bluuebunny/crossref_metadata_2025 huggingface dataset:
Glycopeptide Antibiotic Resistance Genes in Glycopeptide-Producing Organisms
Authors: Marshall C. G. , Lessard I. A. D. , Park I.-S. , Wright G. D.
Published: October 2018

The mechanism of high-level resistance to vancomycin in enterococci consists of the synthesis of peptidoglycan terminating in
d
-alanyl-
d
-lactate instead of the usual
d
-alanyl-
d
-alanine. This alternate cell wall biosynthesis pathway is ensured by the collective actions of three enzymes: VanH, VanA, and VanX. The origin of this resistance mechanism is unknown. We have cloned three genes encoding homologs of VanH, VanA, and VanX from two organisms which produce glycopeptide antibiotics: the A47934 producer
Streptomyces toyocaensis
NRRL 15009 and the vancomycin producer
Amycolatopsis orientalis
C329.2. The predicted amino acid sequences are highly similar to those found in VRE: 54 to 61% identity for VanH, 59 to 63% identity for VanA, and 61 to 64% identity for VanX. Furthermore, the orientations of the genes,
vanH
,
vanA
, and
vanX
, are identical to the orientations found in vancomycin-resistant enterococci. Southern analysis of total DNA from other glycopeptide-producing organisms,
A. orientalis
18098 (chloro-eremomycin producer),
A. orientalis
subsp.
lurida
(ristocetin producer), and
Amycolatopsis coloradensis
subsp.
labeda
(teicoplanin and avoparcin producer), with a probe derived from the
vanH
,
vanA
, and
vanX
cluster from
A. orientalis
C329.2 revealed cross-hybridizing DNA in all strains. In addition, the
vanH
,
vanA
,
vanX
cluster was amplified from all glycopeptide-producing organisms by PCR with degenerate primers complementary to conserved regions in VanH and VanX. Thus, this gene sequence is common to all glycopeptide producers tested. These results suggest that glycopeptide-producing organisms may have been the source of resistance genes in vancomycin-resistant enterococci.
New Trimethoprim-Resistant Dihydrofolate Reductase Cassette,
dfrXV
, Inserted in a Class 1 Integron
Authors: Adrian Peter V. , du Plessis Mignon , Klugman Keith P. , Amyes Sebastian G. B.
Published: October 2018

The nucleotide sequence of a plasmid-borne trimethoprim resistance gene from a commensal fecal
Escherichia coli
isolate revealed a new dihydrofolate reductase gene,
dfrXV
, which occurred as a gene cassette integrated in a site-specific manner in a class 1 integron. The new gene shows 84% nucleotide identity and the predicted protein shows 90% amino acid identity with
dfrI
and DHFR type I, respectively. Genes for spectinomycin resistance,
aadA1
[
ant (3′′)-Ia
], and sulfonamide resistance,
sulI
, were located downstream of
dfrXV
in a manner identical to that in pLMO229.
Human Immunodeficiency Virus Type 1 cDNA Integration: New Aromatic Hydroxylated Inhibitors and Studies of the Inhibition Mechanism
Authors: Farnet C. M. , Wang B. , Hansen M. , Lipford J. R. , Zalkow L. , Robinson W. E. , Siegel J. , Bushman F.
Published: October 2018

Integration of the human immunodeficiency virus type 1 (HIV-1) cDNA is a required step for viral replication. Integrase, the virus-encoded enzyme important for integration, has not yet been exploited as a target for clinically useful inhibitors. Here we report on the identification of new polyhydroxylated aromatic inhibitors of integrase including ellagic acid, purpurogallin, 4,8,12-trioxatricornan, and hypericin, the last of which is known to inhibit viral replication. These compounds and others were characterized in assays with subviral preintegration complexes (PICs) isolated from HIV-1-infected cells. Hypericin was found to inhibit PIC assays, while the other compounds tested were inactive. Counterscreening of these and other integrase inhibitors against additional DNA-modifying enzymes revealed that none of the polyhydroxylated aromatic compounds are active against enzymes that do not require metals (methylases, a pox virus topoisomerase). However, all were cross-reactive with metal-requiring enzymes (restriction enzymes, a reverse transcriptase), implicating metal atoms in the inhibitory mechanism. In mechanistic studies, we localized binding of some inhibitors to the catalytic domain of integrase by assaying competition of binding by labeled nucleotides. These findings help elucidate the mechanism of action of the polyhydroxylated aromatic inhibitors and provide practical guidance for further inhibitor development.
Two 2-Hydroxy-3-Alkyl-1,4-Naphthoquinones with In Vitro and In Vivo Activities against
Toxoplasma gondii
Authors: Khan Anis A. , Nasr Mohamed , Araujo Fausto G.
Published: October 2018

Two 3-alkyl-substituted 2-hydroxy-1,4-naphthoquinones, NSC 113452 (NSC52) and NSC 113455 (NSC55), were evaluated for activity against
Toxoplasma gondii
in vitro and in murine models of acute toxoplasmosis. In vitro, both NSC52 and NSC55 significantly inhibited intracellular replication of
T. gondii
. In vivo, each compound was examined alone and in combination with other drugs currently used for treatment of human toxoplasmosis. Although none of the compounds protected mice against death when administered orally, both were significantly protective when administered intraperitoneally. In addition, a significant increase in survival was observed when suboptimal doses of each compound were used in combination with suboptimal doses of pyrimethamine or sulfadiazine. These results indicate that combinations of NSC52 or NSC55 with pyrimethamine or sulfadiazine have promising activity against
T. gondii
.
Alterations in Topoisomerase IV and DNA Gyrase in Quinolone-Resistant Mutants of
Mycoplasma hominis
Obtained In Vitro
Authors: Bébéar Cécile M. , Renaudin Hélène , Charron Alain , Bové Joseph M. , Bébéar Christiane , Renaudin Joel
Published: October 2018

