bluuebunny/crossref_metadata_2025 huggingface dataset:
Killing of
Chlamydia trachomatis
by Novel Antimicrobial Lipids Adapted from Compounds in Human Breast Milk
Authors: Lampe M. F. , Ballweber L. M. , Isaacs C. E. , Patton D. L. , Stamm W. E.
Published: October 2018

The development of new methods for prevention of sexually transmitted
Chlamydia trachomatis
infection is a top public health priority. Topical self-administered vaginal microbicides represent one such approach in which the organism is eradicated at the time of initial exposure. To this end, we examined the activity of five synthetic lipids adapted from naturally occurring compounds found in human breast milk.
C. trachomatis
serovar D or F elementary bodies were added to serial dilutions of the lipids and incubated for various times. Aliquots were then cultured in monolayers of McCoy cells, and inclusions were counted. A 7.5 mM concentration of 2-
O
-octyl-
sn
-glycerol completely prevented growth of
C. trachomatis
after 120 min of contact with the organism. The remaining lipids, 1-
O
-octyl-, 1-
O
-heptyl-, 2-
O
-hexyl-, and 1-
O
-hexyl-
sn
-glycerol, showed less activity. On electron microscopic examination, the lipids were shown to have disrupted the chlamydial inner membrane, allowing leakage of the cytoplasmic contents from the cell. Lipid activity was unaffected by the presence of 10% human blood or alterations in pH from 4.0 to 8.0, conditions reflecting those sometimes found in the vagina. Our results suggest that these lipids, especially 2-
O
-octyl-
sn
-glycerol, may be effective as topical microbicides in preventing the transmission of
C. trachomatis
. Further efficacy and toxicity studies with these lipids and assessment of their activity against other sexually transmitted disease pathogens are in progress.
Molecular Heterogeneity of the L-1 Metallo-β-Lactamase Family from
Stenotrophomonas maltophilia
Authors: Sanschagrin François , Dufresne Julien , Levesque Roger C.
Published: October 2018

We have determined the nucleotide sequence of the
blaS
gene encoding the carbapenem-hydrolyzing L-1 β-lactamase from
Stenotrophomonas maltophilia
GN12873. Analysis of the DNA and deduced amino acid sequences identified a product of 290 amino acids. Comparisons of the L-1 amino acid sequence with those of other zinc β-lactamases showed 88.6% identity with the L-1 enzyme from
S. maltophilia
IID1275 and less than 20% identity with other class B metalloenzymes.
A New Ketolide, HMR 3004, Active against Streptococci Inducibly Resistant to Erythromycin
Authors: Rosato Adriana , Vicarini Hubert , Bonnefoy Alain , Chantot Jean-François , Leclercq Roland
Published: October 2018

HMR 3004 is a new hydrazono ketolide characterized by a 3-keto function instead of the cladinose moiety. The effect of this antimicrobial agent on inducible and constitutive macrolide-lincosamide-streptogramin B (MLS
B
) resistance was tested in a
lacZ
reporter system under control of several
ermAM
-like attenuator variants. For one constitutively resistant
Streptococcus agalactiae
strain, three inducibly resistant
Streptococcus pneumoniae
strains, and one inducibly resistant
Enterococcus faecalis
strain, the attenuators fused with
lacZ
were cloned into the shuttle plasmid pJIM2246 and the plasmid was introduced into
Staphylococcus aureus
RN4220. For the wild-type attenuators, HMR 3004 was a very weak inducer, unlike its cladinose counterpart RU 6652 and erythromycin. As expected, for the fusion originating from the constitutively resistant
S. agalactiae
strain, the level of uninduced β-galactosidase synthesis was high. For one
S. pneumoniae
attenuator, mutations in the 3′ end of the attenuator that weakened the stem-loop structure that sequesters the ribosome-binding site and start codon for
ermAM
methylase could explain the high level of uninduced β-galactosidase produced. For streptococci, the activity of HMR 3004 correlated with the basal level of β-galactosidase synthesized. The weak inducer activity of HMR 3004 explained its activity against inducibly MLS
B
-resistant
S. pneumoniae
but did not correlate with the moderate activity of the antibiotic against inducibly resistant
E. faecalis.
Moxifloxacin in the Therapy of Experimental Pneumococcal Meningitis
Authors: Schmidt H. , Dalhoff A. , Stuertz K. , Trostdorf F. , Chen V. , Schneider O. , Kohlsdorfer C. , Brück W. , Nau R.
Published: October 2018

The activity of moxifloxacin (BAY 12-8039) against a
Streptococcus pneumoniae
type 3 strain (MIC and minimum bactericidal concentration [MBC] of moxifloxacin, 0.06 and 0.25 μg/ml, respectively; MIC and MBC of ceftriaxone, 0.03 and 0.06 μg/ml, respectively) was determined in vitro and in a rabbit model of meningitis. Despite comparable bactericidal activity, 10 μg of moxifloxacin per ml released lipoteichoic and teichoic acids less rapidly than 10 μg of ceftriaxone per ml in vitro. Against experimental meningitis, 10 mg of moxifloxacin per kg of body weight per ml reduced the bacterial titers in cerebrospinal fluid (CSF) almost as rapidly as ceftriaxone did (mean ± standard deviation, −0.32 ± 0.14 versus −0.39 ± 0.11 Δlog CFU/ml/h). The activity of moxifloxacin could be described by a sigmoid dose-response curve with a maximum effect of −0.33 ΔlogCFU/ml/h and with a dosage of 1.4 mg/kg/h producing a half-maximal effect. Maximum tumor necrosis factor activity in CSF was observed later with moxifloxacin than with ceftriaxone (5 versus 2 h after the initiation of treatment). At 10 mg/kg/h, the concentrations of moxifloxacin in CSF were 3.8 ± 1.2 μg/ml. Adjunctive treatment with dexamethasone at 1 mg/kg prior to the initiation of antibiotic treatment only marginally reduced the concentrations of moxifloxacin in CSF (3.3 ± 0.6 μg/ml). In conclusion, moxifloxacin may qualify for use in the treatment of
S. pneumoniae
meningitis.
Screening for Novel Antimicrobials from Encoded Combinatorial Libraries by Using a Two-Dimensional Agar Format
Authors: Silen Joy L. , Lu Amy T. , Solas Dennis W. , Gore Medini A. , Maclean Derek , Shah Nikil H. , Coffin Jill M. , Bhinderwala Naseema S. , Wang Yongwen , Tsutsui Ken T. , Look Gary C. , Campbell David A. , Hale Ron L. , Navre Marc , DeLuca-Flaherty Camille R.
Published: October 2018

