bluuebunny/crossref_metadata_2025 huggingface dataset:
Roles of Amino Acids 161 to 179 in the PSE-4 Ω Loop in Substrate Specificity and in Resistance to Ceftazidime
Authors: Therrien Christian , Sanschagrin Francois , Palzkill Timothy , Levesque Roger C.
Published: October 2018

The PSE-4 enzyme is a prototype carbenicillin-hydrolyzing enzyme exhibiting high activity against penicillins and early cephalosporins. To understand the mechanism that modulates substrate profiles and to verify the ability of PSE-4 to extend its substrate specificity toward expanded-spectrum cephalosporins, we used random replacement mutagenesis to generate six random libraries from amino acids 162 to 179 in the Ω loop. This region is known from studies with TEM-1 to be implicated in substrate specificity. It was found that the mechanism modulating ceftazidime hydrolysis in PSE-4 was different from that in TEM-1. The specificity of class 2c carbenicillin-hydrolyzing enzymes could not be assigned to the Ω loop of PSE-4. Analysis of the percentage of functional enzymes revealed that the hydrolysis of ampicillin was more affected than hydrolysis of carbenicillin by amino acid substitutions at positions 162 to 164 and 165 to 167.
Targeted Antimicrobial Photochemotherapy
Authors: Soukos Nikolaos S. , Ximenez-Fyvie Laurie Ann , Hamblin Michael R. , Socransky Sigmund S. , Hasan Tayyaba
Published: October 2018

This study explores a new approach for antimicrobial therapy with light activation of targeted poly-
l
-lysine (pL)–chlorin
e6
(c

e6

) conjugates. The goal was to test the hypothesis that these conjugates between pL and c

e6

would efficiently target photodestruction towards gram-positive (
Actinomyces viscosus
) and gram-negative (
Porphyromonas gingivalis
) oral species while sparing an oral epithelial cell line (HCPC-1). Conjugates of c

e6

with pL (average molecular weight, 2,000) having a positive, neutral, or negative charge were prepared. Illumination with red light (λ
max
= 671 nm) from a diode array produced a dose-dependent loss of CFU from the bacteria, under conditions that did not affect the viability of the epithelial cells. For
P. gingivalis
, the cationic conjugate produced 99% killing, while the neutral conjugate killed 91% and the anionic conjugate killed 76% after 1 min of incubation and exposure to red light for 10 min. For
A. viscosus
, the cationic conjugate produced >99.99% killing while HCPC-1 cells remained intact. The importance of the positive charge was shown by the effectiveness of c

e6

-monoethylenediamine monoamide (a monocationic derivative of c

e6

) in killing both bacteria. The clinically employed benzoporphyrin derivative under the same conditions killed epithelial cells while leaving
P. gingivalis
relatively unharmed. A mixture of c

e6

with pL did not show phototoxicity comparable with that of the cationic conjugate. These results were explained by the selective uptake of the conjugates by bacteria (20- to 100-fold) compared to that by mammalian cells, while free c

e6

showed much less selectivity for bacteria (5- to 20-fold). The data suggest that the cationic pL-c

e6

conjugate may have an application for the photodynamic therapy of periodontal disease.
Stability of Cephalosporin Prodrug Esters in Human Intestinal Juice: Implications for Oral Bioavailability
Authors: Stoeckel Klaus , Hofheinz Werner , Laneury Jean Paul , Duchene Patrick , Shedlofsky Steve , Blouin Robert A.
Published: October 2018

The levels of degradation of cefetamet pivoxil (CAT), cefuroxime axetil (CAE), and cefpodoxime proxetil (CPD) in 0.6 M phosphate buffer (pH 7.4) and human intestinal juice (pH 7.4) at 37°C over 24 h were compared. Significant differences in the time courses of degradation and in the patterns of degradation products were observed. (i) The relative proportions of the Δ2- and Δ3-cephalosporins were roughly reversed in the two incubation media. In phosphate buffer, the major degradation product was the Δ2-cephalosporin (CAT = 61%; CAE = 74%; CPD = 85%), while in intestinal juice it was the Δ3-cephalosporin (CAT = 86%; CAE = 75%; CPD = 87%). (ii) Generally, the degradation of the prodrug esters progressed faster in intestinal juice than in phosphate buffer (e.g., for CAT the half-lives [
t
1/2
s] were 0.78 and 4.3 h, respectively). (iii) The two diastereoisomers of CAE and CPD were degraded at different rates in intestinal juice (for the CAE diasteroisomers,
t
1/2
s = 0.37 and 0.93 h; for the CPD diastereoisomers,
t
1/2
s = 0.18 and 0.98 h) but were degraded at similar rates in phosphate buffer (for the CAE diastereoisomers,
t
1/2
= 1.6 h; for the CPD
t
1/2
diastereoisomers, = 2.2 h). It is concluded that (i) the Δ2 isomerization does not significantly affect the bioavailability of prodrug esters since enzymatic hydrolysis in the intestinal fluid proceeds mainly to the active Δ3-cephalosporin and (ii) the high degree of stereoselectivity of the enzymatic ester hydrolysis should make it possible to increase the bioavailabilities of certain prodrug esters (CAE, CPD) by using the more stable diasterioisomer.
Genotypic and Phenotypic Characterization of Human Immunodeficiency Virus Type 1 Variants Isolated from Patients Treated with the Protease Inhibitor Nelfinavir
Authors: Patick A. K. , Duran M. , Cao Y. , Shugarts D. , Keller M. R. , Mazabel E. , Knowles M. , Chapman S. , Kuritzkes D. R. , Markowitz M.
Published: October 2018

