequently reported local skin reactions were erythema, excoriation or flaking, and erosion. Patient-applied 5% imiquimod cream is effective for the treatment of external genital warts and has a favorable safety profile.
Accumulation of Norfloxacin by
Mycobacterium aurum
and
Mycobacterium smegmatis
Authors: Williams Kerstin J. , Chung Gavin A. C. , Piddock Laura J. V.
Published: October 2018

The modified fluorescence method was used to determine the accumulation of norfloxacin by
Mycobacterium aurum
A+ and
Mycobacterium smegmatis
mc
2
155. By using an exogenous norfloxacin concentration of 10 μg/ml, a steady-state concentration (SSC) of 160 to 180 ng of norfloxacin/mg of cells was obtained for
M. aurum
, and an SSC of 120 to 140 ng of norfloxacin/mg of cells obtained for
M. smegmatis
. For both species of mycobacteria, the SSC was achieved within 5 min. The silicon oil method was investigated and gave higher SSCs than the modified fluorescence method. Further studies on the mechanism of norfloxacin accumulation by
M. aurum
were performed. An increase in the pH of the wash buffer from 7.0 to 9.0 did not significantly affect the final SSC obtained. Accumulation was nonsaturated over a norfloxacin concentration range of 0 to 100 μg/ml, and the proton motive force inhibitor 2,4-dinitrophenol (1 and 2 mM), whether it was added before or after norfloxacin was added, had no effect on the final SSC obtained. 2,4-Dinitrophenol also had no effect on norfloxacin accumulation by
M. smegmatis
. Furthermore, norfloxacin accumulation by
M. aurum
was unaffected by the presence of either Tween 80 or subinhibitory concentrations of ethambutol in the growth medium. Therefore, it is proposed that norfloxacin accumulation by mycobacteria occurs by simple, energy-independent diffusion.
Generation and Characterization of a Mutant of Influenza A Virus Selected with the Neuraminidase Inhibitor BCX-140
Authors: Bantia Shanta , Ghate Anita A. , Ananth Sandya L. , Babu Y. Sudhakar , Air Gillian M. , Walsh Gerald M.
Published: October 2018

Influenza neuraminidase (NA) plays an important role in viral replication, and characterization of viruses resistant to NA inhibitors will help elucidate the role of active-site residues. This information will assist in designing better inhibitors targeted to essential active-site residues that cannot generate drug-resistant mutations. In the present study we used the benzoic acid-based inhibitor BCX-140 to select and characterize resistant viruses. BCX-140 binds to the NA active site in an orientation that is opposite that of a sialic acid-based compound, 4-guanidino-2,4-dideoxy-2,3-dehydro-
N
-acetylneuraminic acid (GANA). Thus, the guanidino group of BCX-140 binds to Glu-276, whereas in GANA the guanidino group binds to Glu-119. We passaged influenza A/Singapore/1/57 (H2N2) in Madin-Darby canine kidney cells in the presence of BCX-140, and virus resistant to this inhibitor was selected after six passages. The NA of this mutant was still sensitive to inhibition by BCX-140. However, the mutant virus was resistant to BCX-140 in plaque and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Sequence analysis of hemagglutinin (HA) and NA genes revealed changes in both, although none were in the active site of the NA. Depending on the method of selection of the resistant virus, two types of changes associated with the sialic acid binding site were seen in the HA. One is a change in HA1 of Ala-133 to Thr, a residue close to the binding site, while the other change was Arg-132 of HA1 to Gln, which in HA1 of serotype H3 is a sialic acid contact (Asn-137). Binding studies revealed that both types of resistant viruses had reduced receptor binding affinity compared to that of the wild type. Thus, resistance to BCX-140 was generated by modifying the HA. NA active-site residue 276 may be essential for activity, and thus, it cannot be changed to generate resistance. However, drug-induced changes in the HA can result in a virus that is less dependent on NA activity for growth in cells and, hence, resistant to NA inhibitors.
In Vitro Activity of a New 8-Methoxyquinolone, BAY 12-8039, against
Chlamydia pneumoniae
Authors: Roblin Patricia M. , Hammerschlag Margaret R.
Published: October 2018

The in vitro susceptibilities of 10 strains of
Chlamydia pneumoniae
to a new 8-methoxyquinolone, BAY 12-8039, and to ofloxacin, doxycycline, and erythromycin were determined. The activity of BAY 12-8039 was similar to that of ofloxacin, with a MIC at which 90% of the isolates had no inclusions and a minimal chlamydicidal concentration at which 90% of the isolates had no inclusions after passage of 1.0 μg/ml, but this activity was less than those of doxycycline and erythromycin.
In Vitro Activity of Levofloxacin against a Selected Group of Anaerobic Bacteria Isolated from Skin and Soft Tissue Infections
Authors: Wexler Hannah M. , Molitoris Eric , Molitoris Denise , Finegold Sydney M.
Published: October 2018