A sensitive lawn-based format has been developed to screen bead-tethered combinatorial chemical libraries for antimicrobial activity. This method has been validated with beads linked to penicillin V via a photocleavable chemical linker in several analyses including a spike-and-recover experiment. The lawn-based screen sensitivity was modified to detect antibacterial compounds of modest potency, and a demonstration experiment with a naive combinatorial library of over 46,000 individual triazines was evaluated for antibacterial activity. Numerous hits were identified, and both active and inactive compounds were resynthesized and confirmed in traditional broth assays. This demonstration experiment suggests that novel antimicrobial compounds can be easily identified from very large combinatorial libraries of small, nonpeptidic compounds.
Contribution of β-Lactamases to β-Lactam Susceptibilities of Susceptible and Multidrug-Resistant
Mycobacterium tuberculosis
Clinical Isolates
Authors: Segura C. , Salvadó M. , Collado I. , Chaves J. , Coira A.
Published: October 2018

The β-lactamases in 154 clinical
Mycobacterium tuberculosis
strains were studied. Susceptibilities to β-lactam antibiotics, their combination with clavulanate (2:1), and two fluoroquinolones were determined in 24
M. tuberculosis
strains susceptible to antimycobacterial drugs and in nine multiresistant strains. All 154
M. tuberculosis
isolates showed a single chromosomal β-lactamase pattern (pI 4.9 and 5.1).
M. tuberculosis
β-lactamase hydrolyzes cefotaxime with a maximum rate of 22.5 ± 2.19 IU/liter (strain 1382). Neither amoxicillin, carbenicillin, cefotaxime, ceftriaxone, nor aztreonam was active alone. Except for aztreonam, β-lactam combinations with clavulanate produced better antimycobacterial activity.
In Vitro Susceptibilities of 27 Rickettsiae to 13 Antimicrobials
Authors: Rolain J. M. , Maurin M. , Vestris G. , Raoult D.
Published: October 2018

The MICs of 13 antibiotics (doxycycline, thiamphenicol, rifampin, amoxicillin, gentamicin, co-trimoxazole, ciprofloxacin, pefloxacin, ofloxacin, erythromycin, josamycin, clarithromycin, and pristinamycin) were determined for 27 available rickettsial species or strains. We used two in vitro cell culture methods described previously: the plaque assay and the microplaque colorimetric assay. Our results confirm the susceptibilities of rickettsiae to doxycycline, thiamphenicol, and fluoroquinolones. Beta-lactams, aminoglycosides, and co-trimoxazole were not active. Typhus group rickettsiae were susceptible to all macrolides tested, whereas the spotted fever group rickettsiae,
R. bellii
, and
R. canada
were more resistant, with josamycin, a safe alternative for the treatment of Mediterranean spotted fever, being the most effective compound. Strain Bar 29,
R. massiliae
,
R. montana
,
R. aeschlimannii
, and
R. rhipicephali
, which are members of the same phylogenetic subgroup, were more resistant to rifampin than the other rickettsiae tested. Heterogeneity in susceptibility to rifampin, which we report for the first time, may explain in vivo discrepancies in the effectiveness of this antibiotic for the treatment of rickettsial diseases. We hypothesize that rifampin resistance and erythromycin susceptibility may reflect a divergence during the evolution of rickettsiae.
Antiretroviral Efficacy and Pharmacokinetics of Oral Bis(isopropyloxycarbonyloxymethyl)9-(2-Phosphonylmethoxypropyl)adenine in Mice
Authors: Naesens Lieve , Bischofberger Norbert , Augustijns Patrick , Annaert Pieter , Van den Mooter Guy , Arimilli Murty N. , Kim Choung U. , De Clercq Erik
Published: October 2018

To overcome the low oral bioavailability of the highly potent and selective antiretroviral agent (
R
)-9-(2-phosphonylmethoxypropyl)adenine (PMPA), a new lipophilic ester derivative, i.e., the bis(isopropyloxycarbonyloxymethyl)-ester [bis(POC)-PMPA], was prepared. The usefulness of bis(POC)-PMPA as an oral prodrug for PMPA was investigated in the intestinal mucosa Caco-2 cell monolayer model. The total transport of bis(POC)-PMPA was 2.7%, whereas it was less than 0.1% for PMPA. Bis(POC)-PMPA was considerably metabolized inside the epithelial cells, since the majority of the compound was recovered after transport in the form of the monoester metabolite [mono(POC)-PMPA]. In contrast, bis(POC)-PMPA was relatively resistant to degradation at the luminal side of the Caco-2 cells. Pharmacokinetic studies with mice showed that the oral bioavailability of bis(POC)-PMPA (calculated from the curves of the concentration of free PMPA in plasma) was 20%. Neither bis(POC)-PMPA nor mono(POC)-PMPA could be recovered in plasma, suggesting the efficient release of the active drug PMPA after oral administration of bis(POC)-PMPA. Severe combined immunodeficient (SCID) mice infected with Moloney murine sarcoma virus (MSV) and treated orally with bis(POC)-PMPA for 5 or 10 days (dosages, 50, 100, or 200 mg of PMPA equivalent per kg of body weight per day) showed a significant delay in MSV-induced tumor appearance and tumor-associated death. The antiviral efficacy of oral bis(POC)-PMPA was related to the dosage and treatment period and was not significantly different from that of subcutaneous PMPA given at an equivalent dose. The favorable pharmacokinetic profile, marked antiviral efficacy, and low toxicity make bis(POC)-PMPA an attractive oral prodrug of PMPA that should be further pursued in clinical studies with patients infected with human immunodeficiency virus or hepatitis B virus.
Pharmacokinetic Evaluation of Amphotericin B in Lung Tissue: Lung Lymph Distribution after Intravenous Injection and Airspace Distribution after Aerosolization and Inhalation of Amphotericin B
Authors: Koizumi Tomonobu , Kubo Keishi , Kaneki Toshimichi , Hanaoka Masayuki , Hayano Toshihide , Miyahara Takayuki , Okada Kazuyoshi , Fujimoto Keisaku , Yamamoto Hiroshi , Kobayashi Toshio , Sekiguchi Morie
Published: October 2018

