bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantitative Structure-Activity Relationship Studies of a Series of Sulfa Drugs as Inhibitors of
Pneumocystis carinii
Dihydropteroate Synthetase
Authors: Johnson Theresa , Khan Ikhlas A. , Avery Mitchell A. , Grant Juliana , Meshnick Steven R.
Published: October 2018

Sulfone and sulfanilamide sulfa drugs have been shown to inhibit dihydropteroate synthetase (DHPS) isolated from
Pneumocystis carinii
. In order to develop a pharmacophoric model for this inhibition, quantitative structure-activity relationships (QSAR) for sulfa drugs active against DHPS have been studied. Accurate 50% inhibitory concentrations were collected for 44 analogs, and other parameters, such as partition coefficients and molar refractivity, were calculated. Conventional multiple regression analysis of these data did not provide acceptable QSAR. However, three-dimensional QSAR provided by comparative molecular field analysis did give excellent results. Upon removal of poorly correlated analogs, a data set of 36 analogs, all having a common NHSO
2
group, provided a cross-validated
r
2
value of 0.699 and conventional
r
2
value of 0.964. The resulting pharmacophore model should be useful for understanding and predicting the binding of DHPS by new sulfa drugs.
Antiviral Activities of 9-
R
-2-Phosphonomethoxypropyl Adenine (PMPA) and Bis(isopropyloxymethylcarbonyl)PMPA against Various Drug-Resistant Human Immunodeficiency Virus Strains
Authors: Srinivas Ranga V. , Fridland Arnold
Published: October 2018

9-
R
-2-Phosphonomethoxypropyl adenine (PMPA) is an acyclic nucleoside phosphonate analog that has demonstrated efficacy against human immunodeficiency virus (HIV). We recently described the synthesis, metabolism, and biological activities of bis(isopropyloxymethylcarbonyl)PMPA [bis(poc)PMPA] as an orally bioavailable prodrug for PMPA. Among a large panel of drug-resistant HIV type 1 variants, only the K65R virus was resistant to PMPA. K65R virus also showed reduced susceptibility to bis(poc)PMPA, although the prodrug could still inhibit these viruses at submicromolar, nontoxic concentrations. Among a panel of seven primary clinical isolates from patients with diverse treatment histories, only one isolate showed reduced susceptibility to PMPA and was found to carry three mutations (M41L, T69N, R73K) in its reverse transcriptase catalytic domain.
In Vitro Activity of a New Ketolide Antibiotic, HMR 3647, against
Chlamydia pneumoniae
Authors: Roblin Patricia M. , Hammerschlag Margaret R.
Published: October 2018

The in vitro activities of HMR 3647, roxithromycin, erythromycin, and azithromycin against 19 strains of
Chlamydia pneumoniae
were tested. The MIC at which 90% of the isolates are inhibited and the minimum bactericidal concentration at which 90% of the isolates are killed of HMR 3647 were 0.25 μg/ml (range, 0.015 to 2 μg/ml). Nine recently obtained clinical isolates from children with pneumonia were more susceptible (MICs, 0.015 to 0.0625 μg/ml) than older strains that had been passaged more extensively.
Antimicrobial Susceptibilities of Group B Streptococci Isolated between 1992 and 1996 from Patients with Bacteremia or Meningitis
Authors: Fernandez Marisol , Hickman Melissa E. , Baker Carol J.
Published: October 2018

In vitro testing of 229 group B streptococcal isolates from a variety of patients with invasive infections indicated uniform penicillin G susceptibility. However, 17 (7.4%) isolates were resistant to erythromycin and 8 (3.4%) were resistant to clindamycin. These results support the continued use of penicillin G as the drug of choice for the treatment and prevention of group B streptococcal disease.
Selection and Characterization of β-Lactam–β-Lactamase Inactivator-Resistant Mutants following PCR Mutagenesis of the TEM-1 β-Lactamase Gene
Authors: Vakulenko Sergei B. , Geryk Bruce , Kotra Lakshmi P. , Mobashery Shahriar , Lerner Stephen A.
Published: October 2018

Mechanism-based inactivators of β-lactamases are used to overcome the resistance of clinical pathogens to β-lactam antibiotics. This strategy can itself be overcome by mutations of the β-lactamase that compromise the effectiveness of their inactivation. We used PCR mutagenesis of the TEM-1 β-lactamase gene and sequenced the genes of 20 mutants that grew in the presence of ampicillin-clavulanate. Eleven different mutant genes from these strains contained from 1 to 10 mutations. Each had a replacement of one of the four residues, Met69, Ser130, Arg244, and Asn276, whose substitutions by themselves had been shown to result in inhibitor resistance. None of the mutant enzymes with multiple amino acid substitutions generated in this study conferred higher levels of resistance to ampicillin alone or ampicillin with β-lactamase inactivators (clavulanate, sulbactam, or tazobactam) than the levels of resistance conferred by the corresponding single-mutant enzymes. Of the four enzymes with just a single mutation (Ser130Gly, Arg244Cys, Arg244Ser, or Asn276Asp), the Asn276Asp β-lactamase conferred a wild-type level of ampicillin resistance and the highest levels of resistance to ampicillin in the presence of inhibitors. Site-directed random mutagenesis of the Ser130 codon yielded no other mutant with replacement of Ser130 besides Ser130Gly that produced ampicillin-clavulanate resistance. Thus, despite PCR mutagenesis we found no new mutant TEM β-lactamase that conferred a level of resistance to ampicillin plus inactivators greater than that produced by the single-mutation enzymes that have already been reported in clinical isolates. Although this is reassuring, one must caution that other combinations of multiple mutations might still produce unexpected resistance.
Antimicrobial Therapies for
Helicobacter pylori
Infection in Gnotobiotic Piglets
Authors: Krakowka Steven , Eaton K. A. , Leunk Robert D.
Published: October 2018