The in vitro activity of levofloxacin was compared to the activities of ofloxacin, ciprofloxacin, ampicillin-sulbactam (2:1), cefoxitin, and metronidazole for a selected group of anaerobes (
n
= 175) isolated from skin and soft tissue infections by using the National Committee for Clinical Laboratory Standards-approved Wadsworth method. Ampicillin-sulbactam and cefoxitin inhibited 99% of the strains of this select group, levofloxacin and ofloxacin inhibited 73 and 50%, respectively, at 2 μg/ml, and ciprofloxacin inhibited 51% at 1 μg/ml. The geometric mean MIC of levofloxacin was lower than those of ofloxacin and ciprofloxacin for every group except
Veillonella.
Characteristics and Dynamics of Bacterial Populations during Postantibiotic Effect Determined by Flow Cytometry
Authors: Gottfredsson Magnús , Erlendsdóttir Helga , Sigfússon Ásbjörn , Gudmundsson Sigurdur
Published: October 2018

Changes in bacterial ultrastructure after antibiotic exposure and during the postantibiotic effect (PAE) have been demonstrated by electron microscopy (EM). However, EM is qualitative and subject to individual interpretation. In contrast, flow cytometry gives qualitative and quantitative information. The sizes and nucleic acid contents of
Escherichia coli
and
Pseudomonas aeruginosa
were studied during antimicrobial exposure as well as during the PAE period by staining the organisms with propidium iodide and analyzing them with flow cytometry and fluorescence microscopy. The effects of ampicillin, ceftriaxone, ciprofloxacin, gentamicin, and rifampin were studied for
E. coli
, whereas for
P. aeruginosa
imipenem and ciprofloxacin were investigated. After exposure of
E. coli
to ampicillin, ceftriaxone, and ciprofloxacin, filamentous organisms were observed by fluorescence microscopy. These changes in morphology were reflected by increased forward light scatter (FSC) and nucleic acid content as measured by flow cytometry. For the β-lactams the extent of filamentation increased in a dose-dependent manner after drug removal, resulting in formation of distinct subpopulations of bacteria. These changes peaked at 20 to 35 min, and bacteria returned to normal after 90 min after drug removal. In contrast, the subpopulations induced by ciprofloxacin did not return to normal until >180 min after the end of the classically defined PAE. Rifampin resulted in formation of small organisms with low FSC, whereas no distinctive characteristics were noted after gentamicin exposure. For
P. aeruginosa
an identifiable subpopulation of large globoid cells and increased nucleic acid content was detected after exposure to imipenem. These changes persisted past the PAE, as defined by viability counting. Swollen organisms with increased FSC were detected after ciprofloxacin exposure, even persisting during bacterial growth. In summary, for β-lactam antibiotics and ciprofloxacin, the PAE is characterized by dynamic formation of enlarged cell populations of increased nucleic acid content, whereas rifampin induces a decrease in size and nucleic acid content in the organisms. Flow cytometry is an ideal method for future studies of bacterial phenotypic characteristics during the PAE.
Isolation of Nitrofurantoin-Resistant Mutants of Nitroreductase-Producing
Clostridium
sp. Strains from the Human Intestinal Tract
Authors: Rafii Fatemeh , Hansen Eugene B.
Published: October 2018

Five spontaneous nitrofurantoin-resistant mutants (one each of
Clostridium leptum
,
Clostridium paraputrificum
, two other
Clostridium
spp. strains from the human intestinal microflora, and
Clostridium perfringens
ATCC 3626) were selected by growth on a nitrofurantoin-containing medium. All of the
Clostridium
wild-type and mutant strains produced nitroreductase, as was shown by the conversion of 4-nitrobenzoic acid to 4-aminobenzoic acid. High-performance liquid chromatography (HPLC) analysis of the mutants during incubation with 50 μg of nitrofurantoin per ml showed the gradual disappearance of the nitrofurantoin peak. The nitrofurantoin peak also disappeared when cell-free supernatants instead of cultures of each of the resistant and wild-type bacteria were used, but it persisted if the cell-free supernatants had been inactivated by heat. At least two of the mutants converted nitrofurantoin to metabolites without antibacterial activity, as was shown by a bioassay with a nitrofurantoin-susceptible
Bacillus
sp. strain. Nitrofurantoin at a high concentration (50 μg/ml) continued to exert some toxicity, even on the resistant strains, as was evident from the longer lag phases. This study indicates that
Clostridium
strains can develop resistance to nitrofurantoin while retaining the ability to produce nitroreductase; the mutants metabolized nitrofurantoin to compounds without antibacterial activity.
Steady-State Pharmacokinetics and Electrocardiographic Pharmacodynamics of Clarithromycin and Loratadine after Individual or Concomitant Administration
Authors: Carr Robert A. , Edmonds Anthony , Shi Harry , Locke Charles S. , Gustavson Linda E. , Craft J. Carl , Harris Stuart I. , Palmer Robert
Published: October 2018