Nelfinavir mesylate (formerly AG1343) is a potent and selective inhibitor of human immunodeficiency virus (HIV) protease approved for the treatment of individuals infected with HIV. Nucleotide sequence analysis of protease genes from plasma HIV type 1 (HIV-1) RNA revealed a unique aspartic acid (D)-to-asparagine (N) substitution at residue 30 (D30N) in 25 of 55 patients treated with nelfinavir for a median of 13 weeks. Although the appearance of D30N was occasionally associated with concurrent or sequential emergence of other changes (e.g., at residues 35, 36, 46, 71, 77, and 88), genotypic changes associated with phenotypic resistance to other protease inhibitors were not observed (e.g., at residues 48, 50, 82, and 84) or were only rarely observed (e.g., at residue 90). In phenotypic assays, viral isolates with high-level resistance to nelfinavir remained susceptible to indinavir, saquinavir, ritonavir, and amprenavir (formerly VX-478/141W94). Similar results were observed in phenotypic assays utilizing HIV-1 NL4-3, which contained the D30N substitution alone or in combination with substitutions at other residues (e.g., residues 46, 71, and 88). These data indicate that the initial pathway of resistance to nelfinavir is unique and suggest that individuals failing short courses of nelfinavir-containing regimens may respond to regimens containing other protease inhibitors.
Translation Elongation Factor 2 Is Part of the Target for a New Family of Antifungals
Authors: Capa Laura , Mendoza Alfonso , Lavandera José L. , Gómez de las Heras Federico , García-Bustos José F.
Published: October 2018

Translation elongation factor 2 (EF2), which in
Saccharomyces cerevisiae
is expressed from the
EFT1
and
EFT2
genes, has been found to be targeted by a new family of highly specific antifungal compounds derived from the natural product sordarin. Two complementation groups of mutants resistant to the semisynthetic sordarin derivative GM193663 were found. The major one (21 members) consisted of isolates with mutations on
EFT2
. The minor one (four isolates) is currently being characterized but it is already known that resistance in this group is not due to mutations on
EFT1
, pointing to the complex structure of the functional target for these compounds. Mutations on EF2 clustered, forming a possible drug binding pocket on a three-dimensional model of EF2, and mutant cell extracts lost the capacity to bind to the inhibitors. This new family of antifungals holds the promise to be a much needed and potent addition to current antimicrobial treatments, as well as a useful tool for dissection of the elongation process in ribosomal protein synthesis.
Visceral Leishmaniasis in the BALB/c Mouse: A Comparison of the Efficacy of a Nonionic Surfactant Formulation of Sodium Stibogluconate with Those of Three Proprietary Formulations of Amphotericin B
Authors: Mullen A. B. , Baillie A. J. , Carter K. C.
Published: October 2018

In this study, treatment efficacies of a nonionic surfactant vesicle formulation of sodium stibogluconate (SSG-NIV) and of several formulations of amphotericin B were compared in a murine model of visceral leishmaniasis. Treatment with multiple doses of AmBisome, Abelcet, and Amphocil (total dose, 12.5 mg of amphotericin B/kg of body weight) resulted in a significant suppression of parasite burdens in liver (
P
< 0.0005) and spleen (
P
< 0.0005) compared with those of controls, with Abelcet having the lowest activity. Only AmBisome and Amphocil gave significant suppression of parasites in bone marrow (compared to control values,
P
< 0.005). In the acute-infection model, single-dose treatments of SSG-NIV (296 mg of Sb
V
/kg), SSG solution (296 mg of Sb
V
/kg), or AmBisome (8 mg of amphotericin B/kg) were equally effective against liver parasites (compared to control values,
P
< 0.0005). SSG-NIV and AmBisome treatment also significantly suppressed parasites in bone marrow and spleen (
P
< 0.005), with SSG-NIV treatment being more suppressive (>98% suppression in all three sites). Free-SSG treatment failed to suppress spleen or bone marrow parasites. Infection status influenced treatment outcome. In the chronic-infection model, the AmBisome single-dose treatment was less effective in all three infection sites and the SSG-NIV single-dose treatment was less effective in the spleen. The results of this study suggest that the antileishmanial efficacy of SSG-NIV compares favorably with those of the novel amphotericin B formulations.
Characterization of IS
18
, an Element Capable of Activating the Silent
aac(6′)-Ij
Gene of
Acinetobacter
sp. 13 Strain BM2716 by Transposition
Authors: Rudant Eric , Courvalin Patrice , Lambert Thierry
Published: October 2018

Insertion sequence IS
18
was detected by analysis of the spontaneous aminoglycoside resistant mutant
Acinetobacter
sp. 13 strain BM2716-1. Insertion of the element upstream from the silent acetyltransferase gene
aac(6′)-Ij
created a hybrid promoter that putatively accounts for the expression of the aminoglycoside resistance gene. The 1,074-bp IS
18
element contained partially matched (20 out of 26 bases) terminal inverted repeats, one of which overlapped the 3′ end of a 935-bp open reading frame potentially encoding a protein related to the transposases of the IS
30
family. IS
18
was found in 6 out of 29 strains of
Acinetobacter
sp. 13 but not in 10 strains each of
A. baumannii
and
A. haemolyticus
.
Description of Two New Isolates of
Streptococcus pneumoniae
in Spain That Are Highly Resistant to Cefotaxime
Authors: Ruiz Joaquín , Sempere Marco , Simarro Encarnación , Fenoll Asunción
Published: October 2018

Two strains of
Streptococcus pneumoniae
isolated from sputum and bronchoalveolar samples with high-level resistance to cefotaxime (MIC = 8 to 16 μg/ml) are described. One of them, belonging to serogroup 19, was also highly resistant to penicillin (MIC = 16 μg/ml), while the other, of serogroup 14, was intermediate in its resistance to penicillin (MIC = 0.25 μg/ml). To our knowledge, these are the first two strains to be isolated in Spain with such high levels of resistance to cefotaxime.
DNA Gyrase and Topoisomerase IV Are Dual Targets of Clinafloxacin Action in
Streptococcus pneumoniae
Authors: Pan Xiao-Su , Fisher L. Mark
Published: October 2018