Gnotobiotic piglets infected with
Helicobacter pylori
were treated with various antimicrobials as monotherapy and dual therapy, and the results were compared to those for piglets treated with a triple-therapy regimen (bismuth subsalicyclate at 5.7 mg/kg of body weight, metronidazole at 4.4 mg/kg, and amoxicillin at 6.8 mg/kg four times a day [QID]). Clearance of infection was assessed after 7 days of treatment, and eradication was assessed following 7 days of treatment and a 14-day posttreatment observation interval. Monotherapy with amoxicillin, clarithromycin, and ciprofloxacin cleared and eradicated the organism from porcine stomachs; monotherapy with metronidazole cleared the infection and eradicated it from some piglets. Metronidazole-resistant microbes were recovered from treated piglets which cleared but did not eradicate the infection. Monotherapy with bismuth subsalicylate, erythromycin, nitrofurantoin, and tetracycline in the dosage range of 5.0 to 7.1 mg/kg QID was less than 100% effective in clearance and eradication, in that these drugs cleared and/or eradicated the infection from some of the piglets but did not eradicate the infection from all of the piglets. Monotherapy with an H-2 receptor antagonist (ranitidine) or a proton pump inhibitor (omeprazole) was ineffective at either clearance or eradication. In vivo dose titrations with several effective monotherapies were performed to determine the lowest effective in vivo dose of drug. In piglets, eradication was associated with a statistically significant decline in serum
H. pylori
-specific immunoglobulin M (IgM) antibodies; the titers of both IgA and IgG also declined, but the values were not statistically significant. For many antimicrobials, piglets are more sensitive indicators of clearance and eradication than humans. These data establish the
H. pylori
-infected gnotobiotic piglet as a useful model for the identification of novel antimicrobials for the treatment of this disease and for drug assessment during preclinical evaluations.
Chloramphenicol Resistance in
Clostridium difficile
Is Encoded on Tn
4453
Transposons That Are Closely Related to Tn
4451
from
Clostridium perfringens
Authors: Lyras Dena , Storie Christine , Huggins Andrea S. , Crellin Paul K. , Bannam Trudi L. , Rood Julian I.
Published: October 2018

The chloramphenicol resistance gene
catD
from
Clostridium difficile
was shown to be encoded on the transposons Tn
4453a
and Tn
4453b
, which were structurally and functionally related to Tn
4451
from
Clostridium perfringens
. Tn
4453a
and Tn
4453b
excised precisely from recombinant plasmids, generating a circular form, as is the case for Tn
4451
. Evidence that this process is mediated by Tn
4453
-encoded
tnpX
genes was obtained from experiments which showed that in
trans
these genes complemented a Tn
4451tnpXΔ1
mutation for excision. Nucleotide sequencing showed that the joint of the circular form generated by the excision of Tn
4453a
and Tn
4453b
was similar to that from Tn
4451
. These results suggest that the Tn
4453
-encoded TnpX proteins bind to similar DNA target sequences and function in a manner comparable to that of TnpX from Tn
4451
. Furthermore, it has been shown that Tn
4453a
and Tn
4453b
can be transferred to suitable recipient cells by RP4 and therefore are mobilizable transposons. It is concluded that, like Tn
4451
, they must encode a functional
tnpZ
gene and a target
oriT
or RS
A
site. The finding that related transposable elements are present in
C. difficile
and
C. perfringens
has implications for the evolution and dissemination of antibiotic resistance genes and the mobile elements on which they are found within the clostridia.
In Vitro Activities of Co-Amoxiclav at Concentrations Achieved in Human Serum against the Resistant Subpopulation of HeteroresistantStaphylococcus aureus: a Controlled Study with Vancomycin
Authors: Prieto J. , Aguilar L. , Giménez M. J. , Toro D. , Gómez-Lus M. L. , Dal-Ré R. , Balcabao I. P.
Published: October 2018

The effects of concentrations that simulated those in human serum after a single intravenous dose of amoxicillin (2 g), amoxicillin-clavulanic acid (2,000 and 200 mg, respectively), or vancomycin (500 mg), on the viability and β-lactamase activity of two isogenic (β-lactamase and non-β-lactamase producer) heteroresistantStaphylococcus aureusstrains were studied in an in vitro pharmacodynamic model. A reduction of ≥97% of the initial inoculum was obtained with vancomycin and amoxicillin-clavulanic acid against both strains, with respect to the total bacterial population and the oxacillin-resistant subpopulation. The same pattern was observed with amoxicillin and the β-lactamase-negative strain. β-Lactamase activity in the β-lactamase-positive strain changed over time parallel to viability, decreasing with amoxicillin-clavulanic acid or vancomycin and increasing in the amoxicillin and control groups. Clavulanic acid concentrations achievable in serum that changed over time allowed amoxicillin to act against the β-lactamase-producing methicillin-resistantS. aureusto a similar extent as vancomycin.
Decreased Accumulation or Increased Isoleucyl-tRNA Synthetase Activity Confers Resistance to the Cyclic β-Amino Acid BAY 10-8888 in
Candida albicans
and
Candida tropicalis
Authors: Ziegelbauer Karl
Published: October 2018

BAY 10-8888, a cyclic β-amino acid, exerts its antifungal activity by inhibition of isoleucyl-tRNA synthetase activity after accumulation to a millimolar concentration inside the cell. We have selected and characterized BAY 10-8888-resistant
Candida albicans
mutants. Reduced BAY 10-8888 accumulation as well as increased isoleucyl-tRNA synthetase activity was observed in these mutants. Some of the mutants were cross-resistant to cispentacin, a structurally related β-amino acid, while sensitivities to 5-fluorocytosine and fluconazole remained unchanged in all mutants. All except two in vitro-resistant mutants were pathogenic in a murine candidiasis model, and BAY 10-8888 failed to cure the infection. Furthermore, we have characterized BAY 10-8888 transport and isoleucyl-tRNA synthetase activity in several
Candida tropicalis
strains which showed MICs higher than those of other Candida strains. An analysis of the
C. tropicalis
strains revealed that intracellular concentrations of BAY 10-8888 were in the millimolar range, comparable to those for
C. albicans
. However, these isolates expressed isoleucyl-tRNA synthetase activities about fourfold higher than those for
C. albicans
. To test the possibility of resistance modeling, we determined the correlations between the intracellular concentration of BAY 10-8888, the specific activity of isoleucyl-tRNA synthetase, the number of free, i.e., noninhibited, isoleucyl-tRNA synthetase molecules/cell, and growth, assuming a linear relation. We found significant correlations between growth and the intracellular concentration of BAY 10-8888 and between growth and the number of free isoleucyl-tRNA synthetase molecules/cell, but not between growth and the specific activity of isoleucyl-tRNA synthetase.
Cefepime-Aztreonam: a Unique Double β-Lactam Combination for
Pseudomonas aeruginosa
Authors: Lister Philip D. , Sanders W. Eugene , Sanders Christine C.
Published: October 2018