Mycoplasma hominis
mutants were selected stepwise for resistance to ofloxacin and sparfloxacin, and their
gyrA
,
gyrB
,
parC
, and
parE
quinolone resistance-determining regions were characterized. For ofloxacin, four rounds of selection yielded six first-, six second-, five third-, and two fourth-step mutants. The first-step mutants harbored a single Asp426→Asn substitution in ParE. GyrA changes (Ser83→Leu or Trp) were found only from the third round of selection. With sparfloxacin, three rounds of selection generated 4 first-, 7 second-, and 10 third-step mutants. In contrast to ofloxacin resistance, GyrA mutations (Ser83→Leu or Ser84→Trp) were detected in the first-step mutants prior to ParC changes (Glu84→Lys), which appeared only after the second round of selection. Further analysis of eight multistep-selected mutants of
M. hominis
that were previously described (2) revealed that they carried mutations in ParE (Asp426→Asn), GyrA (Ser83→Leu) and ParE (Asp426→Asn), GyrA (Ser83→Leu) and ParC (Ser80→Ile), or ParC (Ser80→Ile) alone, depending on the fluoroquinolone used for selection, i.e., ciprofloxacin, norfloxacin, ofloxacin, or pefloxacin, respectively. These data indicate that in
M. hominis
DNA gyrase is the primary target of sparfloxacin whereas topoisomerase IV is the primary target of pefloxacin, ofloxacin, and ciprofloxacin.
Antifungal Activity of the Pradimicin Derivative BMS 181184 in the Treatment of Experimental Pulmonary Aspergillosis in Persistently Neutropenic Rabbits
Authors: Gonzalez Corina E. , Groll Andreas H. , Giri Neelam , Shetty Daiva , Al-Mohsen Ibrahim , Sein Tin , Feuerstein Erwin , Bacher John , Piscitelli Stephen , Walsh Thomas J.
Published: October 2018

The activity of the pradimicin derivative BMS 181184 was evaluated in a model of invasive pulmonary aspergillosis in persistently neutropenic rabbits and compared with that of amphotericin B deoxycholate. BMS 181184 at total daily doses of 50 and 150 mg/kg of body weight was at least as effective as amphotericin B at 1 mg/kg once a day in conferring survival and had comparable activity in reducing organism-mediated tissue injury and excess lung weight. Although treatment at all dosing regimens of BMS 181184 resulted in significant reductions in fungal tissue burden compared to untreated controls, equivalence to amphotericin B occurred only at the higher dosage level. Similar observations were made in bronchoalveolar lavage fluid cultures obtained postmortem. Monitoring of the animals through ultrafast computerized tomography scan revealed a marked resolution of pulmonary lesions during treatment with BMS 181184. The compound was well tolerated at all dosing regimens, and no toxicity was noted. Pharmacokinetic studies revealed nonlinear drug disposition with increased clearance at higher dosages and some evidence for extravascular drug accumulation. BMS 181184 had excellent activity in the treatment of experimental invasive pulmonary aspergillosis in persistently neutropenic rabbits, thus underscoring the potential of pradimicin derivatives in therapy of invasive aspergillosis in the neutropenic host.
Influence of Human Serum on Pharmacodynamic Properties of an Investigational Glycopeptide, LY333328, and Comparator Agents against
Staphylococcus aureus
Authors: Zhanel George G. , Kirkpatrick Iain D. C. , Hoban Daryl J. , Kabani Amin M. , Karlowsky James A.
Published: October 2018

The MICs and MBCs of 15 antibiotics for two strains of
Staphylococcus aureus
were determined in Mueller-Hinton broth (MHB) and 90% serum–10% MHB. Subsequent experiments established that highly protein-bound antibiotics (≥80%), such as LY333328, demonstrated higher MICs and MBCs, less killing over an 8-h interval, and shorter postantibiotic effects in 90% serum–10% MHB than in MHB alone. Albumin was demonstrated to be almost solely responsible for changes in the aforementioned pharmacodynamic parameters of LY333328.
Control of
Candida albicans
Murine Vaginitis by Topical Administration of Polycarbophil-Econazole Complex
Authors: Ghelardi E. , Tavanti A. , Lupetti A. , Celandroni F. , Boldrini E. , Campa M. , Senesi S.
Published: October 2018

The complexation of econazole with the mucoadhesive polycarbophil was found to significantly improve the therapeutic benefit of the drug in the topical treatment of experimental vaginal candidiasis in mice, while no difference in the antimycotic activity exerted by econazole and polycarbophil-econazole could be detected in vitro.
In Vitro Activity of the New Glycopeptide LY333328 against Multiply Resistant Gram-Positive Clinical Isolates
Authors: García-Garrote Fernando , Cercenado Emilia , Alcalá Luis , Bouza Emilio
Published: October 2018

The in vitro activity of LY333328 was compared with those of vancomycin and teicoplanin against 425 gram-positive clinical isolates, including a variety of multiply resistant strains. LY333328 at ≤4 μg/ml inhibited all microorganisms tested, including methicillin- and teicoplanin-resistant staphylococci, glycopeptide-resistant enterococci, penicillin- and multiply resistant pneumococci, and
viridans
and beta-hemolytic streptococci.
Acetate-Mediated Growth Inhibition in Sterol 14α-Demethylation-Deficient Cells of
Candida albicans
Authors: Shimokawa Osamu , Nakayama Hiroaki
Published: October 2018

Candida albicans
is a fungus thought to be viable in the presence of a deficiency in sterol 14α-demethylation. We showed in a strain of this species that the deficiency, caused either by a mutation or by an azole antifungal agent, made the cells susceptible to growth inhibition by acetate included in the culture medium. Studies with a mutant demonstrated that the inhibition was complete at a sodium acetate concentration of 0.24 M (20 g/liter) and was evident even at a pH of 8, the latter result indicating the involvement of acetate ions rather than the undissociated form of acetic acid. In fluconazole-treated cells, sterol profiles determined by thin-layer chromatography revealed that the minimum sterol 14α-demethylation-inhibitory concentrations (MDICs) of the drug, thought to be the most important parameter for clinical purposes, were practically identical in the media with and without 0.24 M acetate and were equivalent to the MIC in the acetate-supplemented medium. The acetate-mediated growth inhibition of azole-treated cells was confirmed with two additional strains of
C. albicans
and four different agents, suggesting the possibility of generalization. From these results, it was surmised that the acetate-containing medium may find use in azole susceptibility testing, for which there is currently no method capable of measuring MDICs directly for those fungi whose viability is not lost as a result of sterol 14α-demethylation deficiency. Additionally, the acetate-supplemented agar medium was found to be useful in detecting reversions from sterol 14α-demethylation deficiency to proficiency.
Characterization of Dihydrofolate Reductase Genes from Trimethoprim-Susceptible and Trimethoprim-Resistant Strains of
Enterococcus faecalis
Authors: Coque Teresa M. , Singh Kavindra V. , Weinstock George M. , Murray Barbara E.
Published: October 2018