We examined the response of
Streptococcus pneumoniae
7785 to clinafloxacin, a novel C-8-substituted fluoroquinolone which is being developed as an antipneumococcal agent. Clinafloxacin was highly active against
S. pneumoniae
7785 (MIC, 0.125 μg/ml), and neither
gyrA
nor
parC
quinolone resistance mutations alone had much effect on this activity. A combination of both mutations was needed to register resistance, suggesting that both gyrase and topoisomerase IV are clinafloxacin targets in vivo. The sparfloxacin and ciprofloxacin MICs for the
parC-gyrA
mutants were 16 to 32 and 32 to 64 μg/ml, respectively, but the clinafloxacin MIC was 1 μg/ml, i.e., within clinafloxacin levels achievable in human serum.
S. pneumoniae
7785 mutants could be selected stepwise with clinafloxacin at a low frequency, yielding first-, second-, third-, and fourth-step mutants for which clinafloxacin MICs were 0.25, 1, 6, and 32 to 64 μg/ml, respectively. Thus, high-level resistance to clinafloxacin required four steps. Characterization of the quinolone resistance-determining regions of the
gyrA
,
parC
,
gyrB
, and
parE
genes by PCR,
Hin
fI restriction fragment length polymorphism, and DNA sequence analysis revealed an invariant resistance pathway involving sequential mutations in
gyrA
or
gyrB
, in
parC
, in
gyrA
, and finally in
parC
or
parE
. No evidence was found for other resistance mechanisms. The
gyrA
mutations in first- and third-step mutants altered GyrA hot spots Ser-83 to Phe or Tyr (
Escherichia coli
coordinates) and Glu-87 to Gln or Lys; second- and fourth-step
parC
mutations changed equivalent hot spots Ser-79 to Phe or Tyr and Asp-83 to Ala.
gyrB
and
parE
changes produced novel alterations of GyrB Glu-474 to Lys and of Pro-454 to Ser in the ParE PLRGK motif. Difficulty in selecting first-step gyrase mutants (isolated with 0.125 [but not 0.25] μg of clinafloxacin per ml at a frequency of 5.0 × 10
−10
to 8.5 × 10
−10
) accompanied by the small (twofold) MIC increase suggested only a modest drug preference for gyrase. Given the susceptibility of defined
gyrA
or
parC
mutants, the results suggested that clinafloxacin displays comparable if unequal targeting of gyrase and topoisomerase IV. Dual targeting and the intrinsic potency of clinafloxacin against
S. pneumoniae
and its first- and second-step mutants are desirable features in limiting the emergence of bacterial resistance.
Efficacy of Teicoplanin and Autoradiographic Diffusion Pattern of [
14
C]Teicoplanin in Experimental
Staphylococcus aureus
Infection of Joint Prostheses
Authors: Saleh Mghir Azzam , Claude Crémieux Anne , Bleton Rémy , Ismael Farid , Manteau Michel , Dautrey Sophie , Massias Laurent , Garry Louis , Sales Nicole , Mazière Bernard , Carbon Claude
Published: October 2018

Prosthesis infections are difficult to cure. Infection with methicillin-resistant staphylococci is becoming more common in patients with orthopedic implants. Using a recently developed model of methicillin-resistant
Staphylococcus aureus
(MRSA) infection of a knee prosthesis, we compared the efficacies of teicoplanin and vancomycin. [
14
C]teicoplanin diffusion in this model was also studied by autoradiography. A partial knee replacement was performed with a silicone implant fitting into the intramedullary canal of the tibia, and 10
7
CFU of MRSA was injected into the knee. Treatment with teicoplanin or vancomycin (20 or 60 mg/kg of body weight, respectively, given intramuscularly twice daily) was started 7 days after inoculation and was continued for 7 days. The teicoplanin and vancomycin MICs for MRSA were 1 μg/ml. Mean peak and trough levels in serum were 39.1 and 23.5 μg/ml, respectively, for teicoplanin and 34.4 and 18.5 μg/ml, respectively, for vancomycin. Fifteen days after the end of therapy, the animals were killed and their tibias were removed, pulverized, and quantitatively cultured. Teicoplanin and vancomycin significantly reduced (
P
< 0.05) the bacterial density (2.7 ± 1.3 and 3.3 ± 1.6 log
10
CFU/g of bone, respectively) compared to those for the controls (5.04 ± 1.4 log
10
CFU/g of bone). The bacterial covents of teicoplanin- and vancomycin-treated rabbits were comparable. The [
14
C]teicoplanin autoradiographic diffusion patterns in rabbits with prostheses, two of which were uninfected and two of which were infected, were studied 15 days after inoculation. Sixty minutes after the end of an infusion of 250 μCi of [
14
C]teicoplanin, autoradiography showed that in the infected animals, the highest levels of radioactivity were located around the prosthesis and in the periosteum, bone marrow, and trabecular bone. Radioactivity was less intense in epiphyseal disk cartilage, femoral cartilage, articular ligaments, and muscles and was weak in compact bone. A similar distribution pattern was seen in uninfected rabbits. Thus, teicoplanin may represent an effective alternative therapy for the treatment of these infections.
Subinhibitory Concentrations of Antimicrobial Agents Reduce the Uptake of
Legionella pneumophila
into
Acanthamoeba castellanii
and U937 Cells by Altering the Expression of Virulence-Associated Antigens
Authors: Lück P. Christian , Schmitt Jürgen W. , Hengerer Arne , Helbig Jürgen H.
Published: October 2018

We determined the MICs of ampicillin, ciprofloxacin, erythromycin, imipenem, and rifampin for two clinical isolates of
Legionella pneumophila
serogroup 1 by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay and by quantitative culture. To test the influence of subinhibitory concentrations (sub-MICs) of antimicrobial agents on
Legionella
uptake into
Acanthamoeba castellanii
and U937 macrophage-like cells, both strains were pretreated with 0.25 MICs of the antibiotics for 24 h. In comparison to that for the untreated control, subinhibitory concentrations of antibiotics significantly reduced
Legionella
uptake into the host cells. Measurement of the binding of monoclonal antibodies against several
Legionella
antigens by enzyme-linked immunoassays indicated that sub-MIC antibiotic treatment reduced the expression of the macrophage infectivity potentiator protein (Mip), the Hsp 60 protein, the outer membrane protein (OmpM), an as-yet-uncharacterized protein of 55 kDa, and a few lipopolysaccharide (LPS) epitopes. In contrast, the expression of some LPS epitopes recognized by monoclonal antibodies 8/5 and 30/4 as well as a 45-kDa protein, a 58-kDa protein, and the major outer membrane protein (OmpS) remained unaffected.
Bacteriostatic and Bactericidal In Vitro Activities of Clarithromycin and Erythromycin against Periodontopathic
Actinobacillus actinomycetemcomitans
Authors: Piccolomini R. , Catamo G. , Di Bonaventura G.
Published: October 2018