To evaluate the potential for an interaction between clarithromycin and loratadine, healthy male volunteers (
n
= 24) received each of the following regimens according to a randomized crossover design: 500 mg of clarithromycin orally every 12 h (q12h) for 10 days, 10 mg of loratadine orally q24h for 10 days, and the combination of clarithromycin and loratadine. A washout interval of 14 days separated regimens. The addition of loratadine did not statistically significantly affect the steady-state pharmacokinetics of clarithromycin or its active metabolite, 14(
R
)-hydroxy-clarithromycin. However, the addition of clarithromycin statistically significantly altered the steady-state maximum observed plasma concentration and the area under the plasma concentration-time curve over a dosing interval for loratadine (+36 and +76%, respectively) and for descarboethoxyloratadine (DCL), the active metabolite of loratadine (+69 and +49%, respectively). Clarithromycin probably inhibits the oxidative metabolism of loratadine and DCL by the cytochrome P-450 3A subfamily. Electrocardiograms (
n
= 12) were obtained over 24-h periods at baseline and steady state (day 10). The mean maximum QTc interval and area under the QTc interval-time curve on day 10 were modestly increased (<3%) from baseline for all three regimens, but no QTc interval exceeded 439 ms for any subject. Elevated steady-state concentrations of loratadine and DCL do not appear to be associated with adverse cardiovascular effects related to prolongation of the QTc interval. Loratadine and clarithromycin were well tolerated, alone and in combination.
Flow Cytometric Assessment of the Postantibiotic Effect of Methicillin on
Staphylococcus aureus
Authors: Suller M. T. E. , Lloyd D.
Published: October 2018

The postantibiotic effect (PAE) following a 2-h exposure of
Staphylococcus aureus
NCTC 6571 to methicillin (5× the MIC) was investigated with fluorescent probes, 5-cyano-2,3-di-4-tolyl tetrazolium chloride (CTC), an indicator of respiratory activity, and the membrane potential-sensitive compound bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC
4
(3)]. Counts of the numbers of CFU on solid agar correlated well with information gained from the CTC and DiBAC
4
(3) fluorescence intensity distributions obtained by flow cytometry and revealed that the postantibiotic effect was 3.1 h. Due to the capacity of flow cytometry to provide information on the heterogeneity of a bacterial population, both fluorescent probes identified the emergence of an active subpopulation 4 h after removal of the methicillin, indicating the recovery of a small percentage of the population. After removal of the methicillin and resuspension of the cells in methicillin-free medium, a further decrease in the respiratory activity and the membrane integrity of the population was observed, although the CFU counts hardly varied, indicating continued antibiotic-induced damage. Also, CTC fluorescence measurements identified numerous subpopulations during the PAE period; this suggests that the PAE is complex, with individual organisms exhibiting various degrees of recovery. Flow cytometry thus provides a rapid and sensitive alternative to traditional techniques that have been used to study PAE, with the added advantage that physiological changes can be detected as they arise.
Novel Inhibitory Effects of γ-Glutamylcysteine Ethyl Ester against Human Immunodeficiency Virus Type 1 Production and Propagation
Authors: Kubota Satoshi , Shetty Shubhra , Zhang Huizhong , Kitahara Shigehisa , Pomerantz Roger J.
Published: October 2018

The anti-human immunodeficiency virus type I (anti-HIV-1) effects of γ-glutamylcysteine ethyl ester (γ-GCE; TEI-2306) were examined in vitro. In initial studies using a vigorously HIV-1-producing human T-lymphocytic cell line, γ-GCE displayed a novel biphasic repressive effect on chronic HIV-1 infection that was unlike that of other glutathione prodrugs or other reported antioxidants. In high doses, up to a concentration of 2.5 mM, at which neither glutathione (GSH) nor another GSH precursor has shown inhibitory effects, γ-GCE potently inhibited the production of HIV-1 by a selective cytopathic effect against infected cells, while the viability and growth of uninfected cells were unaffected at the same γ-GCE concentrations. At lower concentrations (200 to 400 μM), γ-GCE significantly repressed the virus production from chronically HIV-1-expressing cells without affecting their viability. The discrepancy of the thresholds of the toxic doses between infected and uninfected cells was found to be more than 10-fold. Relatively high doses of γ-GCE, utilized in acute HIV-1 infection of T-lymphocytic cells, entirely blocked the propagation of HIV-1 and rescued the cells from HIV-1-induced cell death. Furthermore, γ-GCE at such concentrations was found to directly inhibit the infectivity of HIV-1 within 4 h. Repressive effects of γ-GCE on acute HIV-1 infection in human primary human peripheral blood mononuclear cells were also demonstrated. Here, the anti-HIV-1 strategy utilizing γ-GCE is removal of both HIV-1-producing cells and free infectious HIV-1 in vitro, in place of specific immunoclearance in vivo, which might lead to an arrest or slowing of viral propagation in HIV-1-infected individuals.