Enterococci are usually susceptible in vitro to trimethoprim; however, high-level resistance (HLR) (MICs, >1,024 μg/ml) has been reported. We studied
Enterococcus faecalis
DEL, for which the trimethoprim MIC was >1,024 μg/ml. No transfer of resistance was achieved by broth or filter matings. Two different genes that conferred trimethoprim resistance when they were cloned in
Escherichia coli
(MICs, 128 and >1,024 μg/ml) were studied. One gene that coded for a polypeptide of 165 amino acids (MIC, 128 μg/ml for
E. coli
) was identical to
dfr
homologs that we cloned from a trimethoprim-susceptible
E. faecalis
strain, and it is presumed to be the intrinsic
E. faecalis dfr
gene (which causes resistance in
E. coli
when cloned in multiple copies); this gene was designated
dfrE
. The nucleotide sequence 5′ to this
dfr
gene showed similarity to thymidylate synthetase genes, suggesting that the
dfr
and
thy
genes from
E. faecalis
are located in tandem. The
E. faecalis
gene that conferred HLR to trimethoprim in
E. coli
, designated
dfrF
, codes for a predicted polypeptide of 165 amino acids with 38 to 64% similarity with other dihydrofolate reductases from gram-positive and gram-negative organisms. The nucleotide sequence 5′ to
dfrF
did not show similarity to the
thy
sequences. A DNA probe for
dfrF
hybridized under high-stringency conditions only to colony lysates of enterococci for which the trimethoprim MIC was >1,024 μg/ml; there was no hybridization to plasmid DNA from the strain of origin. To confirm that this gene causes trimethoprim resistance in enterococci, we cloned it into the integrative vector pAT113 and electroporated it into RH110 (
E. faecalis
OG1RF::Tn
916
ΔEm) (trimethoprim MIC, 0.5 μg/ml), which resulted in RH110 derivatives for which the trimethoprim MIC was >1,024 μg/ml. These results indicate that
dfrF
is an acquired but probably chromosomally located gene which is responsible for in vitro HLR to trimethoprim in
E. faecalis
.
Increase in Quinolone Resistance in a
Haemophilus influenzae
Strain Isolated from a Patient with Recurrent Respiratory Infections Treated with Ofloxacin
Authors: Vila Jordi , Ruiz Joaquim , Sanchez Ferran , Navarro Ferran , Mirelis Beatriz , de Anta M. Teresa Jimenez , Prats Guillem
Published: October 2018

The increase in the level of quinolone resistance of
Haemophilus influenzae
clinical isolates during ofloxacin therapy of a patient with recurrent respiratory infections was investigated. The first isolate (MIC of ciprofloxacin of 2 μg/ml) and the second isolate (MIC of 32 μg/ml) belonged to the same clone, as shown by pulsed-field gel electrophoresis, and the increase in the resistance level was associated with a substitution in Ser-84 to Arg in the ParC protein. These results emphasize the potential risk of development of quinolone-resistant
H. influenzae
during fluoroquinolone therapy in patients with recurrent respiratory infection.
In Vitro Activities of Voriconazole, Fluconazole, and Itraconazole against 566 Clinical Isolates of
Cryptococcus neoformans
from the United States and Africa
Authors: Pfaller M. A. , Zhang J. , Messer S. A. , Brandt M. E. , Hajjeh R. A. , Jessup C. J. , Tumberland M. , Mbidde E. K. , Ghannoum M. A.
Published: October 2018

We investigated the in vitro activity of voriconazole compared to those of fluconazole and itraconazole against 566 clinical isolates of
Cryptococcus neoformans
from Africa (164) and the United States (402). Isolates were obtained from cerebrospinal fluid (362), blood (139), and miscellaneous sites (65). Voriconazole (MIC at which 90% of the isolates are inhibited [MIC
90
], 0.12 to 0.25 μg/ml) was more active than either itraconazole (MIC
90
, 0.5 μg/ml) or fluconazole (MIC
90
, 8.0 to 16 μg/ml) against both African and U.S. isolates. Isolates inhibited by ≥16 μg of fluconazole per ml were almost all (99%) inhibited by ≤1 μg of voriconazole per ml. These results suggest that voriconazole may be useful in the treatment of cryptococcosis.
A 5-Year Surveillance Study of 44,691 Isolates of
Haemophilus influenzae
Project Beta-Alert 1993–1997
Authors: Derecola A. , Butler D. L. , Kaplan R. L. , Miller L. A. , Poupard J. A.
Published: October 2018

Beta-Alert is a surveillance program developed in 1993 to monitor the percentage of β-lactamase-producing
Haemophilus influenzae
isolates obtained from specimens submitted to regional commercial laboratories. The results of this study demonstrate that levels of β-lactamase producers have remained between 31 and 38% in the United States over the past 5 years.
Two Pharmacodynamic Models for Assessing the Efficacy of Amoxicillin-Clavulanate against Experimental Respiratory Tract Infections Caused by Strains of
Streptococcus pneumoniae
Authors: Woodnutt Gary , Berry Valerie
Published: October 2018

