bluuebunny/crossref_metadata_2025 huggingface dataset:
Phenotypic and Genetic Characterization of Thymidine Kinase from Clinical Strains of Varicella-Zoster Virus Resistant to Acyclovir
Authors: Morfin Florence , Thouvenot Danielle , De Turenne-Tessier Mireille , Lina Bruno , Aymard Michèle , Ooka Tadamasa
Published: October 2018

Varicella-zoster virus (VZV) is a common herpesvirus responsible for disseminated or chronic infections in immunocompromised patients. Effective drugs such as acyclovir (ACV), famciclovir (prodrug of penciclovir), and foscarnet are available to treat these infections. Here we report the phenotypic and genetic characterization of four ACV-resistant VZV strains isolated from AIDS patients and transplant recipients. Sensitivity to six antiviral drugs was determined by an enzyme-linked immunosorbent assay, viral thymidine kinase (TK) activity was measured by comparing [
3
H]thymidine and 1-β-
d
-arabinofuranosyl-[
3
H]thymine as substrates, and the TK gene open reading frame was sequenced. Three strains were found to be TK deficient, and the fourth was a mixed population composed of TK-positive and TK-deficient viruses. Each strain presented a unique TK gene mutation that could account for ACV resistance. In one strain, the deletion of two nucleotides at codon 215 induced a premature stop signal at codon 217. In another strain, a single nucleotide addition at codon 167 resulted in a premature stop signal at codon 206. In both other strains, we identified amino acid substitutions already described in other ACV-resistant VZV strains: either Glu→Gly at residue 48 or Arg→Gly at residue 143. According to our work and data previously reported on resistant VZV strains, there are three areas in the TK gene where 71% of the mutations described to date are located. These areas are putative candidates for a genotypic diagnosis of ACV resistance.
Propagation of TEM- and PSE-Type β-Lactamases among Amoxicillin-Resistant
Salmonella
spp. Isolated in France
Authors: Llanes Catherine , Kirchgesner Veronique , Plesiat Patrick
Published: October 2018

A survey conducted between 1987 and 1994 at the University Hospital of Besançon, France, demonstrated a dramatic increase (from 0 to 42.5%) in the prevalence of amoxicillin resistance among
Salmonella
spp. Of the 96 resistant isolates collected during this period (including 77 Typhimurium), 54 were found to produce TEM-1 β-lactamase, 40 produced PSE-1 (equivalent to CARB-2), one produced PSE-1 plus TEM-2, and one produced OXA-1 in isoelectric focusing and DNA hybridization experiments. Plasmids coding for these β-lactamases were further characterized by (i) profile analysis, (ii) restriction fragmentation pattern analysis, (iii) hybridization with an
spvCD-orfE
virulence probe, and (iv) replicon typing. In addition, isolates of
S. typhimurium
were genotypically compared by pulsed-field gel electrophoresis of
Xba
I-macrorestricted chromosomal DNA. Altogether, these methods showed that 40 of the 41 PSE-1 producers were actually the progeny of a single epidemic
S. typhimurium
strain lysotype DT104. Isolates of that strain were found to harbor RepFIC virulence plasmids with somewhat different restriction profiles, but which all carried the
bla
PSE-1
gene. Of these virulence/resistance plasmids, 15 were transmissible to
Escherichia coli
. TEM-1-producing
S. typhimurium
displayed much greater genotypic and plasmidic diversities, suggesting the acquisition of the
bla
TEM-1
gene from multiple bacterial sources by individual strains. In agreement with this, 32 of the 35
S. typhimurium
plasmids encoding TEM-1 were found to be conjugative. These data show that development of amoxicillin resistance among
Salmonella
, especially in serovar Typhimurium, results from both gene transfers and strain dissemination.
Metabolism and Selective Toxicity of 6-Nitrobenzylthioinosine in
Toxoplasma gondii
Authors: el Kouni Mahmoud H. , Guarcello Vincenzo , Al Safarjalani Omar N. , Naguib Fardos N. M.
Published: October 2018

The purine nucleoside analogue NBMPR {nitrobenzylthioinosine or 6-[(4-nitrobenzyl)thio]-9-β-
d
-ribofuranosylpurine} was selectively phosphorylated to its nucleoside 5′-monophosphate by
Toxoplasma gondii
but not mammalian adenosine kinase (EC
2.7.1.20
). NBMPR was also cleaved in toxoplasma to its nucleobase, nitrobenzylmercaptopurine. However, nitrobenzylmercaptopurine was not a substrate for either adenosine kinase or hypoxanthine-guanine-xanthine phosphoribosyltransferase (EC
2.4.2.8
). Because of this unique and previously unknown metabolism of NBMPR by the parasite, the effect of NBMPR as an antitoxoplasmic agent was tested. NBMPR killed
T. gondii
grown in human fibroblasts in a dose-dependent manner, with a 50% inhibitory concentration of approximately 10 μM and without apparent toxicity to host cells. Doses of up to 100 μM had no significant toxic effect on uninfected host cells. The promising antitoxoplasmic effect of NBMPR led to the testing of other 6-substituted 9-β-
d
-ribofuranosylpurines, which were shown to be good ligands of the parasite adenosine kinase (M. H. Iltzsch, S. S. Uber, K. O. Tankersley, and M. H. el Kouni, Biochem. Pharmacol. 49:1501–1512, 1995), as antitoxoplasmic agents. Among the analogues tested, 6-benzylthioinosine,
p
-nitrobenzyl-6-selenopurine riboside,
N
6
-(
p
-azidobenzyl)adenosine, and
N
6
-(
p
-nitrobenzyl)adenosine, like NBMPR, were selectively toxic to parasite-infected cells. Thus, it appears that the unique characteristics of purine metabolism in
T. gondii
render certain 6-substituted 9-β-
d
-ribofuranosylpurines promising antitoxoplasmic drugs.
Safety and Efficacy of Multilamellar Liposomal Nystatin against Disseminated Candidiasis in Persistently Neutropenic Rabbits
Authors: Groll Andreas H. , Petraitis Vidmantas , Petraitiene Ruta , Field-Ridley Aida , Calendario Myrna , Bacher John , Piscitelli Stephen C. , Walsh Thomas J.
Published: October 2018