An in vitro pharmacokinetic model was used to determine if aztreonam could enhance the pharmacodynamics of cefepime or ceftazidime against an isogenic panel of
Pseudomonas aeruginosa
164, including wild-type (WT), partially derepressed (PD), and fully derepressed (FD) phenotypes. Logarithmic-phase cultures were exposed to peak concentrations achieved in serum with 1- or 2-g intravenous doses, elimination pharmacokinetics were simulated, and viable bacterial counts were measured over three 8-h dosing intervals. In studies with cefepime and cefepime-aztreonam against the PD strain, samples were also filter sterilized, assayed for active cefepime, and assayed for nitrocefin hydrolysis activity before and after overnight dialysis. Against WT strains, the cefepime-aztreonam combination was the most active regimen, but viable counts at 24 h were only 1 log below those in cefepime-treated cultures. Against PD and FD strains, the antibacterial activity of cefepime-aztreonam was significantly enhanced over that of each drug alone, with 3.5 logs of killing by 24 h. Hydrolysis and bioassay studies demonstrated that aztreonam was inhibiting the extracellular cephalosporinase that had accumulated and was thus protecting cefepime in the extracellular environment. In contrast to cefepime-aztreonam, the pharmacodynamics of ceftazidime-aztreonam were not enhanced over those of aztreonam alone. Further pharmacodynamic studies with five other
P. aeruginosa
strains producing increased levels of cephalosporinase demonstrated that the enhanced pharmacodynamics of cefepime-aztreonam were not unique to the isogenic panel. The results of these studies demonstrate that aztreonam can enhance the antibacterial activity of cefepime against derepressed mutants of
P. aeruginosa
producing increased levels of cephalosporinase. This positive interaction appears to be due in part to the ability of aztreonam to protect cefepime from extracellular cephalosporinase inactivation. Clinical evaluation of this combination is warranted.
Antiviral Activity of a Selective Ribonucleotide Reductase Inhibitor against Acyclovir-Resistant Herpes Simplex Virus Type 1 In Vivo
Authors: Duan Jianmin , Liuzzi Michel , Paris William , Lambert Michelle , Lawetz Carol , Moss Neil , Jaramillo Jorge , Gauthier Jean , Déziel Robert , Cordingley Michael G.
Published: October 2018

The present study reports the activity of BILD 1633 SE against acyclovir (ACV)-resistant herpes simplex virus (HSV) infections in athymic nude (
nu/nu
) mice. BILD 1633 SE is a novel peptidomimetic inhibitor of HSV ribonucleotide reductase (RR). In vitro, it is more potent than ACV against several strains of wild-type as well as ACV-resistant HSV mutants. Its in vivo activity was tested against cutaneous viral infections in athymic nude mice infected with the ACV-resistant isolates HSV type 1 (HSV-1)
dl
sptk and PAA
r
5, which contain mutations in the viral thymidine kinase gene and the polymerase gene, respectively. Following cutaneous infection of athymic nude mice, both HSV-1
dl
sptk and PAA
r
5 induced significant, reproducible, and persistent cutaneous lesions that lasted for more than 2 weeks. A 10-day treatment regimen with ACV given topically four times a day as a 5% cream or orally at up to 5 mg/ml in drinking water was partially effective against HSV-1 PAA
r
5 infection with a reduction of the area under the concentration-time curve (AUC) of 34 to 48%. The effects of ACV against HSV-1
dl
sptk infection were not significant when it was administered topically and were only marginal when it was given in drinking water. Treatment under identical conditions with 5% topical BILD 1633 SE significantly reduced the cutaneous lesions caused by both HSV-1
dl
sptk and PAA
r
5 infections. The effect of BILD 1633 SE against HSV-1 PAA
r
5 infections was more prominent and was inoculum and dose dependent, with AUC reductions of 96 and 67% against infections with 10
6
and 10
7
PFU per inoculation site, respectively. BILD 1633 SE also significantly decreased the lesions caused by HSV-1
dl
sptk infection (28 to 51% AUC reduction). Combination therapy with topical BILD 1633 SE (5%) and ACV in drinking water (5 mg/ml) produced an antiviral effect against HSV-1
dl
sptk and PAA
r
5 infections that was more than the sum of the effects of both drugs. This is the first report that a selective HSV RR subunit association inhibitor can be effective against ACV-resistant HSV infections in vivo.
Outer Membrane Profiles of Clonally Related
Klebsiella pneumoniae
Isolates from Clinical Samples and Activities of Cephalosporins and Carbapenems
Authors: Ardanuy Carmen , Liñares Josefina , Domínguez María Angeles , Hernández-Allés Santiago , Benedí Vicente J. , Martínez-Martínez Luis
Published: October 2018