The susceptibilities of 87 periodontitis-associated strains of
Actinobacillus actinomycetemcomitans
to clarithromycin and erythromycin were determined by standard methodology recommended for
Haemophilus influenzae
. For clarithromycin the MIC at which 90% of the isolates were inhibited was ≤2.0 μg/ml and the minimal bactericidal concentration at which 90% of the strains were killed was ≤4.0 μg/ml, suggesting that it would be a candidate for therapeutic trials in patients with periodontitis.
Postantibiotic Effect and Postantibiotic Sub-MIC Effect of Quinupristin-Dalfopristin against Gram-Positive and -Negative Organisms
Authors: Pankuch Glenn A. , Jacobs Michael R. , Appelbaum Peter C.
Published: October 2018

Quinupristin-dalfopristin produced postantibiotic effects (PAEs) and postantibiotic sub-MIC effects of >2 h against 18 gram-positive cocci. Mean pneumococcal and staphylococcal PAEs were 2.8 and 4.7 h, respectively, with shorter PAEs for constitutively than inducibly macrolide-resistant staphylococci. Mean PAEs for vancomycin-susceptible and -resistant
Enterococcus faecium
were 8.5 and 2.6 h, respectively.
High-Level Resistance to Trimethoprim in Clinical Isolates of
Campylobacter jejuni
by Acquisition of Foreign Genes (
dfr1
and
dfr9
) Expressing Drug-Insensitive Dihydrofolate Reductases
Authors: Gibreel Amera , Sköld Ola
Published: October 2018

The pathogenic bacterium
Campylobacter jejuni
has been regarded as endogenously resistant to trimethoprim. The genetic basis of this resistance was characterized in two collections of clinical isolates of
C. jejuni
obtained from two different parts of Sweden. The majority of these isolates were found to carry foreign
dfr
genes coding for resistant variants of the dihydrofolate reductase enzyme, the target of trimethoprim. The resistance genes, found on the chromosome, were
dfr1
and
dfr9
. In about 10% of the strains, the
dfr1
and
dfr9
genes occurred simultaneously. About 10% of the examined isolates were found to be negative for these
dfr
genes and showed a markedly lower trimethoprim resistance level than the other isolates. The
dfr9
and
dfr1
genes were located in the context of remnants of a transposon and an integron, respectively. Two different surroundings for the
dfr9
gene were characterized. One was identical to the right-hand end of the transposon Tn
5393
, and in the other, the
dfr9
gene was flanked by only a few nucleotides of a Tn
5393
sequence. The insertion of the
dfr9
gene into the
C. jejuni
chromosome could have been mediated by Tn
5393
. The frequent occurrence of high-level trimethoprim resistance in clinical isolates of
C. jejuni
could be related to the heavy exposure of food animals to antibacterial drugs, which could lead to the acquisition of foreign resistance genes in naturally transformable strains of
C. jejuni.
Axenically Grown Amastigotes of
Leishmania infantum
Used as an In Vitro Model To Investigate the Pentavalent Antimony Mode of Action
Authors: Sereno D. , Cavaleyra M. , Zemzoumi K. , Maquaire S. , Ouaissi A. , Lemesre J. L.
Published: October 2018

The mechanism(s) of activity of pentavalent antimony [Sb(V)] is poorly understood. In a recent study, we have shown that potassium antimonyl tartrate, a trivalent antimonial [Sb(III)], was substantially more potent than Sb(V) against both promastigotes and axenically grown amastigotes of three
Leishmania
species, supporting the idea of an in vivo metabolic conversion of Sb(V) into Sb(III). We report that amastigotes of
Leishmania infantum
cultured under axenic conditions were poorly susceptible to meglumine [Glucantime; an Sb(V)], unlike those growing inside THP-1 cells (50% inhibitory concentrations [IC
50
s], about 1.8 mg/ml and 22 μg/ml, respectively). In order to define more precisely the mode of action of Sb(V) agents in vivo, we first induced in vitro Sb(III) resistance by direct drug pressure on axenically grown amastigotes of
L. infantum
. Then we determined the susceptibilities of both extracellular and intracellular chemoresistant amastigotes to the Sb(V)-containing drugs meglumine and sodium stibogluconate plus
m
-chlorocresol (Pentostam). The chemoresistant amastigotes LdiR2, LdiR10, and LdiR20 were 14, 26, and 32 times more resistant to Sb(III), respectively, than the wild-type one (LdiWT). In accordance with the hypothesis described above, we found that intracellular chemoresistant amastigotes were resistant to meglumine [Sb(V)] in proportion to the initial level of Sb(III)-induced resistance. By contrast, Sb(III)-resistant cells were very susceptible to sodium stibogluconate. This lack of cross-resistance is probably due to the presence in this reagent of
m
-chlorocresol, which we found to be more toxic than Sb(III) to
L. infantum
amastigotes (IC
50
s, of 0.54 and 1.32 μg/ml, respectively). Collectively, these results were consistent with the hypothesis of an intramacrophagic metabolic conversion of Sb(V) into trivalent compounds, which in turn became readily toxic to the
Leishmania
amastigote stage.
OXA-16, a Further Extended-Spectrum Variant of OXA-10 β-Lactamase, from Two
Pseudomonas aeruginosa
Isolates
Authors: Danel Franck , Hall Lucinda M. C. , Gur Deniz , Livermore David M.
Published: October 2018