We have studied the pharmacokinetics of amphotericin B (AmB) in lung lymph circulation and bronchial-wash fluid after intravenous infusion and inhalation, respectively. For two experiments with awake sheep, we used lung lymph fistulas and tracheotomy. In experiment 1, AmB concentrations in plasma and lung lymph after intravenous infusion of AmB (1 mg/kg of body weight) over 1.5 h were measured. The mean peak in plasma level was 756.0 ± 188.8 ng/ml at 3 h after the start of infusion, and the level then decreased gradually to 194.8 ± 28.9 ng/ml at 24 h. The stable and maximal levels in lung lymph last 5 to 9 h after the start of AmB infusion. The concentrations in lung lymph after 9 h were slightly higher than those in plasma. Thus, the lung lymph-to-plasma ratio of AmB concentrations increased gradually during infusion, and the ratio was more than 1.0 after the end of infusion, suggesting that AmB could be easily moved from plasma to pulmonary interstitium and/or lung lymph circulation. In another experiment, 5 or 30 mg of aerosol AmB was inhaled, and the concentration of AmB in the bronchial-wash fluid was determined by bronchoalveolar lavage. The peak AmB concentration in the fluid was observed at 0.5 h. After that, AmB was slowly eliminated over 24 h. The area under the concentration-time curve for 30 mg of inhaled AmB was higher than that for 5 mg, but maximum concentrations of AmB in serum for 5 and 30 mg were almost similar. These observations identify the pharmacokinetic characteristics of AmB in the lung and may provide a new insight into the strategy for clinical treatment of fungal pneumonia.
Genotypic and Phenotypic Characterization of Human Immunodeficiency Virus Type 1 Variants Isolated from AIDS Patients after Prolonged Adefovir Dipivoxil Therapy
Authors: Mulato A. S. , Lamy P. D. , Miller M. D. , Li W.-X. , Anton K. E. , Hellmann N. S. , Cherrington J. M.
Published: October 2018

Adefovir dipivoxil [bis(pivaloyloxymethyl)-ester prodrug], an orally bioavailable prodrug of adefovir [9-(2-phosphonylmethoxyethyl)adenine], is currently in phase III clinical trials for the treatment of human immunodeficiency virus (HIV). In vitro experiments demonstrated that either a K65R or a K70E mutation in HIV reverse transcriptase (RT) was selected in the presence of adefovir, conferring a 16- or 9-fold decrease in susceptibility to adefovir, respectively. Previous data demonstrated that patients receiving adefovir dipivoxil monotherapy (125 mg daily) for 12 weeks experienced a median decrease in HIV RNA levels of 0.5 log
10
copies/ml and that resistance to adefovir dipivoxil did not arise during that period. In the present investigation, a further study was undertaken to investigate whether RT mutations developed among viruses from patients who completed the 12-week study and who opted to enroll in a maintenance phase of prolonged (6- to 12-month) adefovir dipivoxil therapy (120 mg daily). Concomitant treatment with antiretroviral agents was permitted during the maintenance phase. The median decreases in HIV RNA levels for patients who completed 6 or 12 months of maintenance-phase dosing were 0.6 and 1.14 log
10
copies/ml, respectively. The reductions in the HIV RNA levels were similar among patients who received adefovir dipivoxil with or without concomitant treatment with antiretroviral agents. Viruses from 8 of 29 patients dosed for up to 12 months developed RT mutations that were not present at baseline; these mutations may have been related to adefovir dipivoxil therapy. Viruses from two of the eight patients developed the K70E mutation while the patients were on therapy, but none of the viruses from patients developed the K65R RT substitution. Despite the development of RT mutations, sustained reductions (6 to 12 months) in viral load (≥0.7 log
10
copies/ml decrease from baseline) were observed in all eight patients.
Zidovudine Azido-Reductase in Human Liver Microsomes: Activation by Ethacrynic Acid, Dipyridamole, and Indomethacin and Inhibition by Human Immunodeficiency Virus Protease Inhibitors
Authors: Fayz Shirin , Inaba T.
Published: October 2018

AZT (zidovudine, 3′-azido-3′-deoxythymidine), although metabolized primarily to AZT-glucuronide, is also metabolized to 3′-amino-3′-deoxythmidine (AMT) by reduction of the azide to an amine. The formation of the myelotoxic metabolite AMT has not been well characterized, but inhibition of AMT formation would be of therapeutic benefit. The aim of this study was to identify compounds that inhibit AMT formation. Using human liver microsomes under anaerobic conditions and [2-
14
C]AZT,

K
m

values of AZT azido-reductase, estimated by radio-thin-layer chromatography, were 2.2 to 3.5 mM (
n
= 3). Oxygen completely inhibited this NADPH-dependent reduction. Thirteen of the 28 compounds tested inhibited the formation of AMT. In addition to the CYP3A4 inhibitors ketoconazole, fluconazole, indinavir, ritonavir, and saquinavir, metyrapone strongly inhibited AMT formation. An unexpected finding was the more-than-twofold increase in AMT formation in the presence of ethacrynic acid, dipyridamole, or indomethacin. Such activation of toxic metabolite formation would impair drug therapy.
Antiherpesvirus Activities of (1′
S
,2′
R
)-9-{[1′,2′-Bis(hydroxymethyl)cycloprop-1′-yl]methyl}guanine (A-5021) in Cell Culture
Authors: Iwayama Satoshi , Ono Nobukazu , Ohmura Yuko , Suzuki Katsuya , Aoki Miho , Nakazawa Harumi , Oikawa Miki , Kato Tamamo , Okunishi Masahiko , Nishiyama Yukihiro , Yamanishi Koichi
Published: October 2018