The activity of liposomal nystatin (L-Nys) against subacute disseminated candidiasis was investigated in persistently neutropenic rabbits. Antifungal therapy was administered for 10 days starting 24 h after intravenous inoculation of 10
3
blastoconidia of
Candida albicans
. Responses to treatment were assessed by the quantitative clearance of the organism from blood and tissues. Treatments consisted of L-Nys at dosages of 2 and 4 mg/kg of body weight/day (L-Nys2 and L-Nys4, respectively) amphotericin B deoxycholate at 1 mg/kg/day (D-AmB), and fluconazole at 10 mg/kg/day (Flu). All treatments were given intravenously once daily. Compared to the results for untreated but infected control animals, treatment with L-Nys2, L-Nys4, D-AmB, and Flu resulted in a significant clearance of the residual burden of
C. albicans
from the kidney, liver, spleen, lung, and brain (
P
< 0.0001 by analysis of variance). When the proportion of animals infected at at least one of the five tissue sites studied was evaluated, a dose-dependent response to treatment with L-Nys was found (
P
< 0.05). Compared to D-AmB-treated rabbits, mean serum creatinine and blood urea nitrogen levels at the end of therapy were significantly lower in animals treated with L-Nys2 (
P
< 0.001) and L-Nys4 (
P
< 0.001 and
P
< 0.01, respectively). L-Nys was less nephrotoxic than conventional amphotericin B and had dose-dependent activity comparable to that of amphotericin B for the early treatment of subacute disseminated candidiasis in persistently neutropenic rabbits.
Use of Pharmacodynamic Indices To Predict Efficacy of Combination Therapy In Vivo
Authors: Mouton Johan W. , van Ogtrop Marc L. , Andes David , Craig William A.
Published: October 2018

Although combination therapy with antimicrobial agents is often used, no available method explains or predicts the efficacies of these combinations satisfactorily. Since the efficacies of antimicrobial agents can be described by pharmacodynamic indices (PDIs), such as area under the concentration-time curve (AUC), peak level, and the time that the concentration is above the MIC (time>MIC), it was hypothesized that the same PDIs would be valid in explaining efficacy during combination therapy. Twenty-four-hour efficacy data (numbers of CFU) for
Pseudomonas aeruginosa
in a neutropenic mouse thigh model were determined for various combination regimens: ticarcillin-tobramycin (
n
= 41 different regimens), ceftazidime-netilmicin (
n
= 60), ciprofloxacin-ceftazidime (
n
= 59), netilmicin-ciprofloxacin (
n
= 38) and for each of these agents given singly. Multiple regression analysis was used to determine the importance of various PDIs (time>MIC, time>0.25× the MIC, time>4× the MIC, peak level, AUC, AUC/MIC, and their logarithmically transformed values) during monotherapy and combination therapy. The PDIs that best explained the efficacies of single-agent regimens were time>0.25× the MIC for beta-lactams and log AUC/MIC for ciprofloxacin and the aminoglycosides. For the combination regimens, regression analysis showed that efficacy could best be explained by the combination of the two PDIs that each best explained the response for the respective agents given singly. A regression model for the efficacy of combination therapy was developed by use of a linear combination of the regression models of the PDI with the highest
R
2
for each agent given singly. The model values for the single-agent therapies were then used in that equation, and the predicted values that were obtained were compared with the experimental values. The responses of the combination regimens could best be predicted by the sum of the responses of the single-agent regimens as functions of their respective PDIs (e.g., time>0.25× the MIC for ticarcillin and log AUC/MIC for tobramycin). The relationship between the predicted response and the observed response for the combination regimens may be useful for determination of the presence of synergism. We conclude that the PDIs for the individual drugs used in this study are class dependent and predictive of outcome not only when the drugs are given as single agents but also when they are given in combination. When given in combination, there appears to be a degree of synergism independent of the dosing regimen applied.
Development of a New Experimental Model of Penicillin-Resistant
Streptococcus pneumoniae
Pneumonia and Amoxicillin Treatment by Reproducing Human Pharmacokinetics
Authors: Piroth Lionel , Martin Laurent , Coulon Alexis , Lequeu Catherine , Duong Michel , Buisson Marielle , Portier Henri , Chavanet Pascal
Published: October 2018

The increase of penicillin-resistant
Streptococcus pneumoniae
(PRSP) pneumonia results in a greater risk of antibiotic treatment failure. In vitro data are not sufficient predictors of clinical efficacy, and animal models may be insufficiently contributive, since they often use immunocompromised animals and do not always respect the human pharmacokinetics of antibiotics. We developed an experimental PRSP pneumonia model in immunocompetent rabbits, by using intrabronchial instillation of PRSP (MIC = 4 mg/liter), without any adjuvant. This reproducible model was used to assess amoxicillin efficacy by reproducing human serum pharmacokinetics following 1-g oral or intravenous administrations of amoxicillin every 8 h. Evaluation was performed by using clinical, CT scan, macroscopic, histopathologic, and microbiological criteria. Experimental pneumonia in untreated rabbits was similar to untreated severe human bacteremic untreated pneumonia; in both rabbits and humans, (i) cumulative survival was close to 50%, (ii) red or gray lung congestion and pleuritis were observed, and (iii) lung and spleen concentrations reached 5 and 4 log
10
CFU/g. A 48-h treatment resulted in a significant bacterial clearance in the lungs (1.53 versus 5.07 log
10
CFU/ml,
P
< 0.001) and spleen (1.00 versus 4.40 log
10
CFU/ml,
P
< 10
−6
) and a significant decrease in mortality (0% versus 50%,
P
= 0.02) in treated versus untreated rabbits. No difference was observed on macroscopic and histopathologic lesions between treated and untreated rabbits (
P
= 0.36 and 0.78, respectively). Similar results were obtained by using a fully penicillin-susceptible
S. pneumoniae
strain (MIC = 0.01 mg/liter). Our findings suggest that (i) this new model can be contributive in the evaluation of antibacterial agents and (ii) 1 g of amoxicillin three times a day may be sufficient to treat PRSP pneumonia in immunocompetent humans.
Anti-Kaposi’s Sarcoma and Antiangiogenic Activities of Sulfated Dextrins
Authors: Thornton Mark , Barkley Laura , Mason Justin C. , Shaunak Sunil
Published: October 2018