Two models of respiratory tract infection were used to investigate the pharmacodynamics of amoxicillin-clavulanate against
Streptococcus pneumoniae
. Eight strains of
S. pneumoniae
were used in a mouse model in which the animals were infected intranasally and were then treated with a range of doses and dose intervals. The time that the plasma amoxicillin concentration remained above the MIC (
T
>MIC) correlated well with bacterial killing, such that if
T
>MIC was below 20% there was no effect on bacterial numbers in the lungs. As
T
>MIC increased, the response, in terms of decreased bacterial load, improved and at
T
>MICs of greater than 35 to 40% of the dosing interval, bacteriological cure was maximal. On the basis of equivalent
T
>MICs, these data would suggest that in humans a dosage of 500 mg three times daily (t.i.d.) should have efficacy equal to that of a dosage of 875 mg twice daily (b.i.d.). This hypothesis was evaluated in a rat model in which amoxicillin-clavulanate was given by computer-controlled intravenous infusion to achieve concentrations that approximate the concentrations achieved in the plasma of humans following oral administration of 500/125 mg t.i.d. or 875/125 mg b.i.d. Infusions continued for 3 days and bacterial numbers in the lungs 2 h after the cessation of the infusion were significantly reduced (
P
< 0.01) by both treatments in strains of
S. pneumoniae
for which amoxicillin MICs were below 2 μg/ml. When tested against a strain of
S. pneumoniae
for which the amoxicillin MIC was 4 μg/ml, the simulated 500/125-mg dose was ineffective but the 875/125-mg dose demonstrated a small but significant (
P
< 0.01) reduction in bacterial numbers. These data confirm the findings in the mouse and indicate that amoxicillin-clavulanate administered at 875/125 mg b.i.d. would be as effective clinically as amoxicillin-clavulanate administered at 500/125 mg t.i.d.
Efficacy of High-Dose Amoxicillin-Clavulanate against Experimental Respiratory Tract Infections Caused by Strains of
Streptococcus pneumoniae
Authors: Woodnutt Gary , Berry Valerie
Published: October 2018

The purpose of the present investigation was to determine if the efficacy of amoxicillin-clavulanate against penicillin-resistant
Streptococcus pneumoniae
could be improved by increasing the pediatric amoxicillin unit dose (90 versus 45 mg/kg of body weight/day) while maintaining the clavulanate unit dose at 6.4 mg/kg/day. A rat pneumonia model was used. In that model approximately 6 log
10
CFU of one of four strains of
S. pneumoniae
(amoxicillin MICs, 2 μg/ml [one strain], 4 μg/ml [two strains], and 8 μg/ml [one strain]) were instilled into the bronchi of rats. Amoxicillin-clavulanate was given by computer-controlled intravenous infusion to approximate the concentrations achieved in the plasma of children following the administration of oral doses of 45/6.4 mg/kg/day or 90/6.4 mg/kg/g/day divided every 12 h or saline as a control for a total of 3 days. Infusions continued for 3 days, and 2 h after the cessation of infusion, bacterial numbers in the lungs were significantly reduced by the 90/6.4-mg/kg/day equivalent dosage for strains for which amoxicillin MICs were 2 or 4 μg/ml. The 45/6.4-mg/kg/day equivalent dosage was fully effective only against the strain for which the amoxicillin MIC was 2 μg/ml and had marginal efficacy against one of the two strains for which amoxicillin MICs were 4 μg/ml. The bacterial load for the strain for which the amoxicillin MIC was 8 μg/ml was not reduced with either dosage. These data demonstrate that regimens which achieved concentrations in plasma above the MIC for at least 34% of a 24-h dosing period resulted in significant reductions in the number of viable bacteria, indicating that the efficacy of amoxicillin-clavulanate can be extended to include efficacy against less susceptible strains of
S. pneumoniae
by increasing the amoxicillin dose.
Vancomycin-Dependent
Enterococcus faecalis
Clinical Isolates and Revertant Mutants
Authors: Van Bambeke Françoise , Chauvel Murielle , Reynolds Peter E. , Fraimow Henry S. , Courvalin Patrice
Published: October 2018

Three vancomycin-dependent clinical isolates of
Enterococcus faecalis
of the VanB type were studied by determining (i) the sequence of the
ddl
gene encoding the host
d
-Ala:
d
-Ala ligase and the
vanS
B
-
vanR
B
genes specifying the two-component regulatory system that activates transcription of the
vanB
operon, (ii) the level of expression of resistance genes by using
dd
-dipeptidase activity as a reporter, and (iii) the proportions of the peptidoglycan precursors synthesized. Each strain had a mutation in
ddl
leading to an amino acid substitution (D295 to V; T316 to I) or deletion (DAK251-253 to E) at invariant positions in
d
-Ala:
d
-Ala,
d
-Ala:
d
-Lac, and
d
-Ala:
d
-Ser ligases. These mutations resulted in impaired host
d
-Ala:
d
-Ala ligases since only precursors terminating in
d
-Ala-
d
-Lac were synthesized under vancomycin-inducing conditions. Two types of vancomycin-independent revertants of one isolate were obtained in vitro after growth in the absence of vancomycin: (i) vancomycin-resistant, teicoplanin-susceptible mutants had a 6-bp insertion in the host
ddl
gene, causing the E251-to-EYK change that restored
d
-Ala:
d
-Ala ligase activity, (ii) constitutive vancomycin-resistant, teicoplanin-resistant mutants had substitutions (S232 to F or E247 to K) in the vicinity of the autophosphorylation site of the VanS
B
sensor and produced exclusively precursors ending in
d
-Ala-
d
-Lac. Vancomycin- and teicoplanin-dependent mutants obtained by growth in the presence of teicoplanin had an 18-bp deletion in VanS
B
, affecting residues 402 to 407 and overlapping the G2 ATP binding domain. The rapid emergence of vancomycin-independent revertants in vitro suggests that interruption of vancomycin therapy may not be sufficient to cure patients infected with vancomycin-dependent enterococci.
Type II Topoisomerase Mutations in Ciprofloxacin-Resistant Strains of
Pseudomonas aeruginosa
Authors: Mouneimné Hyam , Robert Jérome , Jarlier Vincent , Cambau Emmanuelle
Published: October 2018