Antiherpetic activity of (1′
S
,2′
R
)-9-{[1′,2′-bis(hydroxymethyl)cycloprop-1′-yl]methyl}guanine (A-5021) was compared with those of acyclovir (ACV) and penciclovir (PCV) in cell cultures. In a plaque reduction assay using a selection of human cells, A-5021 showed the most potent activity in all cells. Against clinical isolates of herpes simplex virus type 1 (HSV-1,
n
= 5) and type 2 (HSV-2,
n
= 6), mean 50% inhibitory concentrations (IC
50
s) for A-5021 were 0.013 and 0.15 μg/ml, respectively, in MRC-5 cells. Corresponding IC
50
s for ACV were 0.22 and 0.30 μg/ml, and those for PCV were 0.84 and 1.5 μg/ml, respectively. Against clinical isolates of varicella-zoster virus (VZV,
n
= 5), mean IC
50
s for A-5021, ACV, and PCV were 0.77, 5.2, and 14 μg/ml, respectively, in human embryonic lung (HEL) cells. A-5021 showed considerably more prolonged antiviral activity than ACV when infected cells were treated for a short time. The selectivity index, the ratio of 50% cytotoxic concentration to IC
50
, of A-5021 was superior to those of ACV and PCV for HSV-1 and almost comparable for HSV-2 and VZV. In a growth inhibition assay of murine granulocyte-macrophage progenitor cells, A-5021 showed the least inhibitory effect of the three compounds. These results show that A-5021 is a potent and selective antiviral agent against HSV-1, HSV-2, and VZV.
Determination of the Excitatory Potencies of Fluoroquinolones in the Central Nervous System by an In Vitro Model
Authors: Schmuck Gabriele , Schürmann Anja , Schlüter Gerhard
Published: October 2018

Fluoroquinolones have been reported to induce central nervous system side effects, including seizures and psychiatric events. Although relatively rare in patients up to now, the proconvulsant activity depends on the chemical structure and might be a critical endpoint of some new representatives of this valuable class of antimicrobials. The electrophysiological determination of field potentials in the CA
1
region of the rat hippocampus slice allowed an assessment of the excitatory potential of fluoroquinolones and might be predictive for their neurotoxic potency in vivo. An optimization of this method and its extension to other fluoroquinolones resulted in a defined rank order. Well-known already-marketed quinolones as well as some fluoroquinolones under evaluation and development were used. The dose range tested was between 0.5 and 4 μmol/liter, which was comparable to the therapeutic concentration in the brain. All tested compounds increased the population spike amplitude in a concentration-dependent manner, and the resulting excitatory potency was highly dependent on the chemical structure, with compounds ranging from least to most excitatory as follows: ofloxacin, ciprofloxacin, nalidixic acid, moxifloxacin (= BAY × 8843), fleroxacin, lomefloxacin, enoxacin, clinafloxacin (much more excitatory than enoxacin), tosufloxacin, trovafloxacin, BAY 15-7828, and BAY × 9181 (much more excitatory than BAY 15-7828). The proposed hippocampus slice model not only is suitable for giving valuable alerts as to convulsive potential during candidate selection but also enables mechanistic investigations. These investigations pointed to the
N
-methyl-
d
-aspartate receptor as the probable target of the fluoroquinolone effects.
Dosing of Aminoglycosides To Rapidly Attain Pharmacodynamic Goals and Hasten Therapeutic Response by Using Individualized Pharmacokinetic Monitoring of Patients with Pneumonia Caused by Gram-Negative Organisms
Authors: Kashuba A. D. M. , Bertino J. S. , Nafziger A. N.
Published: October 2018

Achieving a peak aminoglycoside concentration (
C
max
)/MIC of ≥10 within 48 h of initiation of therapy for pneumonia caused by gram-negative organisms results in a 90% probability of therapeutic response by day 7. Targeting an MIC of 1 μg/ml, empirical aminoglycoside loading doses of 348 (25th- to 75th-percentile range, 275 to 432) mg were calculated to obtain a
C
max
/MIC of 10 in our patient population. Individualized pharmacokinetic monitoring coupled with MIC data should determine subsequent dosing regimens to minimize the potential for toxicity and maximize the probability of clinical response.
Antimicrobial Susceptibility Testing of 59 Strains of
Campylobacter fetus
subsp.
fetus
Authors: Tremblay Carole , Gaudreau Christiane
Published: October 2018

The susceptibilities of 59
Campylobacter fetus
subsp.
fetus
isolates to eight antibiotics were studied by the agar dilution, E-test, and disk diffusion methods. None of the isolates were β-lactamase producers. All were susceptible to ampicillin, gentamicin, imipenem, and meropenem as determined by the three methods, with MICs at which 90% of the isolates are inhibited (MIC
90
s) (determined by agar dilution) of 2, 1, ≤0.06, and 0.12 μg/ml, respectively. Twenty-seven percent of the isolates were resistant to tetracycline, with complete agreement between the agar dilution and disk diffusion results. The MIC
90
s determined by agar dilution were 2 μg/ml for erythromycin, 1 μg/ml for ciprofloxacin, and 8 μg/ml for cefotaxime.
Energy-Dependent Accumulation of Fluoroquinolones in Quinolone-Resistant
Klebsiella pneumoniae
Strains
Authors: Martínez-Martínez Luis , García Isabel , Ballesta Sofía , Benedí Vicente Javier , Hernández-Allés Santiago , Pascual Alvaro
Published: October 2018

