bluuebunny/crossref_metadata_2025_split huggingface dataset:
Sequential Actions of the Two Component Peptides of the Lantibiotic Lacticin 3147 Explain Its Antimicrobial Activity at Nanomolar Concentrations
Authors: Morgan Sheila M. , O'Connor Paula M. , Cotter Paul D. , Ross R. Paul , Hill Colin 
Published: June 2005

ABSTRACT

Lacticin 3147 is a two-peptide (LtnA1 and LtnA2) lantibiotic produced by
Lactococcus lactis
subsp.
lactis
DPC3147 and has inhibitory activity against all gram-positive microorganisms tested. In this study the specific activities of the component peptides (alone or in combination) were determined by using
L. lactis
subsp.
cremoris
HP as the target strain. Lacticin 3147 exhibited an MIC
50
of 7 nM for each component peptide (in combination), suggesting a peptide stoichiometry of 1:1. Interestingly, the LtnA1 peptide demonstrated independent inhibitory activity, with an MIC
50
of 200 nM against
L. lactis
HP. In parallel studies, the single peptide bacteriocin nisin exhibited an MIC
50
of 50 nM against the same target strain. Sequential peptide addition (with an intermediate washing step) demonstrated that LtnA1 must be added before LtnA2 rather than vice versa to observe inhibitory activity. The nanomolar activity of the lacticin peptides suggests the involvement of a docking molecule, speculated to be lipid II. Taken together with the recently determined structure of lacticin 3147 (N. I. Martin, T. Sprules, M. R. Carpenter, P. D. Cotter, C. Hill, R. P. Ross, and J. C. Vederas, Biochemistry, 43:3049-3056, 2004), these data support the hypothesis that the mode of action for lacticin 3147 involves a lipid II binding step (by the mersacidin-like LtnA1 peptide, which would explain its independent inhibitory activity), followed by insertion of the more linear LtnA2 peptide into the target membrane, resulting in pore formation and ultimate cell death.
Microplate Alamar Blue Assay for
Staphylococcus epidermidis
Biofilm Susceptibility Testing
Authors: Pettit Robin K. , Weber Christine A. , Kean Melissa J. , Hoffmann Holger , Pettit George R. , Tan Rui , Franks Kelly S. , Horton Marilyn L. 
Published: June 2005

ABSTRACT

Biofilms are at the root of many infections largely because they are much more antibiotic resistant than their planktonic counterparts. Antibiotics that target the biofilm phenotype are desperately needed, but there is still no standard method to assess biofilm drug susceptibility.
Staphylococcus epidermidis
ATCC 35984 biofilms treated with eight different approved antibiotics and five different experimental compounds were exposed to the oxidation reduction indicator Alamar blue for 60 min, and reduction relative to untreated controls was determined visually and spectrophotometrically. The minimum biofilm inhibitory concentration was defined as ≤50% reduction and a purplish well 60 min after the addition of Alamar blue. All of the approved antibiotics had biofilm MICs (MBICs) of >512 μg/ml (most >4,096 μg/ml), and four of the experimental compounds had MBICs of ≤128 μg/ml. The experimental aaptamine derivative hystatin 3 was used to correlate Alamar blue reduction with 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2
H
-tetrazolium-5-carboxanilide (XTT) reduction and viable counts (CFU/ml) for
S. epidermidis
ATCC 35984, ATCC 12228, and two clinical isolates. For all four strains, Alamar blue results correlated well with XTT (
r
= 0.83 to 0.97) and with CFU/ml results (
r
= 0.85 to 0.94). Alamar blue's stability and lack of toxicity allowed CFU/ml to be determined from the same wells as Alamar blue absorbances. If the described method of microplate Alamar blue biofilm susceptibility testing, which is simple, reproducible, cost-effective, nontoxic, and amenable to high throughput, is applicable to other important biofilm forming species, it should greatly facilitate the discovery of biofilm specific agents.
Emergence of a Novel Mutation in the FLLA Region of Hepatitis B Virus during Lamivudine Therapy
Authors: Pai S. Balakrishna , Bozdayi A. Mithat , Pai Rekha B. , Beker Tolunay , Sarioglu Mustafa , Turkyilmaz Ahmet R. , Grier Jason , Yurdaydin Cihan , Schinazi Raymond F. 
Published: June 2005