Delivery of the sulfated polysaccharide dextrin 2-sulfate by the intraperitoneal route to the lymphatic circulation resulted in a clinically significant improvement in Kaposi’s sarcoma in three patients. Our in vitro studies show that although sulfated dextrins do not interfere with the growth of isolated human umbilical vein endothelial cells, they do inhibit the morphological differentiation of endothelial cells into tubes as well as reduce new vessel formation in a placental angiogenesis assay. The antiangiogenic effect of dextrin 6-sulfate is greater than that of dextrin 2-sulfate and is independent of their anti-human immunodeficiency virus type 1 activities.
Antimicrobial Susceptibility Patterns of Enterococci Causing Infections in Europe
Authors: Schouten M. A. , Voss A. , Hoogkamp-Korstanje J. A. A. ,   The European VRE Study Group
Published: October 2018

In vitro susceptibilities of 4,208 enterococci (83%
Enterococcus faecalis
isolates, 13.6%
Enterococcus faecium
isolates, and 3.4% isolates of other species) from patients in 27 European countries towards 16 antibiotics were determined. High-level resistance to gentamicin varied by country (range, 1 to 49%; mean, 22.6% ± 12.3%) and per species (19.7%
E. faecalis
isolates, 13.6%
E. faecium
isolates, 3.4% by other species). Vancomycin resistance was detected in 0.06%
E. faecalis
, 3.8%
E. faecium
, and 19.1% isolates of other species. All enterococci were susceptible to LY 333328 and everninomicin, and 25% of
E. faecalis
isolates and 85% of other enterococci were susceptible to quinupristin-dalfopristin. The MIC of moxifloxacin and trovafloxacin for ciprofloxacin-susceptible
E. faecalis
at which 90% of the isolates were inhibited was 0.25 to 0.5 μg/ml.
Postantibiotic Effects of Gatifloxacin against Gram-Positive and -Negative Organisms
Authors: Pankuch G. A. , Jacobs M. R. , Appelbaum P. C.
Published: October 2018

Gatifloxacin pneumococcal, staphylococcal and enterococcal postantibiotic effects (PAEs) were 0.5 to 4.0 h, respectively. For
Escherichia coli
and
Pseudomonas aeruginosa
, PAEs were 2.2 to 4.8 h. Pneumococcal, staphylococcal, and enterococcal postantibiotic sub-MIC effects (PA-SMEs) (four times the MICs) were 3.7 to 8.6, 2.3 to 3.8, and 1.6 h, respectively, and
E. coli
and
P. aeruginosa
PA-SMEs were ≥9.6 and 4.4 h, respectively.
Activities of Fluoroquinolones against
Streptococcus pneumoniae
Type II Topoisomerases Purified as Recombinant Proteins
Authors: Morrissey Ian , George John
Published: October 2018

Streptococcus pneumoniae
topoisomerase IV and DNA gyrase have been purified from a fluoroquinolone-susceptible
Streptococcus pneumoniae
strain, from first-step mutants showing low-level resistance to ciprofloxacin, sparfloxacin, levofloxacin, and ofloxacin, and from two clinical isolates showing intermediate- and high-level fluoroquinolone resistance by a gene cloning method that produces recombinant proteins from
Escherichia coli
. The concentrations of ciprofloxacin, sparfloxacin, levofloxacin, or ofloxacin required to inhibit wild-type topoisomerase IV were 8 to 16 times lower than those required to inhibit wild-type DNA gyrase. Furthermore, low-level resistance to these fluoroquinolones was entirely due to the reduced inhibitory activity of fluoroquinolones against topoisomerase IV. For all the laboratory strains, the 50% inhibitory concentration for topoisomerase IV directly correlated with the MIC. We therefore propose that with
S. pneumoniae
, ciprofloxacin, sparfloxacin, levofloxacin, and ofloxacin target topoisomerase IV in preference to DNA gyrase. Sitafloxacin, on the other hand, was found to be equipotent against either enzyme. This characteristic is unique for a fluoroquinolone. A reduction in the sensitivities of both topoisomerase IV and DNA gyrase are required, however, to achieve intermediate- or high-level fluoroquinolone resistance in
S. pneumoniae
.
Involvement of an Active Efflux System in the Natural Resistance of
Pseudomonas aeruginosa
to Aminoglycosides
Authors: Aires Julio Ramos , Köhler Thilo , Nikaido Hiroshi , Plésiat Patrick
Published: October 2018

A mutant, named 11B, hypersusceptible to aminoglycosides, tetracycline, and erythromycin was isolated after Tn
501
insertion mutagenesis of
Pseudomonas aeruginosa
PAO1. Cloning and sequencing experiments showed that 11B was deficient in an, at that time, unknown active efflux system that contains homologs of MexAB. This locus also contained a putative regulatory gene,
mexZ
, transcribed divergently from the efflux operon. Introduction of a recombinant plasmid that carries the genes of the efflux system restored the resistance of 11B to parental levels, whereas overexpression of these genes strongly increased the MICs of substrate antibiotics for the PAO1 host. Antibiotic accumulation studies confirmed that this new system is an energy-dependent active efflux system that pumps out aminoglycosides. Furthermore, this system appeared to function with an outer membrane protein, OprM. While the present paper was being written and reviewed, genes with a sequence identical to our pump genes,
mexXY
of
P. aeruginosa
, have been reported to increase resistance to erythromycin, fluoroquinolones, and organic cations in
Escherichia coli
hosts, although efflux of aminoglycosides was not examined (Mine et al., Antimicrob. Agents Chemother. 43:415–417, 1999). Our study thus shows that the MexXY system plays an important role in the intrinsic resistance of
P. aeruginosa
to aminoglycosides. Although overexpression of MexXY increased the level of resistance to fluoroquinolones, disruption of the
mexXY
operon in
P. aeruginosa
had no detectable effect on susceptibility to these agents.
Erythromycin Inhibits Transcriptional Activation of NF-κB, but not NFAT, through Calcineurin-Independent Signaling in T Cells
Authors: Aoki Yosuke , Kao Peter N.
Published: October 2018