The intracellular accumulation of norfloxacin and pefloxacin in
Klebsiella pneumoniae
was evaluated. The roles of lipopolysaccharide, capsule, and outer membrane proteins were not important for the intrabacterial accumulation of fluoroquinolones in isogenic strains with known outer membrane alterations. In fluoroquinolone-resistant clinical isolates also expressing GyrA alterations, an active efflux leading to decreased accumulation of the drugs enhanced their resistance to these agents.
In Vitro and In Vivo Activities of Moxifloxacin and Clinafloxacin against
Mycobacterium tuberculosis
Authors: Ji Baohong , Lounis Nacer , Maslo Caroline , Truffot-Pernot Chantal , Bonnafous Pascale , Grosset Jacques
Published: October 2018

On 10% oleic acid–albumin–dextrose–catalase-enriched 7H11 agar medium, the MIC at which 90% of the isolates are inhibited for 20 strains of
Mycobacterium tuberculosis
was 0.5 μg of sparfloxacin (SPFX) or moxifloxacin (MXFX) per ml and 1.0 μg of clinafloxacin (CNFX) per ml, indicating that the in vitro activities of SPFX and MXFX were virtually identical and were slightly greater than that of CNFX. However, the in vivo activities of these drugs in a murine tuberculosis model differed considerably. Female Swiss mice were infected intravenously with 6.2 × 10
6
CFU of the H37Rv strain and treated for 4 weeks, beginning the next day after infection, with isoniazid (INH) serving as the positive control. By the criteria of 30-day survival rate, spleen weight, gross lung lesion, and mean number of CFU in the spleen, treatment with CNFX at up to 100 mg/kg of body weight six times weekly displayed no measurable effect against
M. tuberculosis
, whereas both SPFX and MXFX were effective; administration six times weekly of either of the latter two drugs demonstrated dosage-dependent bactericidal effects, as measured by enumeration of CFU in the spleens, and MXFX appeared more bactericidal than the same dosage of SPFX. Of the three fluoroquinolones, only MXFX at 100 mg/kg six times weekly appeared as bactericidal as INH at 25 mg/kg six times weekly. Thus, MXFX may be an important component of the newer combined regimens for treatment of tuberculosis.
Effects of Mutations in GrlA of Topoisomerase IV from
Staphylococcus aureus
on Quinolone and Coumarin Activity
Authors: Fournier Bénédicte , Hooper David C.
Published: October 2018

The
grlA
genes of
Staphylococcus aureus
ISP794 (wild type), MT5224c4 (
grlA
[Phe-80]), MT5224c2 (
grlA
[Pro-116]), and MT111 (
grlA
[Glu-116]) were cloned in pSK950, a shuttle vector, and introduced into
S. aureus
strains derived from strain RN4220. The mutations at position 116 of GrlA (Ala→Pro or Glu) caused an increase in the level of fluoroquinolone resistance and a decrease in the level of coumarin susceptibility, whereas the mutation at position 80 (Ser→Phe) caused only an increase in the level of fluoroquinolone resistance. In multicopy alleles, both types of mutations were codominant for fluoroquinolone resistance, and mutations at position 116 were also codominant for coumarin resistance.
Inhibition of Human Immunodeficiency Virus Type 1 Infection in SCID-hu Thy/Liv Mice by the G-Quartet-Forming Oligonucleotide, ISIS 5320
Authors: Stoddart Cheryl A. , Rabin Linda , Hincenbergs Mara , Moreno Mary e. , Linquist-Stepps Valerie , Leeds Janet M. , Truong Lo Anne , Wyatt J. R. , Ecker David J. , McCune Joseph M.
Published: October 2018

Viral replication was inhibited in a dose-dependent manner after administration of the phosphorothioate oligonucleotide TTGGGGTT (ISIS 5320) to human immunodeficiency virus type 1 (HIV-1)-infected SCID-hu Thy/Liv mice. Potent in vivo antiviral activity was observed against the T-cell-tropic molecular clone NL4-3; the agent was found to have weak activity against one primary HIV-1 isolate, and the agent was inactive against a second primary isolate.
In Vitro Activities of the Ketolide HMR 3647 against Recent Gram-Positive Clinical Isolates and
Haemophilus influenzae
Authors: Barry Arthur L. , Fuchs Peter C. , Brown Steven D.
Published: October 2018

The ketolide HMR 3647 (previously RU 66647) was evaluated against 2,563 recent clinical isolates of gram-positive pathogens and 200
Haemophilus influenzae
isolates. HMR 3647 was active against macrolide-resistant streptococci, including pneumococci, but was not active against macrolide- or lincosamide-resistant staphylococci. Against
H. influenzae
, the potency of HMR 3647 was similar to that of azithromycin.
Fungal β-Tubulin, Expressed as a Fusion Protein, Binds Benzimidazole and Phenylcarbamate Fungicides
Authors: Hollomon Derek W. , Butters Jenny A. , Barker Helen , Hall Len
Published: October 2018

Benzimidazoles are important antitubulin agents used in veterinary medicine and plant disease control. Resistance is a practical problem correlated with single amino acid changes in β-tubulin and is often linked to greater sensitivity to phenylcarbamates. This negative cross-resistance creates opportunities for durable antiresistance strategies. Attempts to understand the molecular basis of benzimidazole resistance have been hampered by the inability to purify tubulin from filamentous fungi. We have overcome some of these problems by expressing β-tubulin as a fusion with a maltose binding protein. This fusion protein is soluble, and we confirm for the first time using a gel filtration assay that benzimidazoles indeed bind to β-tubulin. This binding is reduced by the mutation Glu
198
→Gly
198
, which also confers resistance. Binding of phenylcarbamates is the complete opposite, reflecting their biological activity and the negative cross-resistance. This suggests that the fungicide binding sites fold correctly in the fusion protein.
Sparfloxacin Resistance in Clinical Isolates of
Streptococcus pneumoniae
: Involvement of Multiple Mutations in
gyrA
and
parC
Genes
Authors: Taba Hideki , Kusano Nobuchika
Published: October 2018