We determined the sequences of the quinolone resistance-determining regions of
gyrA
,
gyrB
, and
parC
genes for 30 clinical strains of
Pseudomonas aeruginosa
resistant to ciprofloxacin that were previously complemented by wild-type
gyrA
and
gyrB
plasmid-borne alleles and studied for their coresistance to imipenem (E. Cambau, E. Perani, C. Dib, C. Petinon, J. Trias, and V. Jarlier, Antimicrob. Agents Chemother. 39:2248–2252, 1995). In the present study, we found mutations in type II topoisomerase genes for all strains. Twenty-eight strains had a missense mutation in
gyrA
(codon 83 or 87). Ten of them had an additional mutation in
parC
(codon 80 or 84), including a novel mutation of Ser-80 to Trp, but all were fully complemented by a plasmid-borne wild-type
gyrA
allele. The remaining two strains harbored the first
gyrB
mutation described in
P. aeruginosa
, leading to the substitution of phenylalanine for serine 464. The strains which had two mutations in type II topoisomerase genes (i.e.,
gyrA
and
parC
) were significantly more resistant to fluoroquinolones than those with a single mutation in
gyrA
or
gyrB
(geometric mean MICs of ciprofloxacin, 39.4 versus 10.9 μg/ml,
P
< 0.01; geometric mean MICs of sparfloxacin, 64.0 versus 22.6,
P
< 0.01). No mutant with a
parC
mutation alone was observed, which favors DNA gyrase being the primary target for fluoroquinolones. These results demonstrate that
gyrA
mutations are the major mechanism of resistance to fluoroquinolones for clinical strains of
P. aeruginosa
and that additional mutations in
parC
lead to a higher level of quinolone resistance.
Therapeutic Effect of Clarithromycin on a Transplanted Tumor in Rats
Authors: Sassa Kazuhiko , Mizushima Yutaka , Fujishita Takashi , Oosaki Rokuo , Kobayashi Masashi
Published: October 2018

The therapeutic antitumor effect of clarithromycin (CAM) was examined with the 13762NF mammary adenocarcinoma and F-344 rat system. When CAM treatment at a dosage of 2 mg/kg of body weight orally for 21 days was commenced after inoculation of the tumor, no significant decrease in death rate was observed, although the loss in body weight was less than that in the untreated group. When tumor-bearing (TB) rats were treated with CAM in combination with carboplatin or cyclophosphamide, a significant decrease in the death rate was obtained, although neither treatment alone proved to be effective. A beneficial effect was also observed when CAM treatment was combined with surgical treatment. CAM showed no direct cytotoxicity to this tumor in vitro according to the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Spleen cells obtained from TB rats receiving CAM treatment showed a stronger tumor-neutralizing activity than those from rats which had not received CAM treatment (Winn assay). Enhanced induction of cytotoxic cells to allogeneic tumor was also observed in rats immunized with allogeneic tumor cells together with CAM treatment (
51
Cr release assay). The 13762NF tumor produces transforming growth factor-β (TGF-β), tumor necrosis factor alpha, and matrix metalloproteinase-9, and treatment of tumor cells with CAM in vitro for 24 h significantly inhibited the expression of the genes coding for these proteins (reverse transcription-PCR). Levels of expression of the TGF-β and interleukin-6 genes of spleen cells obtained from CAM-treated TB rats were both significantly lower than those of spleen cells from CAM-untreated TB rats. This study suggests that CAM has biological response modifier activities resulting in a beneficial therapeutic antitumor effect and might be useful for the treatment of human cancers.
Effect of Probenecid on the Renal Excretion Mechanism of a New Carbapenem, DA-1131, in Rats and Rabbits
Authors: Kim So H. , Kim Won B. , Lee Myung G.
Published: October 2018

The effects of probenecid, an anion transport inhibitor, on the renal excretion mechanism of a new anionic carbapenem, DA-1131, were investigated after a 1-min intravenous infusion of DA-1131 at 100 mg/kg of body weight to rabbits and 50 mg/kg to rats with or without probenecid at 50 mg/kg for both species. In control rabbits, the renal clearance (CL
R
) of DA-1131 and the glomerular filtration rate based on creatinine clearance (CL
CR
) were 6.14 ± 2.09 and 2.26 ± 0.589 ml/min/kg, respectively. When considering the less than 10% plasma protein binding of DA-1131 in rabbits, renal tubular secretion of DA-1131 was observed in rabbits. The CL
R
of DA-1131 (3.87 ± 0.543 ml/min/kg) decreased significantly with treatment with probenecid in rabbits, indicating that the renal tubular secretion of DA-1131 was inhibited by probenecid. However, in control rats, the CL
R
of DA-1131 (5.80 ± 1.94 ml/min/kg) was comparable to the CL
CR
(4.29 ± 1.64 ml/min/kg), indicating that DA-1131 was mainly excreted by glomerular filtration in rats. Therefore, it could be expected that the CL
R
of DA-1131 could not be affected by treatment with probenecid in rats; this was proved by a similar CL
R
of DA-1131 with treatment with (6.93 ± 0.675 ml/min/kg) or without (5.80 ± 1.94 ml/min/kg) probenecid. Therefore, the renal secretion of DA-1131 is a factor in rabbits but is not a factor in rats.
Evaluation of Rifapentine in Long-Term Treatment Regimens for Tuberculosis in Mice
Authors: Lenaerts Anne M. J. A. , Chase Sharon E. , Chmielewski Alex J. , Cynamon Michael H.
Published: October 2018

Besides direct bactericidal activity, long-term effectiveness is one of the most important features to consider when developing new drugs for chemotherapy. In this study, we evaluated the ability of rifapentine (RFP), in monotherapy and combination therapy, to completely eradicate a
Mycobacterium tuberculosis
infection and to prevent relapse posttreatment in a Swiss mouse model. The combination of RFP, isoniazid (INH), and pyrazinamide (PZA) administered daily resulted in an apparent clearance of
M. tuberculosis
organisms in the lungs and spleens of infected mice after 10 weeks of treatment. However, 3 months after the cessation of therapy, bacterial regrowth occurred in mice treated for a 12-week period, indicating a relapse of infection. In intermittent treatment regimens of RFP in combination with INH and PZA, sterilization was achieved when mice were treated two to five times per week for 9 weeks. Bacterial growth was still observed in the once-weekly treatment group. Our results show that mouse models can predict important parameters for new drugs. We stress the necessity for long-term posttreatment observation in animal models for the routine evaluation of new drugs for antituberculosis chemotherapy.
Characterization of the Chromosomal
aac(6′)-Iz
Gene of
Stenotrophomonas maltophilia
Authors: Lambert Thierry , Ploy Marie-Cécile , Denis François , Courvalin Patrice
Published: October 2018