Fifteen isolates of
Klebsiella pneumoniae
producing extended-spectrum β-lactamases (ESBLs) isolated during a nosocomial outbreak were studied. The strains belonged to the same clonal type, as shown by pulsed-field gel electrophoretic analysis of chromosomal DNA. All the isolates were resistant to extended-spectrum cephalosporins, aztreonam, gentamicin, and fluoroquinolones and were susceptible to carbapenems, tobramycin, netilmicin, and amikacin. None of the isolates expressed the OmpK36 porin. Eight isolates, for which the MICs of cefoxitin were ≥64 μg/ml, showed a diminished level or no expression of a 35-kDa porin. The MICs of meropenem, cefotaxime, and cefpirome were three to eight times higher for porin-deficient isolates than for isolates expressing the 35-kDa porin, but the MICs of imipenem increased two times for porin-deficient isolates compared to those for isolates expressing the porin. This MIC increase reverted to a level similar to that for the parental strain when porin-deficient isolates were transformed with the gene coding for the
K. pneumoniae
porin OmpK36. It is concluded that the high level of resistance to cefoxitin and the increase in the MICs of meropenem, cefotaxime, and cefpirome for the ESBL-producing
K. pneumoniae
isolates studied are associated with porin deficiency.
Contribution of Outer Membrane Efflux Protein OprM to Antibiotic Resistance in
Pseudomonas aeruginosa
Independent of MexAB
Authors: Zhao Qixun , Li Xian-Zhi , Srikumar Ramakrishnan , Poole Keith
Published: October 2018

A
Pseudomonas aeruginosa
strain carrying an insertion of an ΩHg interposon in the
mexB
gene (
mexB
::ΩHg; strain K879) produced markedly reduced but still detectable levels of OprM, the product of the third gene of the
mexAB-oprM
multidrug efflux operon. By using a
lacZ
transcriptional fusion vector, promoter activity likely responsible for OprM expression in the
mexB
::ΩHg mutant was identified upstream of
oprM
. Introduction of the
oprM
gene, but not the
mexAB
genes, into a
P. aeruginosa
multidrug-susceptible Δ
mexAB-oprM
mutant increased resistance to quinolones, cephalosporins, erythromycin, and tetracycline. A Δ
mexAB-oprM
strain carrying the
oprM
gene accumulated markedly less antibiotic than the deletion strain without
oprM
. Antibiotic accumulation by the MexAB
−
OprM
+
strain was markedly enhanced upon treatment of cells with the uncoupler carbonyl cyanide
m
-chlorophenylhydrazone (CCCP), indicating that MexAB-independent OprM function likely involves an efflux process. Moreover, pretreatment of cells with CCCP prior to the accumulation assay abrogated any differences in accumulation levels between the MexAB
−
OprM
+
and MexAB
−
OprM
−
strains, indicating that reduced drug accumulation by the OprM
+
strain (in the absence of CCCP) cannot be due to OprM-mediated reduction in outer membrane permeability. It appears, therefore, that OprM can be expressed and function in a drug efflux capacity independent of MexAB.
Gene Amplification in
Leishmania tarentolae
Selected for Resistance to Sodium Stibogluconate
Authors: Haimeur Anass , Ouellette Marc
Published: October 2018

Leishmania tarentolae
promastigotes were selected step by step for resistance to sodium stibogluconate (Pentostam). Mutants resistant to antimony-containing drugs and cross-resistant to arsenite were therefore obtained. Amplification of one common locus was observed in several independent sodium stibogluconate-resistant mutants, and the locus amplified was novel. The copy number of the amplified locus was related to the level of resistance to pentavalent antimony. The gene responsible for antimony resistance was isolated by transfection and was shown to correspond to an open reading frame coding for 770 amino acids. The putative gene product did not exhibit significant homology with sequences present in data banks, and the putative role of this protein in antimony resistance is discussed.
Antiproliferative Effects and Mechanism of Action of SCH 56592 against
Trypanosoma
(
Schizotrypanum
)
cruzi
: In Vitro and In Vivo Studies
Authors: Urbina Julio A. , Payares Gilberto , Contreras Lellys M. , Liendo Andreína , Sanoja Cristina , Molina Judith , Piras Marta , Piras Romano , Perez Norma , Wincker Patrick , Loebenberg David
Published: October 2018

We have investigated the antiproliferative effects of SCH 56592, a new experimental triazole, against
Trypanosoma
(
Schizotrypanum
)
cruzi
, the etiological agent of Chagas’ disease in Latin America. SCH 56592 blocked the proliferation of the epimastigote form of the parasite in vitro at 30 nM, a concentration 30- to 100-fold lower than that required with the reference compounds ketoconazole and itraconazole. At that concentration all the parasite’s endogenous sterols (ergosterol, 24-ethyl-cholesta-5,7,22-trien-3β-ol, and its 22-dihydro analogs), were replaced by methylated sterols (lanosterol and 24-methylene-dihydrolanosterol), as revealed by high-resolution gas chromatography coupled with mass spectrometry. This indicated that the primary mechanism of action of the drug was inhibition of the parasite’s sterol C-14α demethylase. Against the clinically relevant intracellular amastigote form, grown in cultured Vero cells at 37°C, the MIC of SCH 56592 was 0.3 nM, again 33- to 100-fold lower than that of ketoconazole or itraconazole. In a murine model of acute Chagas’ disease, SCH 56592 given at ≥ 10 mg/kg of body weight/day for a total of 43 doses allowed 85 to 100% survival and 90 to 100% cure of the surviving animals, as verified by parasitological, serological, and PCR-based tests, while ketoconazole given at 30 mg/kg day allowed 60% survival but only 20% cure. In a murine model of chronic Chagas’ disease, SCH 56592 was again more effective than ketoconazole, providing 75 to 85% protection from death, with 60 to 75% parasitological cures of the surviving animals, while no parasitological cures were observed with ketoconazole. The results indicate that SCH 56592 is the most powerful sterol biosynthesis inhibitor ever tested against
T. cruzi
and may be useful in the treatment of human Chagas’ disease.
Population Pharmacokinetics of Ceftizoxime Administered by Continuous Infusion in Clinically Ill Adult Patients
Authors: Facca Bryan , Frame Bill , Triesenberg Steve
Published: October 2018