Two extended-spectrum mutants of the class D β-lactamase OXA-10 (PSE-2) from
Pseudomonas aeruginosa
isolates obtained in Ankara, Turkey, were described previously and were designated OXA-11 and -14.
P. aeruginosa
906 and 961, isolated at the same hospital, were highly resistant to ceftazidime (MIC ≥ 128 μg/ml) and produced a β-lactamase with a pI of 6.2. The MICs of ceftriaxone, cefoperazone, cefsulodin, and cefepime were 4- to 16-fold above the typical values for
P. aeruginosa
, whereas the MICs of penicillins and cefotaxime were raised only marginally. Ceftazidime MICs were not significantly reduced by clavulanate or tazobactam at 4 μg/ml. Ceftazidime resistance did not transfer conjugatively but was mobilized to
P. aeruginosa
PU21 by plasmid pUZ8. Both isolates gave similar DNA restriction patterns, suggesting that they were replicates; moreover, they yielded identically sized
Bam
HI fragments that hybridized with a
bla
OXA-10
probe. DNA sequencing revealed that both isolates had the same new β-lactamase, designated OXA-16, which differed from OXA-10 in having threonine instead of alanine at position 124 and aspartate instead of glycine at position 157. The latter change is also present in OXA-11 and -14 and seems critical to ceftazidime resistance. Kinetic parameters showed that OXA-16 enzyme was very active against penicillins, cephaloridine, cefotaxime, and ceftriaxone, but hydrolysis of ceftazidime was not detected despite the ability of the enzyme to confer resistance.
Characterization of Human Influenza Virus Variants Selected In Vitro in the Presence of the Neuraminidase Inhibitor GS 4071
Authors: Tai Chun Y. , Escarpe Paul A. , Sidwell Robert W. , Williams Matthew A. , Lew Willard , Wu Huiwei , Kim Choung U. , Mendel Dirk B.
Published: October 2018

An oral prodrug of GS 4071, a potent and selective inhibitor of influenza neuraminidases, is currently under clinical development for the treatment and prophylaxis of influenza virus infections in humans. To investigate the potential development of resistance during the clinical use of this compound, variants of the human influenza A/Victoria/3/75 (H3N2) virus with reduced susceptibility to the neuraminidase inhibitor GS 4071 were selected in vitro by passaging the virus in MDCK cells in the presence of inhibitor. After eight passages, variants containing two amino acid substitutions in the hemagglutinin (A28T in HA1 and R124M in HA2) but no changes in the neuraminidase were isolated. These variants exhibited a 10-fold reduction in susceptibility to GS 4071 and zanamivir (GG167) in an in vitro plaque reduction assay. After 12 passages, a second variant containing these hemagglutinin mutations and a Lys substitution for the conserved Arg292 of the neuraminidase was isolated. The mutant neuraminidase enzyme exhibited high-level (30,000-fold) resistance to GS 4071, but only moderate (30-fold) resistance to zanamivir and 4-amino-Neu5Ac2en, the amino analog of zanamivir. The mutant enzyme had weaker affinity for the fluorogenic substrate 2′-(4-methylumbelliferyl)-α-
d
- N -acetylneuraminic acid and lower enzymatic activity compared to the wild-type enzyme. The viral variant containing the mutant neuraminidase did not replicate as well as the wild-type virus in culture and was 10,000-fold less infectious than the wild-type virus in a mouse model. These results suggest that although the R292K neuraminidase mutation confers high-level resistance to GS 4071 in vitro, its effect on viral virulence is likely to render this mutation of limited clinical significance.
The Oxazolidinone Linezolid Inhibits Initiation of Protein Synthesis in Bacteria
Authors: Swaney Steve M. , Aoki Hiroyuki , Ganoza M. Clelia , Shinabarger Dean L.
Published: October 2018

The oxazolidinones represent a new class of antimicrobial agents which are active against multidrug-resistant staphylococci, streptococci, and enterococci. Previous studies have demonstrated that oxazolidinones inhibit bacterial translation in vitro at a step preceding elongation but after the charging of
N
-formylmethionine to the initiator tRNA molecule. The event that occurs between these two steps is termed initiation. Initiation of protein synthesis requires the simultaneous presence of
N
-formylmethionine-tRNA, the 30S ribosomal subunit, mRNA, GTP, and the initiation factors IF1, IF2, and IF3. An initiation complex assay measuring the binding of [
3
H]
N
-formylmethionyl-tRNA to ribosomes in response to mRNA binding was used in order to investigate the mechanism of oxazolidinone action. Linezolid inhibited initiation complex formation with either the 30S or the 70S ribosomal subunits from
Escherichia coli
. In addition, complex formation with
Staphylococcus aureus
70S tight-couple ribosomes was inhibited by linezolid. Linezolid did not inhibit the independent binding of either mRNA or
N
-formylmethionyl-tRNA to
E. coli
30S ribosomal subunits, nor did it prevent the formation of the IF2–
N
-formylmethionyl-tRNA binary complex. The results demonstrate that oxazolidinones inhibit the formation of the initiation complex in bacterial translation systems by preventing formation of the
N
-formylmethionyl-tRNA–ribosome–mRNA ternary complex.
In Vitro Activity of the New Ketolide HMR3647 in Comparison with Those of Macrolides and Pristinamycins against
Enterococcus
spp
Authors: Torres C. , Zarazaga M. , Tenorio C. , Portillo A. , Saenz Y. , Ruiz F. , Baquero F.
Published: October 2018

Ninety-four erythromycin-susceptible and 107 erythromycin-resistant enterococcal strains (MIC of ≥512 μg/ml) were inhibited by the ketolide HMR3647 at MICs of ≤0.007 to 0.06 and 0.03 to 8 μg/ml, respectively. Eighteen
vanA
-positive isolates and 29 high-level-penicillin-resistant isolates, all of them erythromycin resistant, were inhibited by HMR3647 at an MIC range of 0.015 to 4 μg/ml. The new ketolide has excellent activity against
Enterococcus
species.
Alteration in the GyrA Subunit of DNA Gyrase and the ParC Subunit of DNA Topoisomerase IV in Quinolone-Resistant Clinical Isolates of
Staphylococcus epidermidis
Authors: Li Zhiyu , Deguchi Takashi , Yasuda Mitsuru , Kawamura Takeshi , Kanematsu Emiko , Nishino Yoshinori , Ishihara Satoshi , Kawada Yukimichi
Published: October 2018