Antimicrobial susceptibility testing revealed among 150 clinical isolates of
Streptococcus pneumoniae
4 pneumococcal isolates with resistance to fluoroquinolones (MIC of ciprofloxacin, ≥32 μg/ml; MIC of sparfloxacin, ≥16 μg/ml). Gene amplification and sequencing analysis of
gyrA
and
parC
revealed nucleotide changes leading to amino acid substitutions in both GyrA and ParC of all four fluoroquinolone-resistant isolates. In the case of strains 182 and 674 for which sparfloxacin MICs were 16 and 64 μg/ml, respectively, nucleotide changes were detected at codon 81 in
gyrA
and codon 79 in
parC
; these changes led to an Ser→Phe substitution in GyrA and an Ser→Phe substitution in ParC. Strains 354 and 252, for which sparfloxacin MICs were 128 μg/ml, revealed multiple mutations in both
gyrA
and
parC
. These strains exhibited nucleotide changes at codon 85 leading to a Glu→Lys substitution in GyrA, in addition to Ser-79→Tyr and Lys-137→Asn substitutions in ParC. Moreover, strain 252 showed additional nucleotide changes at codon 93, which led to a Trp→Arg substitution in GyrA. These results suggest that sparfloxacin resistance could be due to the multiple mutations in GyrA and ParC. However, it is possible that other yet unidentified mutations may also be involved in the high-level resistance to fluoroquinolones in
S. pneumoniae
.
Molecular Mode of Action of the Antifungal β-Amino Acid BAY 10-8888
Authors: Ziegelbauer Karl , Babczinski Peter , Schönfeld Wolfgang
Published: October 2018

BAY 10-8888 is a cyclic β-amino acid that is related to cispentacin and that has antifungal activity.
Candida albicans
cells accumulated BAY 10-8888 intracellularly to a concentration about 200 that in the medium when grown in media with a variety of nitrogen sources. In complex growth medium, BAY 10-8888 transport activity was markedly reduced and was paralleled by a decrease in its antifungal activity. Uptake of BAY 10-8888 was mediated by an H
+
-coupled amino acid transporter with specificity for branched-chain amino acids (isoleucine, leucine, and valine) and showed a

K
T

(Michaelis constant of the transport reaction) of 0.95 mM and a
V
max
of 18.9 nmol × min
−1
× 10
7
cells
−1
. Similar to the transport of natural amino acids in
Saccharomyces cerevisiae
, the transport of BAY 10-8888 into the cell was unidirectional. Efflux occurred by diffusion and was not carrier mediated. Inside the cell BAY 10-8888 inhibited specifically isoleucyl-tRNA synthetase, resulting in inhibition of protein synthesis and cell growth. Intracellular isoleucine reversed BAY 10-8888-induced growth inhibition. BAY 10-8888 was not incorporated into proteins. BAY 10-8888 inhibited isoleucyl-tRNA synthetase with the same concentration dependency as protein biosynthesis in intact cells assuming 200-fold accumulation.
In Vitro Effectiveness of Povidone-Iodine on
Acanthamoeba
Isolates from Human Cornea
Authors: Gatti Simonetta , Cevini Claudia , Bruno Antonella , Penso Gabriella , Rama Paolo , Scaglia Massimo
Published: October 2018

Acanthamoeba
keratitis is a severe ocular infection secondary to accidental macro- or microscopic trauma of the cornea. Starting in 1985, a dramatic increase of this infection was recorded along with the spread of contact lens use. This protozoal disease is difficult to treat because of the scarcity of efficacious topical and systemic drugs. We evaluated the in vitro effectiveness of povidone-iodine (PVP-I [Betadine]), an agent with broad antibacterial and antiviral activity, compared to that of chlorhexidine (CXD), a cationic antiseptic, on
Acanthamoeba
isolates from patients with amebic keratitis. The results showed that PVP-I solution from 0.5 to 2.5% has a better antiamebic activity both on trophic and cystic stages of
Acanthamoeba
spp. than does CXD.
Effects of α-Thalassemia on Pharmacokinetics of the Antimalarial Agent Artesunate
Authors: Ittarat Wanida , Looareesuwan Sornchai , Pootrakul Pensri , Sumpunsirikul Petchmanee , Vattanavibool Phantip , Meshnick Steven R.
Published: October 2018

Thalassemia is common in Southeast Asia, where artemisinin derivatives are frequently used in the treatment of malaria. It has been previously reported that artemisinin derivatives can be concentrated by uninfected thalassemic erythrocytes in vitro but not by normal erythrocytes. As a follow-up to this report, we studied the antimalarial kinetics of intravascular artesunate (2.4 mg/kg of body weight) in 10 persons with normal hemoglobins and in 10 patients with thalassemia (2 with α-thalassemia type 1–hemoglobin Constant Spring and 8 with α-thalassemia type 1–α-thalassemia type 2). Concentrations of artesunate and its active metabolites in plasma were measured by bioassay and expressed relative to those of dihydroartemisinin, the major biologically active metabolite. Concentrations of intravascular artesunate in plasma peaked in both the normal individuals and the thalassemic individuals 15 min after injection (the first time point). Plasma drug concentrations at all time intervals, except that at 1 h, were significantly higher in thalassemic subjects than in normal subjects (
P
< 0.05). The area under the concentration-time curve was 9-fold higher (
P
< 0.001) and the volume of distribution at steady state was 15-fold lower (
P
< 0.001) in thalassemic than in normal subjects. In light of the potential neurotoxicity of artemisinin derivatives, these results suggest that thalassemic subjects may need a drug administration regimen different from that of normal patients.
Safety, Pharmacokinetics, and Antiretroviral Activity of Intravenous 9-[2-(
R
)-(Phosphonomethoxy)propyl]adenine, a Novel Anti-Human Immunodeficiency Virus (HIV) Therapy, in HIV-Infected Adults
Authors: Deeks Steven G. , Barditch-Crovo Patricia , Lietman Paul S. , Hwang Frances , Cundy Kenneth C. , Rooney James F. , Hellmann Nicholas S. , Safrin Sharon , Kahn James O.
Published: October 2018