Ceftizoxime is a widely used beta-lactam antimicrobial agent, but pharmacokinetic data for use with clinically ill patients are lacking. We studied the population pharmacokinetics of ceftizoxime in 72 clinically ill patients at a community-based, university-affiliated hospital. A population pharmacokinetic model for ceftizoxime was created by using a prospective observational design. Ceftizoxime was administered by continuous infusion to treat patients with proven or suspected bacterial infections. While the patients were receiving infusions of ceftizoxime, serum samples were collected for pharmacokinetic analysis with the nonlinear mixed-effect modeling program NONMEM. In addition to clearance and volume of distribution, various comorbidities were examined for their influence on the kinetics. All 72 subjects completed the study, and 114 serum samples were collected. Several demographic and comorbidity variables, namely, age, weight, serum creatinine levels, congestive heart failure, and long-term ventilator dependency, had a significant impact on the estimate for ceftizoxime clearance. A mixture model, or two populations for estimation of ceftizoxime clearance, was discovered. One population presented with an additive clearance component of 1.6 liters per h. In addition, a maximizer function for serum creatinine levels was found. In summary, two models for ceftizoxime clearance, mixture and nonmixture, were found and are presented. Clearance for ceftizoxime can be estimated with commonly available clinical information and the models presented. From the clearance estimates, the dose of ceftizoxime to maintain the desired concentration in serum can be determined. Work is needed to validate the model for drug clearance and to evaluate its predictive performance.
Metabolism of 2′,3′-Dideoxy-2′,3′-Didehydro-β-
l
(−)-5-Fluorocytidine and Its Activity in Combination with Clinically Approved Anti-Human Immunodeficiency Virus β-
d
(+) Nucleoside Analogs In Vitro
Authors: Dutschman Ginger E. , Bridges Edward G. , Liu Shwu-Huey , Gullen Elizabeth , Guo Xin , Kukhanova Marina , Cheng Yung-Chi
Published: October 2018

2′,3′-Dideoxy-2′,3′-didehydro-β-
l
(−)-5-fluorocytidine [
l
(−)Fd4C] has been reported to be a potent inhibitor of the human immunodeficiency virus (HIV) in cell culture. In the present study the antiviral activity of this compound in two-drug combinations and its intracellular metabolism are addressed. The two-drug combination of
l
(−)Fd4C plus 2′,3′-didehydro-2′,3′-dideoxythymidine (D4T, or stavudine) or 3′-azido-3′-deoxythymidine (AZT, or zidovudine) synergistically inhibited replication of HIV in vitro. Additive antiviral activity was observed with
l
(−)Fd4C in combination with 2′,3′-dideoxycytidine (ddC, or zalcitabine) or 2′,3′-dideoxyinosine (ddI, or didanosine). This β-
l
(−) nucleoside analog has no activity against mitochondrial DNA synthesis at concentrations up to 10 μM. As we previously reported for other β-
l
(−) nucleoside analogs,
l
(−)Fd4C could protect against mitochondrial toxicity associated with D4T, ddC, and ddI. Metabolism studies showed that this drug is converted intracellularly to its mono-, di-, and triphosphate metabolites. The enzyme responsible for monophosphate formation was identified as cytoplasmic deoxycytidine kinase, and the

K
m

is 100 μM.
l
(−)Fd4C was not recognized in vitro by human mitochondrial deoxypyrimidine nucleoside kinase. Also,
l
(−)Fd4C was not a substrate for deoxycytidine deaminase.
l
(−)Fd4C 5′-triphosphate served as an alternative substrate to dCTP for incorporation into DNA by HIV reverse transcriptase. The favorable anti-HIV activity and protection from mitochondrial toxicity by
l
(−)Fd4C in two-drug combinations favors the further development of
l
(−)Fd4C as an anti-HIV agent.
Detection of Point Mutations inrpoBGene of Rifampin-ResistantRickettsia typhi
Authors: Troyer Jill Michelle , Radulovic Suzana , Andersson Siv G. E. , Azad Abdu F.
Published: October 2018

TherpoBgene of rifampin-resistantRickettsia typhi(Rif mutant) and wild-typeR. typhiwere sequenced and compared. The Rif mutantrpoBhad three nucleotide substitutions, which resulted in amino acid changes at residues 151, 201, and 271 and may be the basis for the rifampin resistance.
Contribution of
rpoB
Mutations to Development of Rifamycin Cross-Resistance in
Mycobacterium tuberculosis
Authors: Williams D. L. , Spring L. , Collins L. , Miller L. P. , Heifets L. B. , Gangadharam P. R. J. , Gillis T. P.
Published: October 2018

The contributions of 23 insertion, deletion, or missense mutations within an 81-bp fragment of
rpoB
, the gene encoding the β-subunit of the DNA-dependent RNA polymerase of
Mycobacterium tuberculosis
, to the development of resistance to rifamycins (rifampin, rifabutin, rifapentine, and KRM-1648) in 29 rifampin-resistant clinical isolates were defined. Specific mutant
rpoB
alleles led to the development of cross-resistance to all rifamycins tested, while a subset of mutations were associated with resistance to rifampin and rifapentine but not to KRM-1648 or rifabutin. To further study the impact of specific
rpoB
mutant alleles on the development of rifamycin resistance, mutations were incorporated into the
rpoB
gene of
M. tuberculosis
H37Rv, contained on a mycobacterial shuttle plasmid, by in vitro mutagenesis. Recombinant
M. tuberculosis
clones containing plasmids with specific mutations in either codon 531 or 526 of
rpoB
exhibited high-level resistance to all rifamycins tested, whereas clones containing a plasmid with a mutation in codon 516 exhibited high-level resistance to rifampin and rifapentine but were susceptible to both rifabutin and KRM-1648. These results provided additional proof of the association of specific
rpoB
mutations with the development of rifamycin resistance and corroborate previous reports of the usefulness of
rpoB
genotyping for predicting rifamycin-resistant phenotypes.
Y-688, a New Quinolone Active against Quinolone-Resistant
Staphylococcus aureus
: Lack of In Vivo Efficacy in Experimental Endocarditis
Authors: Entenza J. M. , Marchetti O. , Glauser M. P. , Moreillon P.
Published: October 2018