The
aac(6′)-Iz
gene of
Stenotrophomonas maltophilia
BM2690 encoding an aminoglycoside 6′-
N
-acetyltransferase was characterized. The gene was identified as a coding sequence of 462 bp corresponding to a protein with a calculated mass of 16,506 Da, a value in good agreement with that of ca. 16,000 found by in vitro coupled transcription-translation. Analysis of the deduced amino acid sequence indicated that the protein was a member of the major subfamily of aminoglycoside 6′-
N
-acetyltransferases. The enzyme conferred resistance to amikacin but not to gentamicin, indicating that it was an AAC(6′) of type I. The open reading frame upstream from the
aac(6′)-Iz
gene was homologous to the
fprA
gene of
Myxococcus xanthus
(61% identity), which encodes a putative pyridoxine (pyridoxamine) 5′-phosphate oxidase. Pulsed-field gel electrophoresis of total DNA from BM2690 and
S. maltophilia
ATTC 13637 digested with
Xba
I,
Dra
I, and
Spe
I followed by hybridization with rRNA and
aac(6′)-Iz
-specific probes indicated that the gene was located in the chromosome. The
aac(6′)-Iz
gene was detected by DNA-DNA hybridization in all 80 strains of
S. maltophilia
tested. The MICs of gentamicin against these strains of
S. maltophilia
were lower than those of amikacin, netilmicin, and tobramycin, indicating that production of AAC(6′)-Iz contributes to aminoglycoside resistance in
S. maltophilia
.
Pharmacokinetics of Isepamicin during Continuous Venovenous Hemodiafiltration
Authors: Breilh Dominique , Allaouchiche Bernard , Jaumain Hélène , Boulétreau Paul , Chassard Dominique , Malbec Isabelle , Ducint Dominique , Saux Marie-Claude
Published: October 2018

The objective of this study was to analyze the pharmacokinetics of isepamicin during continuous venovenous hemodiafiltration. Six patients received 15 mg of isepamicin per kg of body weight. The mean isepamicin concentration peak in serum was 62.88 ± 18.20 mg/liter 0.5 h after the infusion. The elimination half-life was 7.91 ± 0.83 h. The mean total body clearance was 1.75 ± 0.28 liters/h, and dialysate outlet (DO) clearance was 2.76 ± 0.59 liters/h. The mean volume of distribution was 19.83 ± 2.95 liters. The elimination half-life, DO clearance, and volume of distribution were almost constant. In this group of patients, the initial dosage of 15 mg/kg appeared to be adequate, but the dosage interval should be determined by monitoring residual isepamicin concentrations in plasma.
Multicenter Study of In Vitro Susceptibility of the
Bacteroides fragilis
Group, 1995 to 1996, with Comparison of Resistance Trends from 1990 to 1996
Authors: Snydman D. R. , Jacobus N. V. , McDermott L. A. , Supran S. , Cuchural G. J. , Finegold S. , Harrell L. , Hecht D. W. , Iannini P. , Jenkins S. , Pierson Carl , Rihs J. , Gorbach S. L.
Published: October 2018

Antimicrobial resistance, including plasmid-mediated resistance, among the species of the
Bacteroides fragilis
group is well documented. An analysis of the in vitro susceptibility of
B. fragilis
group species referred between 1995 and 1996 as well as during a 7-year (1990 to 1996), prospective, multicenter survey of over 4,000 clinical isolates of
B. fragilis
group species was undertaken to review trends in the percent resistance to and geometric mean MICs of the antibiotics tested. There was a trend toward a decrease in the geometric mean MICs of most β-lactam antibiotics, while the percent resistance to most agents was less affected. Within the species
B. fragilis
, the geometric mean MICs showed significant (
P
< 0.05) decreases for piperacillin-tazobactam, ticarcillin-clavulanate, piperacillin, ticarcillin, ceftizoxime, cefotetan, and cefmetazole; a significant increase was observed for clindamycin and cefoxitin. For the non-
B. fragilis
species, a significant decrease in the geometric mean MICs was observed for meropenem, ampicillin-sulbactam, ticarcillin-clavulanate, piperacillin, ticarcillin, ceftizoxime, and cefmetazole; a significant increase was observed for cefoxitin. Significant increases in percent resistance were observed within the
B. fragilis
strains for ticarcillin and ceftizoxime and within the non-
B. fragilis
isolates for cefotetan. Significant increases in percent resistance among all
B. fragilis
group species were observed for clindamycin, while imipenem showed no significant change in resistance trends. The trend analysis for trovafloxacin was limited to 3 years, since the quinolone was tested only in 1994, 1995, and 1996. During the 7 years analyzed, there was no resistance to metronidazole or chloramphenicol observed. The data demonstrate that resistance among the
B. fragilis
group species has decreased in the past several years, the major exception being clindamycin. The majority of the resistance decrease has been for the β-lactams in
B. fragilis
, compared to other species. The reasons for these changes are not readily apparent.
Decreased Azithromycin Susceptibility of
Neisseria gonorrhoeae
Due to
mtrR
Mutations
Authors: Zarantonelli Leticia , Borthagaray Graciela , Lee Eun-Hee , Shafer William M.
Published: October 2018