ABSTRACT
The emergence of resistance to lamivudine has been one of the major stumbling blocks to successful treatment and control of hepatitis B virus (HBV) infections. The major mechanism of resistance has been attributed to the alteration in the YMDD motif of the HBV polymerase due to an amino acid change of rtM204 to V/I and an accompanying rtL180M conversion. A novel mutation pattern in a patient having clinical breakthrough under lamivudine therapy was discovered. The mutant had a rtL180C/M204I genotype and was detected after 2 years of therapy with lamivudine. To characterize this novel variant, site-directed mutagenesis was performed using a vector construct containing the HBV genome. Transient transfection studies in human hepatoma cells with HBV carrying the new mutant demonstrated that the rtL180C/M204I mutant was resistant to lamivudine up to 10 μM. The resistance profile was comparable to that of the previously reported rtL180 M/M204I-containing virus. These observations were further confirmed by generation of stable cultures transfected with the mutant virus.
Putative VanRS-Like Two-Component Regulatory System Associated with the Inducible Glycopeptide Resistance Cluster of
Paenibacillus popilliae
Authors: Fraimow Henry , Knob Christopher , Herrero Inmaculada A. , Patel Robin 
Published: June 2005

ABSTRACT

Paenibacillus popilliae
contains
vanF
encoding a putative
d
-Ala:
d
-lactate (
d
-Lac) ligase, VanF, as part of the
vanY

F

Z

F

H

F

FX

F

cluster that is similar in structure to the enterococcal
vanA
and
vanB
clusters. Using growth curves, we demonstrated that vancomycin resistance in
P. popilliae
is inducible. Using degenerate oligonucleotides targeted at bacterial cell wall ligases, we identified a second ligase gene with features of a
d
-Ala:
d
-Ala ligase in both
P. popilliae
and the related, vancomycin-susceptible,
Paenibacillus lentimorbus
. The 3,380-bp region upstream of
vanY

F

Z

F

H

F

FX

F

in
P. popilliae
ATCC 14706 was sequenced and found to contain genes encoding a putative two-component regulator, VanR
F
S
F
, similar to VanRS but more closely related to a family of two-component regulators linked to VanY-like carboxypeptidases in several glycopeptide-susceptible
Bacillus
species. This upstream region also included a transposase similar to a transposase found in
Bacillus halodurans
and, in some strains, a 99-bp insertion of unknown function with 95% nucleotide identity to a portion of the Tn
1546
transposase gene. Analysis of glycopeptide resistance-associated clusters from soil and/or insect-dwelling organisms may provide important clues to the molecular evolution of acquired glycopeptide resistance elements in human pathogens.
Biological Activity of an Intravenous Preparation of Human Vaccinia Immune Globulin in Mouse Models of Vaccinia Virus Infection
Authors: Shearer Jeffry D. , Siemann Linda , Gerkovich Mary , House Robert V. 
Published: June 2005