Y-688 is a new fluoroquinolone with increased activity against ciprofloxacin-resistant staphylococci. The MICs of Y-688 and other quinolones were determined for 58 isolates of ciprofloxacin-resistant and methicillin-resistant
Staphylococcus aureus
(MRSA). The MICs at which 50% and 90% of bacteria were inhibited were ≥128 and ≥128 mg/liter, respectively, for ciprofloxacin, 16 and 32 mg/liter, respectively, for sparfloxacin, and 0.25 and 1 mg/liter, respectively, for Y-688. This new quinolone was further tested in rats with experimental endocarditis due to either of two isolates of ciprofloxacin-resistant MRSA (namely, P8/128 and CR1). Infected animals were treated for 3 days with ciprofloxacin, vancomycin, or Y-688. Antibiotics were administered through a computerized pump to simulate human-like pharmacokinetics in the serum of rats. The anticipated peak and trough levels of Y-688 were 4 and 1 mg/liter at 0.5 and 12 h, respectively. Treatment with ciprofloxacin was ineffective. Vancomycin significantly decreased vegetation bacterial counts for both organisms (
P
≲ 0.05). In contrast, Y-688 only marginally decreased vegetation bacterial counts (
P
≳ 0.05). Moreover, several vegetation that failed Y-688 treatment grew staphylococci for which the MICs of the test antibiotic were increased two to eight times. Y-688 also selected for resistance in vitro, and isolates for which the MICs were increased eight times emerged at a frequency of ca. 10
−8
. Thus, in spite of its low MIC for ciprofloxacin-resistant MRSA, Y-688 failed in vivo and its use carried the risk of resistance selection. The fact that ciprofloxacin-resistant staphylococci became rapidly resistant to this potent new drug suggests that the treatment of ciprofloxacin-resistant MRSA with new quinolones might be more problematic than expected.
Structure of CARB-4 and AER-1 CarbenicillinHydrolyzing β-Lactamases
Authors: Sanschagrin François , Bejaoui Noureddine , Levesque Roger C.
Published: October 2018

We determined the nucleotide sequences of

bla
CARB-4

encoding CARB-4 and deduced a polypeptide of 288 amino acids. The gene was characterized as a variant of group 2c carbenicillin-hydrolyzing β-lactamases such as PSE-4, PSE-1, and CARB-3. The level of DNA homology between the
bla
genes for these β-lactamases varied from 98.7 to 99.9%, while that between these genes and

bla
CARB-4

encoding CARB-4 was 86.3%. The

bla
CARB-4

gene was acquired from some other source because it has a G+C content of 39.1%, compared to a G+C content of 67% for typical
Pseudomonas aeruginosa
genes. DNA sequencing revealed that

bla
AER-1

shared 60.8% DNA identity with

bla
PSE-3

encoding PSE-3. The deduced AER-1 β-lactamase peptide was compared to class A, B, C, and D enzymes and had 57.6% identity with PSE-3, including an STHK tetrad at the active site. For CARB-4 and AER-1, conserved canonical amino acid boxes typical of class A β-lactamases were identified in a multiple alignment. Analysis of the DNA sequences flanking

bla
CARB-4

and

bla
AER-1

confirmed the importance of gene cassettes acquired via integrons in
bla
gene distribution.
Cloning and Nucleotide Sequences of the Topoisomerase IV
parC
and
parE
Genes of
Mycoplasma hominis
Authors: Bébéar Cécile M. , Charron Alain , Bové Joseph Marie , Bébéar Christiane , Renaudin Joel
Published: October 2018

The topoisomerase IV
parC
and
parE
genes from the wall-less organism
Mycoplasma hominis
PG21 were cloned and sequenced. The coupled genes are located far from the DNA gyrase genes
gyrA
and
gyrB
. They encode proteins of 639 and 866 amino acids, respectively. As expected, the encoded ParE and ParC proteins exhibit higher homologies with the topoisomerase IV subunits of the gram-positive bacteria
Staphylococcus aureus
and
Streptococcus pneumoniae
than with their
Escherichia coli
counterparts. The conserved regions include the Tyr residue of the active site and the region involved in quinolone resistance (quinolone resistance-determining region [QRDR]) in ParC and the ATP-binding site and the QRDR in ParE.
Prevalence of a Putative Efflux Mechanism among Fluoroquinolone-Resistant Clinical Isolates of
Streptococcus pneumoniae
Authors: Brenwald Nigel P. , Gill Martin J. , Wise Richard
Published: October 2018

Twenty-three norfloxacin-selected first-step mutants of
Streptococcus pneumoniae
showed low-level fluoroquinolone resistance. Their susceptibility to norfloxacin in the presence or absence of reserpine and known efflux pump substrates was determined by an agar dilution method. Five mutants showed four- to eightfold increases in their susceptibility to norfloxacin in the presence of reserpine and four- to eightfold decreases in their susceptibility to acriflavine and ethidium bromide. This phenotype is suggestive of an efflux mechanism of resistance. A representative of these mutants, 1N27, accumulated significantly less ethidium bromide than the parent strain; reserpine abolished these differences. No changes in the quinolone resistance-determining regions of
parC
,
parE
,
gyrA
, or
gyrB
were found in this mutant. By our validated agar dilution method, the efflux phenotype was sought in clinical isolates of
S. pneumoniae
. Of 1,037 clinical isolates examined from the United Kingdom, 273 showed reduced susceptibility to norfloxacin or ciprofloxacin. Of these, 45.4% showed the efflux phenotype. Our findings suggest that an efflux mechanism may be a frequent cause of clinically significant fluoroquinolone resistance in pneumococci.
Pharmacokinetics of [
18
F]Trovafloxacin in Healthy Human Subjects Studied with Positron Emission Tomography
Authors: Fischman Alan J. , Babich John W. , Bonab Ali A. , Alpert Nathaniel M. , Vincent John , Callahan Ronald J. , Correia John A. , Rubin Robert H.
Published: October 2018