9-[2-(
R
)-(Phosphonomethoxy)propyl]adenine (PMPA) is a nucleotide analogue with potent antiretroviral activity in vitro and in simian models. A randomized, double-blind, placebo-controlled, dose-escalation clinical trial of intravenous PMPA monotherapy was conducted in 20 human immunodeficiency virus (HIV)-infected adults with CD4 cell counts of ≥200 cells/mm
3
and plasma HIV RNA levels of ≥10,000 copies/ml. Two dose levels were evaluated (1 and 3 mg/kg of body weight/day). Ten subjects were enrolled at each dose level (eight randomized to receive PMPA and two randomized to receive placebo). On day 1, a single dose of PMPA or placebo was administered by intravenous infusion. Beginning on study day 8, PMPA or placebo was administered once daily for an additional 7 consecutive days. All subjects tolerated dosing without significant adverse events. Mean peak serum PMPA concentrations were 2.7 ± 0.9 and 9.1 ± 2.1 μg/ml in the 1- and 3-mg/kg cohorts, respectively. Serum concentrations declined in a biexponential fashion, with a terminal half-life of 4 to 8 h. At 3 mg/kg/day, a single infusion of PMPA resulted in a 0.4 log
10
median decline in plasma HIV RNA by study day 8. Following 7 consecutive days of study drug administration thereafter, the median changes in plasma HIV RNA from baseline were −1.1, −0.6, and 0.1 log
10
in the 3-mg/kg/day, 1-mg/kg/day, and placebo dose groups, respectively. Following the final dose in the 3-mg/kg/day cohort, the reduction in HIV RNA was sustained for 7 days before returning toward baseline. Further studies evaluating an oral prodrug of PMPA are under way.
Safety, Tolerance, and Pharmacokinetics of a Small Unilamellar Liposomal Formulation of Amphotericin B (AmBisome) in Neutropenic Patients
Authors: Walsh Thomas J. , Yeldandi Vijay , McEvoy Maureen , Gonzalez Corina , Chanock Stephen , Freifeld Alison , Seibel Nita I. , Whitcomb Patricia O. , Jarosinski Paul , Boswell Garry , Bekersky Ihor , Alak Ala , Buell Donald , Barret John , Wilson Wyndham
Published: October 2018

The safety, tolerance, and pharmacokinetics of a small unilamellar liposomal formulation of amphotericin B (AmBisome) administered for empirical antifungal therapy were evaluated for 36 persistently febrile neutropenic adults receiving cancer chemotherapy and bone marrow transplantation. The protocol was an open-label, sequential-dose-escalation, multidose pharmacokinetic study which enrolled a total of 8 to 12 patients in each of the four dosage cohorts. Each cohort received daily doses of either 1.0, 2.5, 5.0, or 7.5 mg of amphotericin B in the form of AmBisome/kg of body weight. The study population consisted of patients between the ages of 13 and 80 years with neutropenia (absolute neutrophil count, <500/mm
3
) who were eligible to receive empirical antifungal therapy. Patients were monitored for safety and tolerance by frequent laboratory examinations and the monitoring of infusion-related reactions. Efficacy was assessed by monitoring for the development of invasive fungal infection. The pharmacokinetic parameters of AmBisome were measured as those of amphotericin B by high-performance liquid chromatography. Noncompartmental methods were used to calculate pharmacokinetic parameters. AmBisome administered as a 1-h infusion in this population was well tolerated and was seldom associated with infusion-related toxicity. Infusion-related side effects occurred in 15 (5%) of all 331 infusions, and only two patients (5%) required premedication. Serum creatinine, potassium, and magnesium levels were not significantly changed from baseline in any of the dosage cohorts, and there was no net increase in serum transaminase levels. AmBisome followed a nonlinear dosage relationship that was consistent with reticuloendothelial uptake and redistribution. There were no breakthrough fungal infections during empirical therapy with AmBisome. AmBisome administered to febrile neutropenic patients in this study was well tolerated, was seldom associated with infusion-related toxicity, was characterized by nonlinear saturation kinetics, and was effective in preventing breakthrough fungal infections.
Evaluation of Several Dosing Regimens of Cefepime, with Various Simulations of Renal Function, against Clinical Isolates of
Pseudomonas aeruginosa
in a Pharmacodynamic Infection Model
Authors: Cappelletty Diane M.
Published: October 2018

The objectives of this study were as follows: (i) to examine the killing activity of 2-g doses of cefepime against two clinical isolates (mucoid and nonmucoid) of
Pseudomonas aeruginosa
in a pharmacodynamic in vitro infection model, (ii) to compare the percentage of time above the MIC (T > MIC) for each of the regimens against
P. aeruginosa
, and (iii) to evaluate the area under the bactericidal curve for each regimen. Cefepime was administered at intervals of 8, 12, and 24 h with and without tobramycin, and two different levels of renal function were simulated: normal (creatinine clearance [CL
CR
] = 90 ml/min) and decreased (CR
CL
= 60 ml/min). Also, the killing activity of cefepime with and without tobramycin was compared to the killing activity of ceftazidime (2 g every 8 h) with and without tobramycin. The T > MIC was 100% in the central chamber except for the regimen in which cefepime was administered every 12 h and the CL
CR
was 90 ml/min, which provided concentrations above the MIC for 92% of the dosing interval against the C31 (mucoid; MIC of cefepime, 4 μg/ml) isolate and for 75% of the interval against the C34 (nonmucoid; MIC of cefepime, 8 μg/ml) isolate. All cefepime and ceftazidime monotherapy simulations resulted in 99.9% killing of the nonmucoid isolate within 4 to 8 h and within 4 to 6 h, respectively. Against the mucoid isolate, 99.9% killing was achieved only with combination therapy. The results of this study indicate that cefepime dosed at 2 g every 12 h under conditions of normal renal function and every 24 h with decreased creatinine clearance (60 ml/min) is effective both as monotherapy and in combination therapy against a nonmucoid strain of
P. aeruginosa
. With cefepime MICs of 4 and 8 μg/ml, the single-agent regimens provided T > MIC values in the central chamber for 92 and ≥75% of the dosing interval against the mucoid and nonmucoid isolates, respectively. Cefepime dosed at 2 g every 12 h, with a creatinine clearance of 90 ml/min, and every 24 h, with a creatinine clearance of 60 ml/min, resulted in killing activity equivalent to that of ceftazidime dosed at 2 g every 8 h. None of the monotherapies provided adequate killing of the mucoid strain of
P. aeruginosa
despite drug concentrations being above the MIC for ≥92% of all dosing intervals. Finally, combination therapy with tobramycin and either cefepime or ceftazidime enhanced the killing of both the mucoid and nonmucoid
P. aeruginosa
isolates.
Pharmacokinetics of Quinupristin-Dalfopristin in Continuous Ambulatory Peritoneal Dialysis Patients
Authors: Johnson Curtis A. , Taylor Claude A. , Zimmerman Stephen W. , Bridson William E. , Chevalier Paul , Pasquier Olivier , Baybutt Robert I.
Published: October 2018