ABSTRACT

The biological activity of a new intravenous (i.v.) preparation of human vaccinia immune globulin (VIGIV) was evaluated in two mouse models of vaccinia virus (VV) infection. In a mouse tail lesion model, female CD-1 mice were inoculated i.v. with 7 × 10
4
PFU of VV to produce >10 lesions per tail 8 days later. In a mouse lethality model, female severe combined immunodeficient (SCID) mice were inoculated i.v. with 3 × 10
4
PFU of VV to produce 100% mortality within 45 days. The ability of VIGIV to reduce tail lesion formation in CD-1 mice and mortality in SCID mice was determined by (i) pretreatment of a lethal VV dose with VIGIV prior to i.v. inoculation into SCID mice and (ii) i.v. administration of VIGIV to CD-1 and SCID mice the day before and up to 8 days after VV infection. VIGIV reduced the proportion of CD-1 mice with >10 tail lesions in a dose-related manner when VIGIV was given 1 day before and up to 1 day after VV inoculation. The pretreatment of VV with VIGIV prolonged survival and decreased mortality. VIGIV (100 and 400 mg/kg) prolonged survival when given up to 4 days after VV inoculation, and the 400-mg/kg dose reduced the mortality rate by 80% when given the day before or immediately after VV inoculation. The biological activity of VIGIV was demonstrated in both the immunocompetent and immunocompromised murine models. The timing of treatment relative to VV inoculation appeared to be important for the demonstration of VIGIV's biological activity.
Antistaphylococcal Effect Related to the Area under the Curve/MIC Ratio in an In Vitro Dynamic Model: Predicted Breakpoints versus Clinically Achievable Values for Seven Fluoroquinolones
Authors: Firsov Alexander A. , Lubenko Irene Y. , Vostrov Sergey N. , Portnoy Yury A. , Zinner Stephen H. 
Published: June 2005

ABSTRACT

Prediction of the relative efficacies of different fluoroquinolones is often based on the ratios of the clinically achievable area under the concentration-time curve (AUC) to the MIC, usually with incorporation of the MIC
50
or the MIC
90
and with the assumption of antibiotic-independent patterns of the AUC/MIC-response relationships. To ascertain whether this assumption is correct, the pharmacodynamics of seven pharmacokinetically different quinolones against two clinical isolates of
Staphylococcus aureus
were studied by using an in vitro model. Two differentially susceptible clinical isolates of
S. aureus
were exposed to two 12-h doses of ciprofloxacin (CIP) and one dose of gatifloxacin (GAT), gemifloxacin (GEM), grepafloxacin (GRX), levofloxacin (LVX), moxifloxacin (MXF), and trovafloxacin (TVA) over similar AUC/MIC ranges from 58 to 932 h. A specific bacterial strain-independent AUC/MIC relationship with the antimicrobial effect (

I
E

) was associated with each quinolone. Based on the

I
E

-log AUC/MIC relationships, breakpoints (BPs) that are equivalent to a CIP AUC/MIC ratio of 125 h were predicted for GRX, MXF, and TVA (75 to 78 h), GAT and GEM (95 to 103 h) and LVX (115 h). With GRX and LVX, the predicted BPs were close to those established in clinical settings (no clinical data on other quinolones are available in the literature). To determine if the predicted AUC/MIC BPs are achievable at clinical doses, i.e., at the therapeutic AUCs (AUC
ther
s), the AUC
ther
/MIC
50
ratios were studied. These ratios exceeded the BPs for GAT, GEM, GRX, MXF, TVA, and LVX (750 mg) but not for CIP and LVX (500 mg). AUC/MIC ratios above the BPs can be considered of therapeutic potential for the quinolones. The highest ratios of AUC
ther
/MIC
50
to BP were achieved with TVA, MXF, and GEM (2.5 to 3.0); intermediate ratios (1.5 to 1.6) were achieved with GAT and GRX; and minimal ratios (0.3 to 1.2) were achieved with CIP and LVX.
The L74V Mutation in Human Immunodeficiency Virus Type 1 Reverse Transcriptase Counteracts Enhanced Excision of Zidovudine Monophosphate Associated with Thymidine Analog Resistance Mutations
Authors: Miranda Luis R. , Götte Matthias , Kuritzkes Daniel R. 
Published: June 2005