Tissue pharmacokinetics of trovafloxacin, a new broad-spectrum fluoroquinolone antimicrobial agent, were measured by positron emission tomography (PET) with [
18
F]trovafloxacin in 16 healthy volunteers (12 men and 4 women). Each subject received a single oral dose of trovafloxacin (200 mg) daily beginning 5 to 8 days before the PET measurements. Approximately 2 h after the final oral dose, the subject was positioned in the gantry of the PET camera, and 1 h later 10 to 20 mCi of [
18
F]trovafloxacin was infused intravenously over 1 to 2 min. Serial PET images and blood samples were collected for 6 to 8 h, starting at the initiation of the infusion. Drug concentrations were expressed as the percentage of injected dose per gram, and absolute concentrations were estimated by assuming complete absorption of the final oral dose. In most tissues, there was rapid accumulation of the radiolabeled drug, with high levels achieved within 10 min after tracer infusion. Peak concentrations of more than five times the MIC at which 90% of the isolates are inhibited (MIC
90
) for most members of
Enterobacteriaceae
and anaerobes (>10-fold for most organisms) were achieved in virtually all tissues, and the concentrations remained above this level for more than 6 to 8 h. Particularly high peak concentrations (micrograms per gram; mean ± standard error of the mean [SEM]) were achieved in the liver (35.06 ± 5.89), pancreas (32.36 ± 20.18), kidney (27.20 ± 10.68), lung (22.51 ± 7.11), and spleen (21.77 ± 11.33). Plateau concentrations (measured at 2 to 8 h; micrograms per gram; mean ± SEM) were 3.25 ± 0.43 in the myocardium, 7.23 ± 0.95 in the lung, 11.29 ± 0.75 in the liver, 9.50 ± 2.72 in the pancreas, 4.74 ± 0.54 in the spleen, 1.32 ± 0.09 in the bowel, 4.42 ± 0.32 in the kidney, 1.51 ± 0.15 in the bone, 2.46 ± 0.17 in the muscle, 4.94 ± 1.17 in the prostate, and 3.27 ± 0.49 in the uterus. In the brain, the concentrations (peak, ∼2.63 ± 1.49 μg/g; plateau, ∼0.91 ± 0.15 μg/g) exceeded the MIC
90
s for such common causes of central nervous system infections as
Streptococcus pneumoniae
(MIC
90
, <0.2 μg/ml),
Neisseria meningitidis
(MIC
90
, <0.008 μg/ml), and
Haemophilus influenzae
(MIC
90
, <0.03 μg/ml). These PET results suggest that trovafloxacin will be useful in the treatment of a broad range of infections at diverse anatomic sites.
Topoisomerase Mutations in Trovafloxacin-Resistant
Staphylococcus aureus
Authors: Fitzgibbon Joseph E. , John Joseph F. , Delucia Jennifer L. , Dubin Donald T.
Published: October 2018

A total of 201
Staphylococcus aureus
isolates were surveyed for susceptibility to ciprofloxacin and trovafloxacin. Of 66 methicillin-resistant isolates, 89% were ciprofloxacin resistant and 6% were also trovafloxacin resistant. Trovafloxacin-resistant strains had unusual patterns of quinoline resistance mutations in DNA topoisomerase genes, including two mutations in the A subunit (encoded by
grlA
) of topoisomerase IV.
The M539V Polymerase Variant of Human Hepatitis B Virus Demonstrates Resistance to 2′-Deoxy-3′-Thiacytidine and a Reduced Ability to Synthesize Viral DNA
Authors: Ladner Stephanie K. , Miller Thomas J. , King Robert W.
Published: October 2018

The cytosine analog 2′-deoxy-3′-thiacytidine (3TC) has been shown to be an effective treatment for chronic hepatitis B virus (HBV) infection. However, several liver transplant patients who were undergoing treatment with 3TC for HBV infection experienced a breakthrough of virus while on 3TC. The predominant virus found in these patients’ sera contained either a valine or isoleucine for the methionine in the highly conserved YMDD nucleotide binding site in the HBV polymerase. To determine the biological relevance of the Met-to-Val substitution, we mutated a plasmid that contained a cDNA copy of the HBV pregenomic RNA such that when virus replication occurred during transient transfection of HepG2 cells, an M539V polymerase variant was produced. We found that in transiently transfected cells, this variant was approximately 330-fold less sensitive to the antiviral effects of 3TC and produced 7-fold less viral DNA than the wild type.
Iron-Limited Biofilms of
Candida albicans
and Their Susceptibility to Amphotericin B
Authors: Baillie George S. , Douglas L. Julia
Published: October 2018

Biofilms of
Candida albicans
were grown in vitro under iron limitation and at a low growth rate to simulate conditions for implant-associated biofilms in vivo. Their properties were compared with those of glucose-limited biofilms grown under analogous conditions. At steady state, the adherent cell populations of iron-limited biofilms were double those of glucose-limited biofilms, although the growth rates were similar (0.038 to 0.043 h
−1
). Both biofilm types were resistant to amphotericin B, but daughter cells from iron-limited biofilms were significantly more susceptible to the drug than those from glucose-limited biofilms.
Pharmacokinetics and Distribution over the Blood-Brain Barrier of Zalcitabine (2′,3′-Dideoxycytidine) and BEA005 (2′,3′-Dideoxy-3′-Hydroxymethylcytidine) in Rats, Studied by Microdialysis
Authors: Borg Natalia , Ståhle Lars
Published: October 2018