Single-dose azithromycin therapy has recently been used in Uruguay for the treatment of uncomplicated gonococcal infections. As part of an active surveillance study to monitor the emergence of antibiotic resistance in gonococcal isolates, we examined the levels of azithromycin susceptibility in 51 consecutive isolates obtained from males with uncomplicated gonococcal urethritis. Isolates with decreased susceptibility to azithromycin (MICs, 0.25 to 0.5 μg/ml) were common, and these isolates often displayed cross-resistance to hydrophobic antimicrobial agents (erythromycin and Triton X-100). Resistance to erythromycin and Triton X-100 is frequently due to overexpression of the
mtrCDE
-encoded efflux pump mediated by mutations in the
mtrR
gene, which encodes a transcriptional repressor that modulates expression of the
mtrCDE
operon. Accordingly, we questioned whether clinical isolates that express decreased azithromycin susceptibility harbor
mtrR
mutations. Promoter mutations that would decrease the level of expression of
mtrR
as well as a missense mutation at codon 45 in the
mtrR
-coding region that would result in a radical amino acid replacement within the DNA-binding motif of MtrR were found in these strains. When these mutations were transferred into azithromycin-susceptible strain FA19 by transformation, the susceptibility of gonococci to azithromycin was decreased by nearly 10-fold. The
mtrCDE
-encoded efflux pump system was responsible for this property since insertional inactivation of the
mtrC
gene resulted in enhanced susceptibility of gonococci to azithromycin. We conclude that the
mtrCDE
-encoded efflux pump can recognize azithromycin and that the emergence of gonococcal strains with decreased susceptibility to azithromycin can, in part, be explained by
mtrR
mutations.
In Vitro Induction of Human Immunodeficiency Virus Type 1 Variants Resistant to Phosphoralaninate Prodrugs of
Z
-Methylenecyclopropane Nucleoside Analogues
Authors: Yoshimura Kazuhisa , Feldman Ron , Kodama Eiichi , Kavlick Mark F. , Qiu Yao-Ling , Zemlicka Jiri , Mitsuya Hiroaki
Published: October 2018

Two methylenecyclopropane nucleoside analogues with a phenylphosphoralaninate moiety, QYL-685 and QYL-609, exert potent and specific activities against human immunodeficiency virus type 1 strain LAI (HIV-1
LAI
) and HIV-2 in vitro. In this study, we induced HIV-1 variants resistant to QYL-685 by exposing HIV-1
LAI
to increasing concentrations of QYL-685. After 16 passages, the virus (HIV-1
P16
) was less sensitive to QYL-685 (104-fold), QYL-609 (>41-fold), and (−)-β-2′,3′-dideoxy-3′-thiacytidine (3TC) (>1,100-fold) than was HIV-1
LAI
and contained an M184I mutation. Two infectious clones, HIV-1
M184I
and HIV-1
M184V
, were resistant to QYL-685, QYL-609, and 3TC, confirming that the M184I mutation was responsible for the observed resistance. Viral-fitness analyses (competitive HIV-1 replication assays) revealed that in the absence of drugs, M184I and M184V conferred a replication disadvantage on the virus compared to the replication efficiency of the wild-type infectious clone (HIV-1
wt
). However, in the presence of QYL-685 (4 μM), HIV-1
M184I
and HIV-1
M184V
showed greater fitness than HIV-1
wt
. These data may provide structural and virological relevance with regard to the emergence of M184I and M184V substitutions in HIV-1.
Association of Alterations in ParC and GyrA Proteins with Resistance of Clinical Isolates of
Enterococcus faecium
to Nine Different Fluoroquinolones
Authors: Brisse Sylvain , Fluit Ad C. , Wagner Ulrich , Heisig Peter , Milatovic Dana , Verhoef Jan , Scheuring Sybille , Köhrer Karl , Schmitz Franz-Josef
Published: October 2018

The
parC
and
gyrA
genes of 73 ciprofloxacin-resistant and 6 ciprofloxacin-susceptible
Enterococcus faecium
clinical isolates were partly sequenced. Alterations in ParC and GyrA, possibly in combination with other resistance mechanisms, severely restricted the in vitro activities of the nine quinolones tested. For all isolates, clinafloxacin and sitafloxacin showed the best activities.
Effects of Cilastatin on the Pharmacokinetics of a New Carbapenem, DA-1131, in Rats, Rabbits, and Dogs
Authors: Kim So H. , Kwon Jong W. , Kim Won B. , Lee Myung G.
Published: October 2018

DA-1131, a new carbapenem antibiotic, undergoes renal metabolism by renal dehydropeptidase I (DHP-I), located on the brush border of the proximal tubular cell. Species differences with regard to the effects of cilastatin, a renal DHP-I inhibitor, were investigated after a 1-min intravenous infusion of DA-1131, with or without cilastatin, to rats, rabbits, and dogs. After intravenous infusion, the nonrenal clearance (CL
NR
) of DA-1131 was significantly slower in rats (3.00 versus 8.01 ml/min/kg) and rabbits (2.41 versus 6.77 ml/min/kg) when the drug was coadministered with cilastatin; this could be due to the slower metabolism of DA-1131 by rat and rabbit kidney DHP-I. This indicated that renal metabolism of DA-1131 by renal DHP-I was inhibited by cilastatin. However, coadministration with cilastatin to dogs did not affect the CL
NR
of DA-1131.
Potent Bacteriolytic Activity of Ritipenem Associated with a Characteristic Profile of Affinities for Penicillin-Binding Proteins of
Haemophilus influenzae
Authors: Inui Takashi , Oshida Tadahiro , Endo Toshio , Matsushita Tadahiro
Published: October 2018

Ritipenem is highly bacteriolytic against
Haemophilus influenzae
. Bacterial lysis was shown after treatments with ritipenem and cefsulodin at their MICs and after treatments with fropenem and cefdinir at four times their MICs, indicated by decreases in the culture turbidities and by morphological changes of the destroyed cells. These β-lactams were preferentially bound to penicillin-binding protein (PBP) 1b. Ritipenem, fropenem, and cefsulodin exhibited poor affinities to PBPs 3a and 3b, but cefdinir showed high affinities to these PBPs. Microscopic examinations revealed that selective PBP 3 inhibitors, such as aztreonam and cefotaxime, inhibited lysis induced by ritipenem. These results suggest that the preferential inactivation of PBP 1b could be essential to induce the lysis of
H. influenzae
cells and that binding to PBPs 3a and 3b may interfere with lysis.
Potentiation of Artemisinin Activity against Chloroquine-Resistant
Plasmodium falciparum
Strains by Using Heme Models
Authors: Benoit-Vical Françoise , Robert Anne , Meunier Bernard
Published: October 2018