ABSTRACT
Thymidine analog mutations (TAMs) in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) confer resistance to zidovudine (AZT) by increasing the rate of ATP-dependent phosphorolysis of the terminal nucleotide monophosphate (primer unblocking). By contrast, the L74V mutation, which confers resistance to didanosine, sensitizes HIV-1 to AZT and partially restores AZT susceptibility when present together with one or more TAMs. To compare rates of primer unblocking in RTs carrying different clusters of TAMs and to explore the biochemical mechanism by which L74V affects AZT susceptibility, ATP-mediated rescue of AZT-blocked DNA synthesis was assayed using a series of purified recombinant RTs. Rates of primer unblocking were higher in the 67N/70R/219Q RT than in the 41L/210W/215Y enzyme and were similar to rates observed with an RT carrying six TAMs (41L/67N/70R/210W/215Y/219Q). The presence of 74V in an otherwise wild-type RT reduced the rate of primer unblocking to a degree similar to that observed with the M184V mutation for lamivudine resistance, which also sensitizes HIV-1 to AZT. Introduction of 74V into RTs carrying TAMs partially counteracted the effect of TAMs on the rate of primer unblocking. The effect of 74V was less marked than that of the 184V mutation in the 67N/70R/219Q and 41L/210W/215Y RTs but similar in the RT carrying six TAMs. These results demonstrate that L74V enhances AZT susceptibility by reducing the extent of its removal by ATP-dependent phosphorolysis and provides further evidence for a common mechanism by which mutations conferring resistance to didanosine and lamivudine sensitize HIV-1 to AZT.
Impaired Rescue of Chain-Terminated DNA Synthesis Associated with the L74V Mutation in Human Immunodeficiency Virus Type 1 Reverse Transcriptase
Authors: Frankel Fernando A. , Marchand Bruno , Turner Dan , Götte Matthias , Wainberg Mark A. 
Published: June 2005

ABSTRACTThe L74V and M184V mutations in the reverse transcriptase (RT) gene of human immunodeficiency virus type 1 (HIV-1) are frequently associated with resistance to the nucleoside reverse transcriptase inhibitors abacavir, didanosine, and lamivudine. Yet viruses containing any of these mutations often display hypersusceptibility to zidovudine (ZDV). Two distinct mechanisms have been described to explain HIV-1 drug resistance. One of these involves diminished rates of incorporation of the nucleotide analogue by mutated RT, while the other mechanism involves increased rates of phosphorolytic excision of the drug-terminated primer. To understand the biochemical mechanisms responsible for the hypersensitization of L74V-containing viruses to ZDV, we studied the efficiency of excision of ZDV-monophosphate (ZDV-MP)-terminated primers by recombinant wild-type and mutated HIV-1 RTs in cell-free assays. We observed that the L74V mutation in RT caused reductions in ATP-dependent removal of ZDV-MP from newly synthesized viral DNA. In addition, we determined that the L74V and M184V mutations did not affect the ratio between the populations of RT-DNA/DNA complexes found at pre- and posttranslocational stages; however, they might have affected proper alignment between incorporated chain terminator and pyrophosphate donor, substrate orientation, affinity for ATP, and/or primer-template substrate. Finally, we confirmed previous findings that L74V-containing viruses display diminished replication capacity and that this is associated with reduced levels of synthesis of early reverse-transcribed viral DNA molecules.
Antimicrobial Activity of Novel Dendrimeric Peptides Obtained by Phage Display Selection and Rational Modification
Authors: Pini Alessandro , Giuliani Andrea , Falciani Chiara , Runci Ylenia , Ricci Claudia , Lelli Barbara , Malossi Monica , Neri Paolo , Rossolini Gian Maria , Bracci Luisa 
Published: June 2005

ABSTRACT

A large 10-mer phage peptide library was panned against whole
Escherichia coli
cells, and an antimicrobial peptide (QEKIRVRLSA) was selected. The peptide was synthesized in monomeric and dendrimeric tetrabranched form (multiple antigen peptide [MAP]), which generally allows a dramatic increase of peptide stability to peptidases and proteases. The antibacterial activity of the dendrimeric peptide against
E. coli
was much higher than that of the monomeric form. Modification of the original sequence, by residue substitution or sequence shortening, produced three different MAPs, M4 (QAKIRVRLSA), M5 (KIRVRLSA), and M6 (QKKIRVRLSA) with enhanced stability to natural degradation and antimicrobial activity against a large panel of gram-negative bacteria. The MICs of the most potent peptide, M6, were as low as 4 to 8 μg/ml against recent clinical isolates of multidrug-resistant
Pseudomonas aeruginosa
and members of the
Enterobacteriaceae
. The same dendrimeric peptides showed high stability to blood proteases, low hemolytic activity, and low cytotoxic effects on eukaryotic cells, making them promising candidates for the development of new antibacterial drugs.
Correlation between Ratio of Serum Doxycycline Concentration to MIC and Rapid Decline of Antibody Levels during Treatment of Q Fever Endocarditis
Authors: Rolain Jean-Marc , Boulos Areen , Mallet Marie-Noëlle , Raoult Didier 
Published: June 2005