We examined 22 clinical isolates of
Staphylococcus epidermidis
to analyze the association of alterations in GyrA and ParC with fluoroquinolone resistance. The simultaneous presence of GyrA and ParC alterations was associated with a high level of fluoroquinolone resistance in the clinical isolates of
S. epidermidis
.
Immunomodulating Effects of HMR 3004 on Pulmonary Inflammation Caused by Heat-Killed
Streptococcus pneumoniae
in Mice
Authors: Duong Michel , Simard Marie , Bergeron Yves , Ouellet Nathalie , Côté-Richer Mélanie , Bergeron Michel G.
Published: October 2018

We investigated the influence of HMR 3004, a new ketolide antibiotic, on the pulmonary inflammation induced by heat-killed fluorescein isothiocyanate-labeled
Streptococcus pneumoniae
. HMR 3004 downregulated (
P
< 0.05) the pneumococcus-induced release of interleukin-6 (IL-6), IL-1β, and nitric oxide in bronchoalveolar lavage fluid. The drug limited (
P
< 0.05) neutrophil recruitment to lung tissues and alveoli but did not interfere with phagocytosis. HMR 3004 totally abrogated lung edema. By reducing inflammation in addition to possessing antimicrobial properties, HMR 3004 may participate in improving the outcome of bacterial pneumonia.
Comparative Efficacy of Trovafloxacin in Experimental Endocarditis Caused by Ciprofloxacin-Sensitive, Methicillin-Resistant
Staphylococcus aureus
Authors: Kim Young S. , Liu Quingxiang , Chow Lucien L. , Chambers Henry F. , Täuber Martin G.
Published: October 2018

The new fluoroquinolone trovafloxacin was tested against a ciprofloxacin-sensitive, methicillin-resistant
Staphylococcus aureus
strain in the rabbit model of endocarditis. Trovafloxacin was more effective than vancomycin (CFU/g of vegetation, 2.65 ± 1.87 versus 4.54 ± 2.80 [mean ± standard deviation]; P < 0.05) or ampicillin-sulbactam plus rifampin (4.9 ± 1.1 CFU/g). The addition of ampicillin-sulbactam to trovafloxacin tended to reduce titers further.
Inhibition of RNA Synthesis as a Therapeutic Strategy against
Aspergillus
and
Fusarium
: Demonstration of In Vitro Synergy between Rifabutin and Amphotericin B
Authors: Clancy Cornelius J. , Yu Yue C. , Lewin Alfred , Nguyen M. Hong
Published: October 2018

We investigated the in vitro antifungal activity of amphotericin B, alone and in combination with rifabutin, an inhibitor of bacterial RNA polymerase, against 26 clinical isolates of
Aspergillus
and 25 clinical isolates of
Fusarium
. Synergy or additivism between these drugs was demonstrated against all isolates tested. Amphotericin B MICs were reduced upon combination with rifabutin from a mean of 0.65 μg/ml to a mean of 0.16 μg/ml against
Aspergillus
, and from a mean of 0.97 μg/ml to a mean of 0.39 μg/ml against
Fusarium
(
P
< 0.000001 for both). Similarly, the MICs of rifabutin were reduced upon combination with amphotericin B from a mean of >32 μg/ml to a mean of 1.1 μg/ml against both fungi (
P
< 0.000001 for both). These positive interactions were corroborated by a colony count study with two
Fusarium
isolates, for which treatment with the combination of subinhibitory concentrations of amphotericin B (at concentrations 2- and 4-fold less than the MIC) and rifabutin (at concentrations ranging from 4- to 64-fold less than the MIC) resulted in 3.2-log reductions in colony counts compared to those after treatment with either drug alone. Inhibition of RNA synthesis was shown to be the mechanism of antifungal activity. These results suggest that inhibition of fungal RNA synthesis might be a potential target for antifungal therapy.
Introduction of the
mec
Element (Methicillin Resistance) into
Staphylococcus aureus
Alters In Vitro Functional Activities of Fibrinogen and Fibronectin Adhesins
Authors: Vaudaux Pierre E. , Monzillo Vincenza , Francois Patrice , Lew Daniel P. , Foster Tim J. , Berger-Bächi Brigitte
Published: October 2018

Some methicillin-resistant strains of
Staphylococcus aureus
are defective in the production of major surface components such as protein A, clumping factor, or other important adhesins to extracellular matrix components which may play a role in bacterial colonization and infection. To evaluate the impact of methicillin resistance (
mec
) determinants on bacterial adhesion mediated by fibrinogen or fibronectin adhesins, we compared the in vitro attachment of two genetically distinct susceptible strains (NCTC8325 and Newman) to protein-coated surfaces with that of isogenic methicillin-resistant derivatives. All strains containing an intact
mec
element in their chromosomes were found to be defective in adhesion to fibrinogen and fibronectin immobilized on polymethylmethacrylate coverslips, regardless of the presence or absence of additional mutations in the
femA
,
femB
, or
femC
gene, known to decrease expression of methicillin resistance in
S. aureus
. Western ligand affinity blotting or immunoblotting of cell wall-associated adhesins revealed similar contents of fibrinogen- or fibronectin-binding proteins in methicillin-resistant strains compared to those of their methicillin-susceptible counterparts. In contrast to methicillin-resistant strains carrying a
mec
element in their genomes, methicillin-resistant strains constructed in vitro, by introducing the
mecA
gene on a plasmid, retained their adhesion phenotypes. In conclusion, the chromosomal insertion of the
mec
element into genetically defined strains of
S. aureus
impairs the in vitro functional activities of fibrinogen or fibronectin adhesins without altering their production. This effect is unrelated to the activity of the
mecA
gene.
Plasmid-Mediated Resistance to Expanded-Spectrum Cephalosporins among
Enterobacter aerogenes
Strains
Authors: Pitout Johann D. D. , Thomson Kenneth S. , Hanson Nancy D. , Ehrhardt Anton F. , Coudron Philip , Sanders Christine C.
Published: October 2018