The molecular mechanism of the anti-inflammatory effect of erythromycin (EM) was investigated at the level of transcriptional regulation of cytokine gene expression in T cells. EM (>10−6M) significantly inhibited interleukin-8 (IL-8) expression but not IL-2 expression from T cells induced with 20 ng of phorbol 12-myristate 13-acetate (PMA) per ml plus 2 μM calcium ionophore (P-I). In electrophoretic mobility shift assays EM at 10−7to 10−5M concentrations inhibited nuclear factor kappa B (NF-κB) DNA-binding activities induced by P-I. Reporter gene assays also showed that EM (10−5M) inhibited IL-8 NF-κB transcription by 37%. The inhibitory effects of EM on transcriptional activation of IL-2 and DNA-binding activity of nuclear factor of activated T cells (NFAT) were not seen in T cells. On the other hand, FK506, which is also a macrolide derivative, inhibited transcriptional activation of both NF-κB and NFAT more strongly than EM did. The mechanism of EM inhibition of transactivation of NF-κB was further investigated in transiently transfected T cells that express calcineurin A and B subunits. Expression of calcineurin did not render transactivation of NF-κB in T cells more resistant to EM, while the inhibitory effect of FK506 on transactivation of NF-κB was attenuated. These findings indicate that EM is capable of inhibiting expression of the IL-8 gene in T cells through transcriptional inhibition and that this inhibition is mediated through a non-calcineurin-dependent signaling event in T lymphocytes.
Specific Inhibition of the Eubacterial DNA Ligase by Arylamino Compounds
Authors: Ciarrocchi Giovanni , MacPhee Donald G. , Deady Les W. , Tilley Leann
Published: October 2018

All known DNA ligases catalyze the formation of a phosphodiester linkage between adjacent termini in double-stranded DNA via very similar mechanisms. The ligase family can, however, be divided into two classes: eubacterial ligases, which require NAD
+
as a cofactor, and other ligases, from viruses, archaea, and eukaryotes, which use ATP. Drugs that discriminate between DNA ligases from different sources may have antieubacterial activity. We now report that a group of arylamino compounds, including some commonly used antimalarial and anti-inflammatory drugs and a novel series of bisquinoline compounds, are specific inhibitors of eubacterial DNA ligases. Members of this group of inhibitors have different heterocyclic ring systems with a common amino side chain in which the two nitrogens are separated by four carbon atoms. The potency, but not the specificity of action, is influenced by the DNA-binding characteristics of the inhibitor, and the inhibition is noncompetitive with respect to NAD
+
. The arylamino compounds appear to target eubacterial DNA ligase in vivo, since a
Salmonella
Lig
−
strain that has been rescued with the ATP-dependent T4 DNA ligase is less sensitive than the parental
Salmonella
strain.
In Vitro Susceptibilities of
Neisseria gonorrhoeae
to Fatty Acids and Monoglycerides
Authors: Bergsson Gudmundur , Steingrímsson Ólafur , Thormar Halldor
Published: October 2018

The susceptibility of
Neisseria gonorrhoeae
to several medium-chain fatty acids and their 1-monoglycerides was tested at a short inactivation time of 1 min. The results indicate that monocaprin, a monoglyceride of capric acid (10 carbon atoms, no double bonds), causes the fastest and most effective killing of all strains of
N. gonorrhoeae
tested.
Molecular Characterization of
rpoB
Mutations Conferring Cross-Resistance to Rifamycins on Methicillin-Resistant
Staphylococcus aureus
Authors: Wichelhaus Thomas A. , Schäfer Volker , Brade Volker , Böddinghaus Boris
Published: October 2018

Mutations of the
rpoB
gene conferring resistance to rifampin were analyzed in 40 methicillin-resistant
Staphylococcus aureus
isolates obtained from six countries. Interestingly, the majority of clinical isolates showed multiple mutations within
rpoB
. The amino acid substitution 481His→Asn was the most prevalent one, capable of conferring low-level resistance on its own. Cross-resistance to rifampin, rifabutin, and rifapentine was demonstrated for all mutants identified. The level of resistance to rifamycins correlated with both the mutation position and type of amino acid substitution.
Potentiation of Vancomycin-Induced Histamine Release by Muscle Relaxants and Morphine in Rats
Authors: Shuto Hideki , Sueyasu Masanori , Otsuki Shuji , Hara Tomoko , Tsuruta Yuki , Kataoka Yasufumi , Oishi Ryozo
Published: October 2018

The intravenous injection of vancomycin sometimes causes anaphylactoid reactions, in which histamine release may play a major role. These reactions are more frequently manifested when vancomycin is injected into anesthetized patients. We examined the vancomycin-induced histamine release and the interaction of vancomycin with muscle relaxants or opioid in rats. In an in vitro study with rat peritoneal mast cells, treatment with vancomycin at concentrations of greater than 1.25 mM produced significant histamine release. Tubocurarine, vecuronium, pancuronium, succinylcholine, and morphine up to concentrations of 0.25, 1, 5, 30, and 5 mM, respectively, produced no significant histamine release. However, the nonsignificant histamine release induced by 0.5 mM vancomycin was clearly enhanced by combining vancomycin with any of these agents. In the in vivo study, the intravenous injection of vancomycin significantly increased the plasma histamine levels in rats when vancomycin was injected at 200 mg/kg of body weight (63.2 ± 34.0 ng/ml [mean ± standard deviation]) but not when it was injected at 100 mg/kg (30.8 ± 20.2 ng/ml) compared with that in the saline-treated rats (22.5 ± 11.4 ng/ml). Although the subcutaneous administration of morphine (10 mg/kg) never increased the plasma histamine levels, the intravenous injection of vancomycin (100 mg/kg) 30 min after this morphine treatment markedly increased the plasma histamine levels (56.0 ± 26.9 ng/ml). These findings provide experimental evidence that the combination of muscle relaxants or an opioid with vancomycin may increase the risk of anaphylactoid reactions by enhancing the release of histamine.
Biochemical Characterization of Novel Tetrahydrofuranyl 1β-Methylcarbapenems: Stability to Hydrolysis by Renal Dehydropeptidases and Bacterial β-Lactamases, Binding to Penicillin Binding Proteins, and Permeability Properties
Authors: Yang Youjun , Testa Raymond T. , Bhachech Niraja , Rasmussen Beth A. , Bush Karen
Published: October 2018