The influence of different metalloporphyrin derivatives on the antimalarial activity of artemisinin was studied with two chloroquine-resistant strains of
Plasmodium falciparum
(FcB1-Colombia and FcM29-Cameroon) cultured in human erythrocytes. This potentiation study indicates that the manganese complex of
meso
-tetrakis(4-sulfonatophenyl)porphyrin has a significant synergistic effect on the activity of artemisinin against both
Plasmodium
strains.
Dose Range Evaluation of Liposomal Nystatin and Comparisons with Amphotericin B and Amphotericin B Lipid Complex in Temporarily Neutropenic Mice Infected with an Isolate of
Aspergillus fumigatus
with Reduced Susceptibility to Amphotericin B
Authors: Denning David W. , Warn Peter
Published: October 2018

Using an isolate of
Aspergillus fumigatus
that is less susceptible in vivo to amphotericin B than most other isolates, we compared different doses of liposomal nystatin (L-nystatin), liposomal amphotericin B (L-amphotericin), and amphotericin B lipid complex (ABLC) with amphotericin B deoxycholate. Four experiments with intravenously infected neutropenic mice were conducted. A dose of L-nystatin at 10 mg/kg of body weight was toxic (the mice had fits or respiratory arrest). The optimal dosage of L-nystatin was 5 mg/kg daily on days 1, 2, 4, and 7 (90% survival). This was superior to L-amphotericin (5 mg/kg [
P
= 0.24] and 1 mg/kg [
P
< 0.0001]), ABLC (5 mg/kg [
P
= 0.014] and 1 mg/kg [
P
< 0.0001]), and amphotericin B deoxycholate (5 mg/kg [
P
= 0.008]). In terms of liver and kidney cultures, L-nystatin (5 mg/kg) was superior to all other regimens (
P
= 0.0032 and <0.0001, respectively). Higher doses of L-amphotericin (25 and 50 mg/kg) in one earlier experiment were more effective (100% survival) than 1 mg of L-amphotericin per kg and amphotericin deoxycholate (5 mg/kg) in terms of mortality and both liver and kidney culture results and to L-amphotericin (5 mg/kg) in terms of liver and kidney culture results only. ABLC (25 mg/kg) given daily for 7 days was superior to ABLC (50 mg/kg [
P
= 0.03]) but not to ABLC at 5 mg/kg or amphotericin B deoxycholate in terms of mortality, although it was in terms of liver and kidney culture results. No dose-response for amphotericin B (5 and 1 mg/kg) was demonstrable. In conclusion, in this stringent model, high doses of L-amphotericin and ABLC could overcome reduced susceptibility to amphotericin B deoxycholate, but all were inferior to 5- to 10-fold lower doses of L-nystatin.
Synergistic Fungistatic Effects of Lactoferrin in Combination with Antifungal Drugs against Clinical
Candida
Isolates
Authors: Kuipers M. E. , de Vries H. G. , Eikelboom M. C. , Meijer D. K. F. , Swart P. J.
Published: October 2018

Because of the rising incidence of failures in the treatment of oropharyngeal candidosis in the case of severely immunosuppressed patients (mostly human immunodeficiency virus [HIV]-infected patients), there is need for the development of new, more effective agents and/or compounds that support the activity of the common antifungal agents. Since lactoferrin is one of the nonspecific host defense factors present in saliva that exhibit antifungal activity, we studied the antifungal effects of human, bovine, and iron-depleted lactoferrin in combination with fluconazole, amphotericin B, and 5-fluorocytosine in vitro against clinical isolates of
Candida
species. Distinct antifungal activities of lactoferrin were observed against clinical isolates of
Candida
. The MICs generally were determined to be in the range of 0.5 to 100 mg · ml
−1
. Interestingly, in the combination experiments we observed pronounced cooperative activity against the growth of
Candida
by using lactoferrin and the three antifungals tested. Only in a limited concentration range was minor antagonism detected. The use of lactoferrin and fluconazole appeared to be the most successful combination. Significant reductions in the minimal effective concentrations of fluconazole were found when it was combined with a relatively low lactoferrin concentration (1 mg/ml). Such combinations still resulted in complete growth inhibition, while synergy of up to 50% against several
Candida
species was observed. It is concluded that the combined use of lactoferrin and antifungals against severe infections with
Candida
is an attractive therapeutic option. Since fluconazole-resistant
Candida
species have frequently been reported, especially in HIV-infected patients, the addition of lactoferrin to the existing fluconazole therapy could postpone the occurrence of species resistance against fluconazole. Clinical studies to further elucidate the potential utility of this combination therapy have been initiated.
Competition of Various β-Lactam Antibiotics for the Major Penicillin-Binding Proteins of
Helicobacter pylori
: Antibacterial Activity and Effects on Bacterial Morphology
Authors: DeLoney Cindy R. , Schiller Neal L.
Published: October 2018

The penicillin-binding proteins (PBPs) of helical (log-phase)
Helicobacter pylori
ATCC 43579 were identified by using biotinylated ampicillin. The major PBPs had apparent molecular masses of 47, 60, 63, and 66 kDa; an additional minor PBP of 95 to 100 kDa was also detected. The relative affinities of various β-lactams for these PBPs were tested by competitive-binding assays. Only PBP63 appeared to be significantly bound to each of the competing antibiotics, whereas PBP66 strongly bound mezlocillin, oxacillin, amoxicillin, and ceftriaxone. Whereas most of the β-lactams significantly bound two or more PBPs, aztreonam specifically targeted PBP63. The influence of sub-MICs of these β-lactams on the morphologies of log-phase
H. pylori
was observed at both the phase-contrast and transmission electron microscopy levels. Each of the eight β-lactams examined induced blebbing and sphere formation, whereas aztreonam was the only antibiotic studied which induced pronounced filamentation in
H. pylori
. Finally, studies comparing the PBPs of helical (log-phase) cultures with those of coccoid (7-, 14-, and 21-day-old) cultures of
H. pylori
revealed that the major PBPs at 60 and 63 kDa seen in the helical form were almost undetectable in the coccoid forms, whereas PBP66 remained the major PBP in the coccoid forms, although somewhat reduced in level compared to the helical form. PBP47 was present in both forms at approximately equal concentrations. These studies thus identified the major PBPs in both helical and coccoid forms of
H. pylori
and compared the relative affinities of seven different β-lactams for the PBPs in the helical forms and their effects on bacterial morphology.