Resistance to expanded-spectrum cephalosporins commonly develops in
Enterobacter aerogenes
during therapy due to selection of mutants producing high levels of the chromosomal Bush group 1 β-lactamase. Recently, resistant strains producing plasmid-mediated extended-spectrum β-lactamases (ESBLs) have been isolated as well. A study was designed to investigate ESBL production among 31 clinical isolates of
E. aerogenes
from Richmond, Va., with decreased susceptibility to expanded-spectrum cephalosporins and a positive double-disk potentiation test. Antibiotic susceptibility was determined by standard disk diffusion and agar dilution procedures. β-Lactamases were investigated by an isoelectric focusing overlay technique which simultaneously determined isoelectric points (pIs) and substrate or inhibitor profiles. Decreased susceptibility to cefotaxime, ceftazidime, and aztreonam (MIC range, 1 to 64 μg/ml) was detected and associated with resistance to gentamicin and trimethoprim-sulfamethoxazole. All strains produced an inducible Bush group 1 β-lactamase (pI 8.3). Twenty-nine of the 31 isolates also produced an enzyme similar to SHV-4 (pI 7.8), while 1 isolate each produced an enzyme similar to SHV-3 (pI 6.9) and to SHV-5 (pI 8.2). The three different SHV-derived ESBLs were transferred by transconjugation to
Escherichia coli
C600N and amplified by PCR. Plasmid profiles of the clinical isolates showed a variety of different large plasmids. Because of the linkage of resistance to aminoglycosides and trimethoprim-sulfamethoxazole with ESBL production, it is possible that the usage of these drugs was responsible for selecting plasmid-mediated resistance to extended-spectrum cephalosporins in
E. aerogenes
. Furthermore, it is important that strains such as these be recognized, because they can be responsible for institutional spread of resistance genes.
Oral Administration of a Prodrug of the Influenza Virus Neuraminidase Inhibitor GS 4071 Protects Mice and Ferrets against Influenza Infection
Authors: Mendel Dirk B. , Tai Chun Y. , Escarpe Paul A. , Li Weixing , Sidwell Robert W. , Huffman John H. , Sweet Clive , Jakeman Kenneth J. , Merson James , Lacy Steven A. , Lew Willard , Williams Matthew A. , Zhang Lijun , Chen Ming S. , Bischofberger Norbert , Kim Choung U.
Published: October 2018

We have recently described GS 4071, a carbocyclic transition-state analog inhibitor of the influenza virus neuraminidase, which has potent inhibitory activity comparable to that of 4-guanidino-Neu5Ac2en (GG167; zanamivir) when tested against influenza A virus replication and neuraminidase activity in vitro. We now report that GS 4071 is active against several strains of influenza A and B viruses in vitro and that oral GS 4104, an ethyl ester prodrug which is converted to GS 4071 in vivo, is active in the mouse and ferret models of influenza virus infection. Oral administration of 10 mg of GS 4104 per kg of body weight per day caused a 100-fold reduction in lung homogenate viral titers and enhanced survival in mice infected with influenza A or B viruses. In ferrets, a 25-mg/kg dose of GS 4104 given twice daily reduced peak viral titers in nasal washings and eliminated constitutional responses to influenza virus infection including fever, increased nasal signs (sneezing, nasal discharge, mouth breathing), and decreased activity. Consistent with our demonstration that the parent compound is highly specific for influenza virus neuraminidases, no significant drug-related toxicity was observed after the administration of oral dosages of GS 4104 of up to 800 mg/kg/day for 14 days in nonclinical toxicology studies with rats. These results indicate that GS 4104 is a novel, orally active antiviral agent with the potential to be used for the prophylaxis and treatment of influenza A and B virus infections.
Relationship between Antimalarial Drug Activity, Accumulation, and Inhibition of Heme Polymerization in
Plasmodium falciparum
In Vitro
Authors: Hawley Shaun R. , Bray Patrick G. , Mungthin Mathirut , Atkinson Jill D. , O’Neill Paul M. , Ward Stephen A.
Published: October 2018

We have investigated the contribution of drug accumulation and inhibition of heme polymerization to the in vitro activities of a series of antimalarial drugs. Only those compounds exhibiting structural relatedness to the quinolines inhibited heme polymerization. We could find no direct correlation between in vitro activity against chloroquine-susceptible or chloroquine-resistant isolates and either inhibition of heme polymerization or cellular drug accumulation for the drugs studied. However, in vitro activity against a chloroquine-susceptible isolate but not a chloroquine-resistant isolate showed a significant correlation with inhibition of heme polymerization when the activity was normalized for the extent of drug accumulation. The importance of these observations to the rational design of new quinoline-type drugs and the level of agreement of these conclusions with current views on quinoline drug action and resistance are discussed.
Pharmacokinetics and Bioavailability of the Anti-Human Immunodeficiency Virus Nucleotide Analog 9-[(
R
)2-(Phosphonomethoxy)Propyl]Adenine (PMPA) in Dogs
Authors: Cundy Kenneth C. , Sueoka Cathy , Lynch Geoffrey R. , Griffin Linda , Lee William A. , Shaw Jeng-Pyng
Published: October 2018

The pharmacokinetics, bioavailability, and metabolism of the anti-human immunodeficiency virus nucleotide analog 9[(
R
)-2-(phosphonomethoxy)propyl]adenine (PMPA) were determined in beagle dogs following intravenous, intraperitoneal, and oral administration. Fasted male beagle dogs (
n
= 5) were pretreated with pentagastrin and received PMPA (10 mg/kg of body weight) by the intravenous and oral routes with a washout period of 1 week between doses. A further group of male dogs received PMPA as a single dose via the intravenous (1 mg/kg;
n
= 5) and the intraperitoneal (10 mg/kg;
n
= 3) routes, with 1-week washout period between doses. The concentrations of PMPA in plasma and urine were determined over 48 h postdosing by fluorescence derivatization and high-performance liquid chromatography (HPLC). The potential for metabolism or biliary excretion of PMPA was evaluated in a dog with a chronic indwelling bile cannula. Urine, feces, and bile were collected at intervals over 48 h following the intravenous administration of [
14
C]PMPA (10 mg/kg; 55 μCi/kg). The concentrations of PMPA in plasma after intravenous injection were best described by an open two-compartment model with a terminal half-life of approximately 10 h. PMPA was excreted unchanged in urine (70%); recovery in feces (0.42%) or bile (0.26%) was negligible. The plasma clearance of PMPA (0.28 ± 0.05 liter/h/kg) was substantially greater than the glomerular filtration rate in this species, suggesting active tubular secretion of PMPA. No metabolites of [
14
C]PMPA were observed in urine, feces, or bile on the basis of HPLC with radioactive flow detection. The remainder of the dose was probably excreted unchanged in urine beyond 48 h postdosing. The mean ± standard deviation observed bioavailabilities of PMPA following oral and intraperitoneal administration at 10 mg/kg were 17.1% ± 1.88% and 73.5% ± 10.5%, respectively.
Activities of Isoniazid Alone and in Combination with Other Drugs against
Mycobacterium avium
Infection in Beige Mice
Authors: Fattorini Lanfranco , Xiao Yan , Mattei Maurizio , Li Yongjun , Iona Elisabetta , Ricci Maria Luisa , Thoresen Ove Fredrik , Creti Roberta , Orefici Graziella
Published: October 2018