Quinupristin-dalfopristin may be useful for treatment of organisms causing peritoneal dialysis-related peritonitis, including methicillin-resistant coagulase-negative staphylococci, methicillin-resistant
Staphylococcus aureus
, and vancomycin-resistant enterococci. The pharmacokinetic profiles of single intravenous doses of this combination streptogramin antibiotic of 7.5 mg/kg of body weight were characterized for eight noninfected patients receiving continuous ambulatory peritoneal dialysis. Comparison was made to pharmacokinetic profiles determined for eight healthy volunteers matched by age, sex, and race. Drug was measured in dialysate up to 6 h following the dose. Plasma and dialysate were assayed for parent compounds and metabolites. Mean pharmacokinetic parameters were compared between groups. No statistically significant differences were observed between groups for maximal concentrations in plasma, times to maximal concentration, areas under the curve, distribution volumes, rates of total body clearance, or half-lives in plasma for quinupristin and dalfopristin. No statistically significant differences were observed in maximal concentrations in plasma, times to maximal concentration, areas under the curve, or half-lives for cysteine, the glutathione conjugates of quinupristin, or the pristinamycin IIA metabolite of dalfopristin. The measurements in dialysate of the parent and most metabolites were below the expected MICs. Dialysis clearance was insignificant. Quinupristin-dalfopristin was well tolerated in both groups, causing only mild adverse events that resolved prior to discharge from the study. The disposition of quinupristin, dalfopristin, or their primary metabolites following a single dose was unaltered in patients receiving peritoneal dialysis. Intravenous dosing of this antibiotic combination is unlikely to be adequate for the treatment of peritonitis associated with peritoneal dialysis.
In Vitro Activities of Six New Fluoroquinolones against
Brucella melitensis
Authors: Trujillano-Martín Ignacio , García-Sánchez Enrique , Martínez Isaías Montes , Fresnadillo María José , García-Sánchez José Elías , García-Rodríguez José Ángel
Published: October 2018

We have tested the in vitro activities of eight fluoroquinolones against 160
Brucella melitensis
strains. The most active was sitafloxacin (MIC at which 90% of the isolates are inhibited [MIC
90
], 0.12 μg/ml). In decreasing order, the activities (MIC
90
s) of the rest of the tested fluoroquinolones were as follows: levofloxacin, 0.5 μg/ml; ciprofloxacin, trovafloxacin, and moxifloxacin, 1 μg/ml; and ofloxacin, grepafloxacin, and gatifloxacin, 2 μg/ml.
Molecular Basis of Rifampin Resistance inStreptococcus pneumoniae
Authors: Padayachee Thanugarani , Klugman Keith P.
Published: October 2018

Rifampin resistance among South African clinical isolates ofStreptococcus pneumoniaewas shown to be due to missense mutations within therpoBgene. Sequence analysis of 24 rifampin-resistant isolates revealed the presence of mutations within cluster I as well as novel mutations in an area designated pneumococcus cluster III. Of the 24 isolates characterized, only 1 resistant isolate did not contain any mutations in the regions sequenced. Either the cluster I or the cluster III mutations separately conferred MICs of 32 to 128 μg/ml. Clinical isolate 55, for which the MIC was 256 μg/ml, was noted to contain 9 of the 10 mutations identified, which included the cluster I and cluster III mutations. As inEscherichia coli, it is possible that cluster I (amino acids 406 to 434) and cluster III (amino acids 523 to 600) ofS. pneumoniaeinteract to form part of the antibiotic binding site, thus accounting for the very high MIC observed for isolate 55. PCR products containing cluster I or cluster III mutations were able to transform rifampin-susceptibleS. pneumoniaeto resistance. Although many of the isolates studied displayed identical sequences, pulsed-field gel electrophoresis analysis revealed that the isolates were not of clonal origin.
Multiple Novel Inhibitors of the NorA Multidrug Transporter of
Staphylococcus aureus
Authors: Markham Penelope N. , Westhaus Eric , Klyachko Katya , Johnson Michael E. , Neyfakh Alex A.
Published: October 2018

The multidrug transporter NorA contributes to the resistance of
Staphylococcus aureus
to fluoroquinolone antibiotics by promoting their active extrusion from the cell. Previous studies with the alkaloid reserpine, the first identified inhibitor of NorA, indicate that the combination of a chemical NorA inhibitor with a fluoroquinolone could improve the efficacy of this class of antibiotics. Since reserpine is toxic to humans at the concentrations required to inhibit NorA, we sought to identify new inhibitors of NorA that may be used in a clinical setting. Screening of a chemical library yielded a number of structurally diverse inhibitors of NorA that were more potent than reserpine. The new inhibitors act in a synergistic manner with the most widely used fluoroquinolone, ciprofloxacin, by substantially increasing its activity against both NorA-overexpressing and wild-type
S. aureus
isolates. Furthermore, the inhibitors dramatically suppress the emergence of ciprofloxacin-resistant
S. aureus
upon in vitro selection with this drug. Some of these new inhibitors, or their derivatives, may prove useful for augmentation of the antibacterial activities of fluoroquinolones in the clinical setting.