Microdialysis was applied to sample the unbound drug concentration in the extracellular fluid in brain and muscle of rats given zalcitabine (2′,3′-dideoxycytidine;
n
= 4) or BEA005 (2′,3′-dideoxy-3′-hydroxymethylcytidine;
n
= 4) (50 mg/kg of body weight given subcutaneously). Zalcitabine and BEA005 were analyzed by high-pressure liquid chromatography with UV detection. The maximum concentration of zalcitabine in the dialysate (
C
max
) was 31.4 ± 5.1 μM (mean ± standard error of the mean) for the brain and 238.3 ± 48.1 μM for muscle. The time to
C
max
was found to be from 30 to 45 min for the brain and from 15 to 30 min for muscle. Zalcitabine was eliminated from the brain and muscle with half-lives 1.28 ± 0.64 and 0.85 ± 0.13 h, respectively. The ratio of the area under the concentration-time curve (AUC) (from 0 to 180 min) for the brain and the AUC for muscle (AUC ratio) was 0.191 ± 0.037. The concentrations of BEA005 attained in the brain and muscle were lower than those of zalcitabine, with
C
max
s of 5.7 ± 1.4 μM in the brain and 61.3 ± 12.0 μM in the muscle. The peak concentration in the brain was attained 50 to 70 min after injection, and that in muscle was achieved 30 to 50 min after injection. The half-lives of BEA005 in the brain and muscle were 5.51 ± 1.45 and 0.64 ± 0.06 h, respectively. The AUC ratio (from 0 to 180 min) between brain and muscle was 0.162 ± 0.026. The log octanol/water partition coefficients were found to be −1.19 ± 0.04 and −1.47 ± 0.01 for zalcitabine and BEA005, respectively. The degrees of plasma protein binding of zalcitabine (11% ± 4%) and BEA005 (18% ± 2%) were measured by microdialysis in vitro. The differences between zalcitabine and BEA005 with respect to the AUC ratio (
P
= 0.481), half-life in muscle (
P
= 0.279), and level of protein binding (
P
= 0.174) were not statistically significant. The differences were statistically significant in the case of the half-life in the brain (
P
= 0.032), clearance (
P
= 0.046), volume of distribution (
P
= 0.027) in muscle, and octanol/water partition coefficient (
P
= 0.019).
Granulocyte Colony-Stimulating Factor Enhances Endotoxin-Induced Decrease in Biliary Excretion of the Antibiotic Cefoperazone in Rats
Authors: Nadai Masayuki , Matsuda Izumi , Wang Li , Itoh Akio , Naruhashi Kazumasa , Nabeshima Toshitaka , Asai Masaki , Hasegawa Takaaki
Published: October 2018

We have recently reported that endotoxin (lipopolysaccharide [LPS]) derived fromKlebsiella pneumoniaedramatically decreased the biliary excretion of the β-lactam antibiotic cefoperazone (CPZ), which is primarily excreted into the bile via the anion transport system, in rats. The present study was designed to investigate the effect of human recombinant granulocyte colony-stimulating factor (G-CSF), which is reported to be beneficial in experimental models of inflammation, on the pharmacokinetics and biliary excretion of CPZ in rats. CPZ (20 mg/kg of body weight) was administered intravenously 2 h after the intravenous injection of LPS (250 μg/kg). G-CSF was injected subcutaneously at 12 μg/kg for 3 days and was administered intravenously at a final dose of 50 μg/kg 1 h before LPS injection. Peripheral blood cell numbers were also measured. LPS dramatically decreased the systemic and biliary clearances of CPZ and the bile flow rate. Pretreatment with G-CSF enhanced these decreases induced by LPS. The total leukocyte numbers were increased in rats pretreated with G-CSF compared to the numbers in the controls, while the total leukocyte numbers were decreased (about 3,000 cells/μl) by treatment with LPS. Pretreatment with G-CSF produces a deleterious effect against the LPS-induced decrease in biliary secretion of CPZ, and leukocytes play an important role in that mechanism.
Pharmacodynamic Analysis of the Activity of Quinupristin-Dalfopristin against Vancomycin-Resistant
Enterococcus faecium
with Differing MBCs via Time-Kill-Curve and Postantibiotic Effect Methods
Authors: Aeschlimann Jeffrey R. , Rybak Michael J.
Published: October 2018

Quinupristin-dalfopristin (Q-D) is a new water-soluble, semisynthetic antibiotic that is derived from natural streptogramins and that is combined in a 30:70 ratio. A number of studies have described the pharmacodynamic properties of this drug, but most have investigated only staphylococci or streptococci. We evaluated the relationship between Q-D, quinupristin (Q), and/or dalfopristin (D) susceptibility parameters and antibacterial activities against 22 clinical isolates of vancomycin-resistant
Enterococcus faecium
(VREF) by using the concentration-time-kill-curve method and by measuring postantibiotic effects. Q-D, Q, and D MICs and minimum bactericidal concentrations (MBCs) ranged from 0.125 to 1 and 0.25 to 64, 8 to 512 and >512, and 2 to 8 and 8 to 512 μg/ml, respectively. There were no significant relationships between susceptibilities to the individual components and the susceptibilities to the Q-D combination product. In the time-kill-curves studies, Q-D at a concentration of 6 μg/ml was at least bacteriostatic against all VREF tested. There was increased activity against more susceptible isolates when the isolates were grouped either by Q-D MBCs or by Q MICs. By multivariate regression analyses, the percent change in the inoculum from that at the baseline was significantly correlated with the Q MIC (
R
= 0.74;
P
= 0.008) and the Q-D concentration-to-MBC ratio (
R
= 0.58;
P
= 0.02) and was inversely correlated with the Q-D MBC-to-MIC ratio (
R
= 0.68;
P
= 0.003). A strong correlation existed between the killing rate and the Q-D concentration-to-MBC ratio (
R
= 0.99;
P
< 0.0001). Time to 99.9% killing was best correlated with the Q-D MBC (
R
= 0.96;
P
< 0.0001). The postantibiotic effect ranged from 0.2 to 3.2 h and was highly correlated with the Q-D concentration-to-MBC ratio (
R
= 0.96;
P
< 0.0001) and was less highly correlated with the Q MIC (
R
= 0.42;
P
= 0.04). Further study of these relationships with in vitro or in vivo infection models that simulate Q-D pharmacokinetics should further define the utility of these pharmacodynamic parameters in the prediction of Q-D activity for the treatment of VREF infections in humans.