The biochemical properties of tetrahydrofuranyl (THF) carbapenems, carbapenems with THF substituents, were evaluated with respect to enzyme stability, binding to penicillin-binding proteins (PBPs), and penetration into gram-negative organisms. THF carbapenems showed increased stability to hog renal dehydropeptidases (DHPs) compared to that of imipenem or meropenem and were more stable to human DHP than imipenem (<10% hydrolysis compared to that for imipenem). THF carbapenems were stable to hydrolysis by all serine β-lactamases tested. CL 191,121, a prototype THF carbapenem, was more stable to hydrolysis by carbapenem-hydrolyzing serine β-lactamases such as IMI-1 and Sme-1 than imipenem, with a relative
k
cat
value of <20% for imipenem. Similar to imipenem and meropenem, THF carbapenems were not stable to the metallo β-lactamases CcrA and L1. However, CL 191,121 bound to all
Staphylococcus aureus
PBPs at concentrations that were less than or equal to the MICs. The THF carbapenems bound to PBPs from
Escherichia coli
and
Pseudomonas aeruginosa
, with the highest affinities being for PBPs 2 and 4, as noted with imipenem. The affinities for PBPs 1a and 1b in
E. coli
were reduced for the THF carbapenems compared to that for imipenem, even though the MICs of the THF carbapenems for
E. coli
strains were lower than those of imipenem. The penetrability of the THF carbapenems into
Serratia marcescens
S6, which produces the Sme-1 carbapenem-hydrolyzing β-lactamase, was 2.4 to 7.8 times less than that of imipenem. Compounds CL 190,294 and CL 188,624 showed good penetrability, with permeability coefficient values comparable to those of the rapidly penetrating agents cephaloridine, imipenem, meropenem, and biapenem. Decreased penetration into wild-type
P. aeruginosa
was suggested by the high MICs of the THF carbapenems (MICs, 16 to 32 μg/ml), despite equivalent or better binding to
P. aeruginosa
PBPs than that of imipenem. However, the MICs of the THF carbapenems for wild-type
P. aeruginosa
compared to that for an OprD2 mutant generally varied no more than 2-fold, but those of imipenem and other carbapenems differed 16-fold. These data indicated that THF carbapenems do not appear to enter through protein OprD2. In conclusion, the THF carbapenems exhibited stability to hydrolysis by renal DHPs and serine β-lactamases, exhibited strong binding to essential PBPs from
E. coli
and
S. aureus
, and penetrated gram-negative enteric bacteria at rates comparable to those for meropenem and biapenem.
Proteoglycan and Collagen Biochemical Variations during Fluoroquinolone-Induced Chondrotoxicity in Mice
Authors: Simonin Marie-Agnès , Gegout-Pottie Pascale , Minn Alain , Gillet Pierre , Netter Patrick , Terlain Bernard
Published: October 2018

Although fluoroquinolone antibacterials have a broad therapeutic use, with a relatively low incidence of severe side effects, they have been reported to induce lesions in the cartilage of growing animals by a mechanism that remains unclear. This study was undertaken to determine the potentially deleterious effect of a high dose of pefloxacin (400 mg/kg of body weight) on two main constituents of cartilage in mice, i.e., proteoglycans and collagen. Variations in levels of proteoglycan anabolism measured by in vivo [
35
S]sulfate incorporation into cartilage and oxidative modifications of collagen assessed by detection of carbonyl derivatives were monitored after administration of pefloxacin. Treatment of mice with 1 day of pefloxacin treatment significantly decreased the rate of biosynthesis of proteoglycan for the first 24 h. However, no difference was observed after 48 h. The decrease in proteoglycan synthesis was accompanied by a marked drop in serum sulfate concentration and a concomitant increase in urinary sulfate excretion. The decrease in proteoglycan synthesis, also observed ex vivo, may suggest a direct effect of pefloxacin on this process, rather than it being a consequence of a low concentration of sulfate. On the other hand, treatment with pefloxacin for 10 days induced oxidative damage to collagen. In conclusion, this study demonstrates, for the first time, that pefloxacin administration to mice leads to modifications in the metabolism and integrity of extracellular proteins, such as collagen and proteoglycans, which may account for the side effects observed. These results offer new insights to explain quinolone-induced disorders in growing articular cartilage.
Characterization of a
Mycobacterium smegmatis
Mutant Lacking Penicillin Binding Protein 1
Authors: Billman-Jacobe Helen , Haites Ruth E. , Coppel Ross L.
Published: October 2018

The
ponA
gene of
Mycobacterium smegmatis
encodes a 95-kDa penicillin binding protein, PBP1, that is similar to PBP1s of
Mycobacterium tuberculosis
and
Mycobacterium leprae
. Transposon disruption of
ponA
in
M. smegmatis
resulted in a PBP1-deficient mutant that was sensitive to β-lactam antibiotics, was more permeable to glycine, and grew slowly in liquid culture.
Mechanism Underlying Levofloxacin Uptake by Human Polymorphonuclear Neutrophils
Authors: Vazifeh Doina , Bryskier André , Labro Marie-Thérèse
Published: October 2018