ABSTRACT

Endocarditis is the major clinical manifestation of chronic Q fever. Although doxycycline along with hydroxychloroquine remains the mainstay of medical therapy for Q fever endocarditis, there are wide variations in the rapidity of the patient's decline of antibody levels during such therapy. We undertook a retrospective examination of whether there was any correlation between the ratio of serum concentration to MIC of doxycycline and response to treatment in patients with Q fever endocarditis. Included herein are 16 patients from whom
Coxiella burnetii
was isolated from cardiac valve materials. Serology and measurement of doxycycline and hydroxychloroquine serum levels were performed and recorded after 1 year of treatment. The MIC of doxycycline for
C. burnetii
isolates was determined using the shell vial assay in a real-time quantitative PCR assay. At the completion of a yearlong therapy with doxycycline-hydroxychloroquine, all those that showed a low decline of antibody levels (
n
= 6) (i.e., <2-fold decrease in antibody titer to phase I
C. burnetii
antigen) had a ratio of serum doxycycline concentration to MIC between 0.5 and 1. In contrast, those having a ratio of ≥1 showed a rapid decline of phase I antibody levels (
n
= 9;
P
< 0.05). The only patient who died had a serum doxycycline-to-MIC ratio of <0.5, and the isolate of
C. burnetii
cultured from this patient was resistant to doxycycline (MIC = 8 μg/ml). The ratio of serum doxycycline concentration to MIC should be monitored during the course of therapy in patients with Q fever endocarditis.
Alteration of Fatty Acid and Sterol Metabolism in Miltefosine-Resistant
Leishmania donovani
Promastigotes and Consequences for Drug-Membrane Interactions
Authors: Rakotomanga M. , Saint-Pierre-Chazalet M. , Loiseau P. M. 
Published: June 2005

ABSTRACT

Miltefosine (hexadecylphosphocholine [HePC]) is the first orally active drug approved for the treatment of visceral leishmaniasis. In order to investigate the biochemical modifications occurring in HePC-resistant (HePC-R)
Leishmania donovani
promastigotes, taking into account the lipid nature of HePC, we investigated their fatty acid and sterol metabolisms. We found that the content of unsaturated phospholipid alkyl chains was lower in HePC-R parasite plasma membranes than in those of the wild type, suggesting a lower fluidity of HePC-R parasite membranes. We also demonstrated that HePC insertion within an external monolayer was more difficult when the proportion of unsaturated phospholipids decreased, rendering the HePC interaction with the external monolayer of HePC-R parasites more difficult. Furthermore, HePC-R parasite membranes displayed a higher content of short alkyl chain fatty acids, suggesting a partial inactivation of the fatty acid elongation enzyme system in HePC-R parasites. Sterol biosynthesis was found to be modified in HePC-R parasites, since the 24-alkylated sterol content was halved in HePC-R parasites; however, this modification was not related to HePC sensitivity. In conclusion, HePC resistance affects three lipid biochemical pathways: fatty acid elongation, the desaturase system responsible for fatty acid alkyl chain unsaturation, and the C-24-alkylation of sterols.
Reduced Susceptibility of
Staphylococcus aureus
to Vancomycin and Platelet Microbicidal Protein Correlates with Defective Autolysis and Loss of Accessory Gene Regulator (
agr
) Function
Authors: Sakoulas George , Eliopoulos George M. , Fowler Vance G. , Moellering Robert C. , Novick Richard P. , Lucindo Natalie , Yeaman Michael R. , Bayer Arnold S. 
Published: June 2005