Monotherapy with isoniazid or amikacin or clarithromycin or combinations of two of these drugs showed nil to modest therapeutic activity in beige mice infected with
Mycobacterium avium
. However, the combination of all three, isoniazid-amikacin-clarithromycin, markedly reduced CFUs in both spleens and lungs after 91 days of infection.
Cefepime versus Ceftriaxone for Empiric Treatment of Hospitalized Patients with Community-Acquired Pneumonia
Authors: Zervos M. , Nelson M. ,   the Cefepime Study Group
Published: October 2018

Effective empiric treatment of pneumonia requires antibiotic coverage against gram-negative and gram-positive pathogens, including drug-resistant isolates. We compared the safety and efficacy of intravenous (i.v.) cefepime (2 g administered every 12 h) to those of i.v. ceftriaxone (1 g administered every 12 h) for the empiric treatment of hospitalized patients with community-acquired pneumonia. Of the 115 patients randomized to the study, 86 (cefepime recipients,
n
= 40; ceftriaxone recipients,
n
= 46) were evaluated for clinical efficacy (clinically evaluated patients). Favorable clinical outcomes (cure or improvement) were comparable among clinically evaluated patients in the cefepime and ceftriaxone treatment arms (95.0 versus 97.8%, respectively; 95% confidence interval for treatment difference [data for ceftriaxone group minus data for cefepime group], −5.1 to +10.8%). The most common bacteria isolated from patients in both treatment groups were
Streptococcus pneumoniae
,
Haemophilus influenzae
, and
Staphylococcus aureus
. In clinically evaluated patients with a microbiologic response, all (100%) of the 32 pathogens from cefepime-treated patients and 97.4% (38 of 39) of the pathogens from ceftriaxone-treated patients were eradicated (documented or presumed eradication). The one persistent infection in the ceftriaxone group was caused by
Pseudomonas fluorescens
. Both treatments were well tolerated. Our data thus suggest that cefepime and ceftriaxone have comparable safety and efficacy for the treatment of pneumonia in hospitalized patients.
Effect of Dirithromycin onHaemophilus influenzaeInfection of the Respiratory Mucosa
Authors: Rutman Andrew , Dowling Ruth , Wills Peter , Feldman Charles , Cole Peter J. , Wilson Robert
Published: October 2018

Macrolides have properties other than their antibiotic action which may benefit patients with airway infections. We have investigated the effect of dirithromycin (0.125 to 8.0 μg/ml) on the interaction ofHaemophilus influenzaewith respiratory mucosa in vitro using human nasal epithelium, adenoid tissue, and bovine trachea. Dirithromycin did not affect the ciliary beat frequency of the nasal epithelium or the transport of mucus on bovine trachea, but dirithromycin (1 μg/ml) did reduce the slowing of the ciliary beat frequency and the damage to the nasal epithelium caused byH. influenzaebroth culture filtrate. Amoxicillin (2 μg/ml) did not reduce the effects of theH. influenzaebroth culture filtrate.H. influenzaeinfection of the organ cultures for 24 h caused mucosal damage and the loss of ciliated cells. Bacteria adhered to damaged epithelium and to a lesser extent to mucus and unciliated cells. Incubation ofH. influenzaewith dirithromycin at sub-MICs (0.125 and 0.5 μg/ml) prior to infection of the organ cultures did not reduce the mucosal damage caused by bacterial infection. By contrast, incubation of adenoid tissue with dirithromycin (0.125 to 1.0 μg/ml) for 4 h prior to assembling the organ culture reduced the mucosal damage caused by subsequentH. influenzaeinfection by as much as 50%. The number of bacteria adherent to the mucosa was reduced, although the tissue that had been incubated with dirithromycin (0.125 and 0.5 μg/ml) did not inhibit bacterial growth. This was achieved by a reduction in the amount of damaged epithelium to whichH. influenzaeadhered and a reduction in the density of bacteria adhering to mucus. We conclude that dirithromycin at concentrations achievable in vivo markedly reduces the mucosal damage caused byH. influenzaeinfection due to a cytoprotective effect.
In Vivo Activity of HSR-903, a New Fluoroquinolone, against Respiratory Pathogens
Authors: Yoshizumi Satoshi , Domon Haruki , Miyazaki Shuichi , Yamaguchi Keizo
Published: October 2018

The in vivo activity of HSR-903, a new fluoroquinolone, against major bacteria which cause respiratory tract infections was evaluated. HSR-903 was active against experimental respiratory tract infections in mice challenged with penicillin-susceptible and penicillin-resistant
Streptococcus pneumoniae
and
Haemophilus influenzae
strains. Treatment with HSR-903 reduced the bacterial numbers in infected murine lungs. In accord with the pulmonary clearance results, the rates of survival for mice treated with HSR-903, sparfloxacin, levofloxacin, ciprofloxacin, and benzylpenicillin were 50, 30, 10, 0, and 0%, respectively, 14 days after being infected with penicillin-resistant
S. pneumoniae
. A pharmacokinetic study with pneumonic mice showed that the levels of HSR-903 in the lungs were seven to eight times higher than those in the plasma. These results indicate that clinical studies of HSR-903 against respiratory tract infections may be warranted.