The mechanism of radiolabeled levofloxacin ([
3
H]levofloxacin) uptake by human polymorphonuclear neutrophils (PMNs) was investigated by a classical velocity centrifugation technique. PMNs were incubated with levofloxacin for 5 to 180 min under various conditions before centrifugation through an oil cushion. Radioactivity was measured in the cell pellet to determine the amount of cell-associated drug. The uptake of levofloxacin was moderate with a cellular concentration/extracellular concentration ratio of about 4 to 6. Levofloxacin accumulated in PMNs parallel to the extracellular concentration, without saturation, over the range of 2.5 to 200 mg/liter (linear regression analysis:
r
= 0.92;
P
< 0.001). The activation energy was low (36 ± 7.2 kJ/mol). Levofloxacin uptake was increased in Ca
2+
-depleted, EGTA-containing medium by approximately 33% (
P
= 0.022), while Ni
2+
, a Ca
2+
channel inhibitor, inhibited it in a concentration-dependent manner, with the concentration that inhibited 50% of control uptake being approximately 2.65 mM. Verapamil (an
l
-type Ca
2+
channel inhibitor) and other pharmacologic agents which modify Ca
2+
homeostasis did not modify levofloxacin uptake. Interestingly, Ca
2+
and Mg
2+
inhibited levofloxacin uptake in a concentration-dependent manner. EGTA, Ni
2+
, and verapamil did not modify levofloxacin efflux; thapsigargin, a Ca
2+
pool-releasing agent, modestly increased the intracellular retention of levofloxacin. In addition, contrary to other fluoroquinolones, probenecid at 1 to 10 mM did not modify either levofloxacin uptake or efflux. These data are consistent with a mechanism of passive accumulation of levofloxacin in PMNs. Extracellular Ca
2+
and Mg
2+
may influence the structural conformation of levofloxacin or the lipophilicity of PMN membranes, thus explaining their effect on levofloxacin uptake.
Genetic Variation and Susceptibilities to Protease Inhibitors among Subtype B and F Isolates in Brazil
Authors: Tanuri Amilcar , Vicente Ana C. P. , Otsuki Koko , Ramos Carlos A. , Ferreira Orlando C. , Schechter Mauro , Janini Luis M. , Pieniazek Danuta , Rayfield Mark A.
Published: October 2018

The genetic variation of the human immunodeficiency virus type 1 (HIV-1) protease gene (
prt
) permits the classification of HIV-1 strains into five distinct protease subtypes, which follow the
gag
subtyping patterns. The susceptibilities of non-B-subtype strains to protease inhibitors (PIs) and other antiretroviral drugs remain largely unknown. Subtype F is the main non-B strain contributing to the Brazilian epidemic, accounting for 15 to 20% of these infections. In this work, we report the findings on 81 isolates from PI-naive Brazilian patients collected between 1993 and 1997. In addition, the relevant PI resistance mutations and their phenotypes were determined in vitro for 15 of these patients (B = 9 and F = 6). Among these, the subtype F samples evidenced high sensitivities in vitro to ritonavir and indinavir, with MICs at which 50 and 90% of the isolates are inhibited similar to those of both the Brazilian and the U.S. subtype B isolates. Analysis of the 81 Brazilian
prt
sequences demonstrated that the subtype F consensus sequence differs from the U.S. and Brazilian subtype B consensus in eight positions (I15V, E35D, M36I, R41K, R57K, Q61N, L63P, and L89M). The frequency of critical PI resistance substitutions (amino acid changes D30N, V82A/F/T, I84V, N88D, and L90M) among Brazilian isolates is very low (mean, 2.5%), and the associated secondary substitutions (amino acid positions 10L, 20K, 36M, 46M, 48G, 54I, 63P, 71A, and 77A) are infrequent. These observations document the relative rarity of resistance to PIs in the treatment of patients infected with HIV-1 subtype F in South America.
Characterization of SFO-1, a Plasmid-Mediated Inducible Class A β-Lactamase from
Enterobacter cloacae
Authors: Matsumoto Yoshimi , Inoue Matsuhisa
Published: October 2018

Enterobacter cloacae
8009 produced an inducible class A β-lactamase which hydrolyzed cefotaxime efficiently. It also hydrolyzed other β-lactams except cephamycins and carbapenems. The activity was inhibited by clavulanic acid and imipenem. The
bla
gene was transferable to
Escherichia coli
by electroporation of plasmid DNA. The molecular mass of the β-lactamase was 29 kDa and its pI was 7.3. All of these phenotypic characteristics of the enzyme except for inducible production resemble those of some extended-spectrum class A β-lactamases like FEC-1. The gene encoding this β-lactamase was cloned and sequenced. The deduced amino acid sequence of the β-lactamase was homologous to the AmpA sequences of the
Serratia fonticola
chromosomal enzyme (96%), MEN-1 (78%),
Klebsiella oxytoca
chromosomal enzymes (77%), TOHO-1 (75%), and FEC-1 (72%). The conserved sequences of class A β-lactamases, including the S-X(T)-X(S)-K motif, in the active site were all conserved in this enzyme. On the basis of the high degree of homology to the β-lactamase of
S. fonticola
, the enzyme was named SFO-1. The
ampR
gene was located upstream of the
ampA
gene, and the AmpR sequence of SFO-1 had homology with the AmpR sequences of the chromosomal β-lactamases from
Citrobacter diversus
(80%),
Proteus vulgaris
(68%), and
Pseudomonas aeruginosa
(60%). SFO-1 was also inducible in
E. coli
. However, a transformant harboring plasmid without intact
ampR
produced a small amount of β-lactamase constitutively, suggesting that AmpR works as an activator of
ampA
of SFO-1. This is the first report from Japan describing an inducible plasmid-mediated class A β-lactamase in gram-negative bacteria.
Therapeutic Effects of Benzoxazinorifamycin KRM-1648 Administered Alone or in Combination with a Half-Sized Secretory Leukocyte Protease Inhibitor or the Nonsteroidal Anti-Inflammatory Drug Diclofenac Sodium againstMycobacterium aviumComplex Infection in Mice
Authors: Sano Chiaki , Shimizu Toshiaki , Sato Katsumasa , Kawauchi Hideyuki , Kawahara Shin , Tomioka Haruaki
Published: October 2018