ABSTRACT

Loss of
agr
function, vancomycin exposure, and abnormal autolysis have been linked with both development of the GISA phenotype and low-level resistance in vitro to thrombin-induced platelet microbicidal proteins (tPMPs). We examined the potential in vitro interrelationships among these parameters in well-characterized, isogenic laboratory-derived and clinical
Staphylococcus aureus
isolates. The laboratory-derived
S. aureus
strains included RN6607 (
agr
II-positive parent) and RN6607V (vancomycin-passaged variant; hetero-GISA), RN9120 (RN6607
agr
::
tetM
;
agr
II knockout parent), RN9120V (vancomycin-passaged variant), and RN9120-GISA (vancomycin passaged, GISA). Two serial isolates from a vancomycin-treated patient with recalcitrant, methicillin-resistant
S. aureus
(MRSA) endocarditis were also studied: A5937 (
agr
II-positive initial isolate) and A5940 (
agr
II-defective/hetero-GISA isolate obtained after prolonged vancomycin administration). In vitro tPMP susceptibility phenotypes were assessed after exposure of strains to either 1 or 2 μg/ml. Triton X-100- and vancomycin-induced lysis profiles were determined spectrophotometrically. For
agr
II-intact strain RN6607, vancomycin exposure in vitro was associated with modest increases in vancomycin MICs and reduced killing by tPMP, but no change in lysis profiles. In contrast, vancomycin exposure of
agr
II-negative RN9120 yielded a hetero-GISA phenotype and was associated with defects in lysis and reduced in vitro killing by tPMP. In the clinical isolates, loss of
agr
II function during prolonged vancomycin therapy was accompanied by emergence of the hetero-GISA phenotype and reduced tPMP killing, with no significant change in lysis profiles. An association was identified between loss of
agr
II function and the emergence of hetero-GISA phenotype during either in vitro or in vivo vancomycin exposure. In vitro, these events were associated with defective lysis and reduced susceptibility to tPMP. The precise mechanism(s) underlying these findings is the subject of current investigations.
Population Structure of
Enterococcus faecium
Causing Bacteremia in a Spanish University Hospital: Setting the Scene for a Future Increase in Vancomycin Resistance?
Authors: Coque Teresa M. , Willems Rob J. L. , Fortún Jesús , Top Janetta , Diz Sergio , Loza Elena , Cantón Rafael , Baquero Fernando 
Published: June 2005

ABSTRACT

Over an 8-year period (1995 to 2002), 86
Enterococcus faecium
blood isolates from 84 patients, of which 54 were ampicillin resistant (AREF) and 32 were ampicillin susceptible (ASEF), were studied in a university hospital (1,200 beds; serving a population of 600,000) in Spain, a country characterized by a near-absence of resistance to vancomycin and very high rates of ampicillin resistance among enterococci. Clonal relatedness by pulsed-field gel electrophoresis (PFGE), antibiotic susceptibility, presence of the virulence/epidemicity genes
esp
Efm
and
hyl
Efm
, and identification of
purK
alleles were studied. A group of isolates was also analyzed by amplified fragment length polymorphism (AFLP) and multilocus sequence typing. Medical charts (30 variables collected) were reviewed for 60/84 patients. ASEF showed high clonal diversity (32 PFGE types, 11
purK
alleles, 4 AFLP genogroups), did not harbor putative virulence genes, and had no specific association with hospital acquisition. AREF isolates belonged to a clonal complex (CC) of genetically related strains (
purK
-
1
, AFLP genogroup C), occasionally harboring putative virulence traits, and were from patients with particular risk factors. Within this CC, previously associated with vancomycin-resistant
E. faecium
isolates causing outbreaks worldwide (W. L. Homan et al., J. Clin. Microbiol.
40:
1963-1971, 2002), a great genetic diversity of antibiotic resistance and virulence/epidemicity profiles was found. Associations between
esp
and a >7-day hospital stay and between
purK
-
1
, hospital location, and nosocomial acquisition were noted (
P
< 0.001). These findings reflect the importance of local environmental differences in the evolution of this CC, suggesting that the emergence of vancomycin resistance among AREF strains in Spain may be a question of time.