The effects of half-sized secretory leukocyte protease inhibitor or diclofenac sodium administered alone or in combination with the benzoxazinorifamycin KRM-1648 on the therapeutic efficacy of KRM-1648 againstMycobacterium aviumcomplex (MAC) in mice were studied. Neither of the two anti-inflammatory drugs affected the efficacy of KRM-1648, while they exerted significant modulating effects on tumor necrosis factor alpha production by MAC-infected macrophages.
Updated Sequence Information for TEM β-Lactamase Genes
Authors: Goussard Sylvie , Courvalin Patrice
Published: October 2018

The sequences of the promoter regions and of the structural genes for 13 penicillinase, extended-spectrum, and inhibitor-resistant TEM-type β-lactamases have been determined, and an updated
bla
TEM
gene nomenclature is proposed.
Human Herpesvirus 8 DNA Load in Leukocytes of Human Immunodeficiency Virus-Infected Subjects: Correlation with the Presence of Kaposi’s Sarcoma and Response to Anticytomegalovirus Therapy
Authors: Boivin Guy , Gaudreau Annie , Toma Emil , Lalonde Richard , Routy Jean-Pierre , Murray Gilles , Handfield Julie , Bergeron Michel G.
Published: October 2018

Specific human herpesvirus 8 (HHV-8) DNA sequences were found in leukocytes of 12 of 29 (41.4%) AIDS subjects with Kaposi’s sarcoma (KS), whereas they were found in 4 of 43 (9.3%) AIDS subjects without KS (
P
= 0.003), although the peak HHV-8 DNA load in PCR-positive subjects with KS (mean, 425 copies per 0.2 μg of DNA) did not significantly differ from the one found in PCR-positive patients without KS (mean, 218 copies). The use of intravenous ganciclovir or foscarnet therapy to treat cytomegalovirus disease did not affect the HHV-8 DNA load in seven patients for whom serial samples were analyzed.
RecA-Mediated Gene Conversion and Aminoglycoside Resistance in Strains Heterozygous for rRNA
Authors: Prammananan Therdsak , Sander Peter , Springer Burkhard , Böttger Erik C.
Published: October 2018

Clinical resistance to aminoglycosides in general is due to enzymatic drug modification. Mutational alterations of the small ribosomal subunit rRNA have recently been found to mediate acquired resistance in bacterial pathogens in vivo. In this study we investigated the effect of 16S rRNA heterozygosity (wild-type [wt] and mutant [mut] operons at position 1408 [1408
wt
/1408
mut
]) on aminoglycoside resistance. Using an integrative vector, we introduced a single copy of a mutated rRNA operon (1408 A→G) into
Mycobacterium smegmatis
, which carries two chromosomal wild-type rRNA operons; the resultant transformants exhibited an aminoglycoside-sensitive phenotype. In contrast, introduction of the mutated rRNA operon into an
M. smegmatis rrnB
knockout strain carrying a single functional chromosomal wild-type rRNA operon resulted in aminoglycoside-resistant transformants. Subsequent analysis by DNA sequencing and RNase protection assays unexpectedly demonstrated a homozygous mutant genotype, rRNA
mut
/rRNA
mut
, in the resistant transformants. To investigate whether RecA-mediated gene conversion was responsible for the aminoglycoside-resistant phenotype in the rRNA
wt
/rRNA
mut
strains,
recA
mutant strains were generated by allelic exchange techniques. Transformation of the
recA rrnB M. smegmatis
mutant strains with an integrative vector expressing a mutated rRNA operon (
Escherichia coli
position 1408 A→G) resulted in transformants with an aminoglycoside-sensitive phenotype. Subsequent analysis showed stable heterozygosity at 16S rRNA position 1408 with a single wild-type allele and a single resistant allele. These results demonstrate that rRNA-mediated mutational resistance to aminoglycosides is recessive.
Efflux-Mediated Aminoglycoside and Macrolide Resistance in
Burkholderia pseudomallei
Authors: Moore Richard A. , DeShazer David , Reckseidler Shauna , Weissman Ania , Woods Donald E.
Published: October 2018

Burkholderia pseudomallei
, the causative agent of melioidosis, is intrinsically resistant to a wide range of antimicrobial agents including β-lactams, aminoglycosides, macrolides, and polymyxins. We used Tn
5
-OT182 to mutagenize
B. pseudomallei
to identify the genes involved in aminoglycoside resistance. We report here on the identification of AmrAB-OprA, a multidrug efflux system in
B. pseudomallei
which is specific for both aminoglycoside and macrolide antibiotics. We isolated two transposon mutants, RM101 and RM102, which had 8- to 128-fold increases in their susceptibilities to the aminoglycosides streptomycin, gentamicin, neomycin, tobramycin, kanamycin, and spectinomycin. In addition, both mutants, in contrast to the parent, were susceptible to the macrolides erythromycin and clarithromycin but not to the lincosamide clindamycin. Sequencing of the DNA flanking the transposon insertions revealed a putative operon consisting of a resistance, nodulation, division-type transporter, a membrane fusion protein, an outer membrane protein, and a divergently transcribed regulator protein. Consistent with the presence of an efflux system, both mutants accumulated [
3
H]dihydrostreptomycin, whereas the parent strain did not. We constructed an
amr
deletion strain,
B. pseudomallei
DD503, which was hypersusceptible to aminoglycosides and macrolides and which was used successfully in allelic exchange experiments. These results suggest that an efflux system is a major contributor to the inherent high-level aminoglycoside and macrolide resistance found in
B. pseudomallei
.
In Vitro Activities of Cephalosporins and Quinolones againstEscherichia coliStrains Isolated from Diarrheic Dairy Calves
Authors: Orden José Antonio , Ruiz-Santa-Quiteria José Antonio , García Silvia , Cid Dolores , de la Fuente Ricardo
Published: October 2018

The in vitro activities of several cephalosporins and quinolones against 195 strains ofEscherichia coliisolated from dairy calves affected by neonatal diarrhea were determined. One hundred thirty-seven of these strains produced one or more potential virulence factors (F5, F41, F17, cytotoxic necrotizing factor, verotoxin, and theeaegene), but the remaining 58 strains did not produce any of these factors. From 11 to 18% of theE. colistrains were resistant to cephalothin, nalidixic acid, enoxacin, and enrofloxacin. However, cefuroxime, cefotaxime, and cefquinome were highly effective against theE. coliisolates tested. Some significant differences (P< 0.05) in resistance to quinolones between the strains producing potential virulence factors and nonfimbriated, nontoxigenic,eae-negative strains were found. Thus,eae-positive, necrotoxigenic, and verotoxigenic (except for nalidixic acid)E. colistrains were significantly more sensitive to nalidixic acid, enoxacin, and enrofloxacin than nonfimbriated, nontoxigenic,eae-negative strains. Moreover,eae-positive strains were significantly more sensitive to enoxacin and enrofloxacin than F5-positive strains. Thus, the results of this study suggest that the bovineE. colistrains that produce some potential virulence factors are more sensitive to quinolones than those that do not express these factors.
Alternative Oxidase Inhibitors Potentiate the Activity of Atovaquone against
Plasmodium falciparum
Authors: Murphy Anina D. , Lang-Unnasch Naomi
Published: October 2018

Recent evidence suggests that the malaria parasite
Plasmodium falciparum
utilizes a branched respiratory pathway including both a cytochrome chain and an alternative oxidase. This branched respiratory pathway model has been used as a basis for examining the mechanism of action of two antimalarial agents, atovaquone and proguanil. In polarographic assays, atovaquone immediately reduced the parasite oxygen consumption rate in a concentration-dependent manner. This is consistent with its previously described role as an inhibitor of the cytochrome bc
1
complex. Atovaquone maximally inhibited the rate of
P. falciparum
oxygen consumption by 73% ± 10%. At all atovaquone concentrations tested, the addition of the alternative oxidase inhibitor, salicylhydroxamic acid, resulted in a further decrease in the rate of parasite oxygen consumption. At the highest concentrations of atovaquone tested, the activities of salicylhydroxamic acid and atovaquone appear to overlap, suggesting that at these concentrations, atovaquone partially inhibits the alternative oxidase as well as the cytochrome chain. Drug interaction studies with atovaquone and salicylhydroxamic acid indicate atovaquone’s activity against
P. falciparum
in vitro is potentiated by this alternative oxidase inhibitor, with a sum fractional inhibitory concentration of 0.6. Propyl gallate, another alternative oxidase inhibitor, also potentiated atovaquone’s activity, with a sum fractional inhibitory concentration of 0.7. Proguanil, which potentiates atovaquone activity in vitro and in vivo, had a small effect on parasite oxygen consumption in polarographic assays when used alone or in the presence of atovaquone or salicylhydroxamic acid. This suggests that proguanil does not potentiate atovaquone by direct inhibition of either branch of the parasite respiratory chain.
Lack of Absorption of Didanosine after Rectal Administration in Human Immunodeficiency Virus-Infected Patients
Authors: Wintergerst Uwe , Rolinski B. , Sölder B. , Bogner J. R. , Wolf E. , Jäger H. , Roscher A. A. , Belohradsky B. H.
Published: October 2018

The feasibility of rectal administration of didanosine (DDI) was studied in six human immunodeficiency virus-infected patients. After oral intake of a DDI solution (100 mg/m
2
of body surface area) combined with an antacid (Maalox), pharmacokinetic parametric values were in accordance with previously published data; the mean ± standard deviation for terminal half-life was 59.5 ± 15.0 min, that for peak concentration was 5.2 ± 3.9 μmol/liter, and that for the area under the time-concentration curve (AUC) was 494 ± 412 min · μmol/liter. After rectal administration of a similarly prepared DDI solution (100 mg/m
2
of body surface area), plasma DDI levels were below the detection limit (0.1 μmol/liter) at all time points in five of the six patients, and in the remaining patient the AUC after rectal application was only 5% of that after oral administration. We conclude that oral administration of DDI cannot be easily replaced by rectal application.
Efficacy of RD3-0028 Aerosol Treatment against Respiratory Syncytial Virus Infection in Immunosuppressed Mice
Authors: Sudo Kenji , Watanabe Wataru , Konno Kenji , Sato Ryu , Kajiyashiki Tsuyoshi , Shigeta Shiro , Yokota Tomoyuki
Published: October 2018

RD3-0028, a benzodithiin compound, has antiviral activity against respiratory syncytial virus (RSV) in cell culture. We used a mouse model of RSV infection to determine the in vivo effect of RD3-0028. Cyclophosphamide (CYP)-treated, immunosuppressed mice were inoculated intranasally. The lungs of the mice were removed on day 4. The virus titers of the lungs of RD3-0028-treated mice were compared to the virus titers of the lungs of virus-inoculated, untreated control mice. In an effort to increase the therapeutic effectiveness of this compound, RD3-0028 was administered by aerosol to RSV-infected mice by using a head-exposure system. Aerosols generated from reservoirs containing RD3-0028 (7 mg/ml) administered for 2 h twice daily for 3 days significantly reduced the pulmonary titer of RSV-infected mice. It is clear that the minimal effective dose of RD3-0028 for RSV-infected mice is significantly less than that of ribavirin, the only compound currently available for use against RSV disease. Furthermore, the RD3-0028 aerosol administration appeared to protect the lungs of infected, CYP-treated mice against tissue damage, as evidenced by the preservation of the lung architecture and a reduction in pulmonary inflammatory infiltrates. RD3-0028 aerosol was not toxic for mice at the therapeutic dose. The present study demonstrates the effectiveness of aerosol administration of RD3-0028 for RSV-infected mice.