bluuebunny/crossref_metadata_2025 huggingface dataset:
MepR, a Repressor of the
Staphylococcus aureus
MATE Family Multidrug Efflux Pump MepA, Is a Substrate-Responsive Regulatory Protein
Authors: Kaatz Glenn W. , DeMarco Carmen E. , Seo Susan M.
Published: March 2006

The
mepRAB
gene cluster of
Staphylococcus aureus
encodes a MarR family repressor (MepR; known to repress
mepA
expression), a MATE family multidrug efflux pump (MepA), and a protein of unknown function (MepB). In this report, we show that MepR also is autoregulatory, repressing the expression of its own gene. Exposure of strains containing a
mepR
::
lacZ
fusion with
mepR
provided in
trans
under the control of an inducible promoter, or a
mepA
::
lacZ
fusion alone, to subinhibitory concentrations of MepA substrates resulted in variably increased expression mainly of
mepA
. Mobility shift assays revealed that MepR binds upstream of
mepR
and
mepA
, with an apparently higher affinity for the
mepA
binding site. MepA substrates abrogated MepR binding to each site in a differential manner, with the greatest effect observed on the MepR-
mepA
operator interaction. DNase I footprinting identified precise binding sites which included promoter motifs, inverted repeats, and transcription start sites for
mepR
and
mepA
, as well as a conserved GTTAG motif, which may be a signature recognition sequence for MepR. Analogous to other multidrug efflux pump regulatory proteins such as QacR, the substrate-MepR interaction likely results in its dissociation from its
mepA
, and in a more limited fashion its
mepR
, operator sites and relief of its repressive effect. The enhanced effect of substrates on
mepA
compared to
mepR
expression, and on the MepR-
mepA
operator interaction, results in significant relief of
mepA
and relative maintenance of
mepR
repression, leading to increased MepA protein unimpeded by MepR when the need for detoxification exists.
In Vitro Analysis of IS
Ecp1B
-Mediated Mobilization of Naturally Occurring β-Lactamase Gene
bla
CTX-M
of
Kluyvera ascorbata
Authors: Lartigue Marie-Frédérique , Poirel Laurent , Aubert Daniel , Nordmann Patrice
Published: March 2006

IS
Ecp1B
has been reported to be associated with and to mobilize the emerging expanded-spectrum β-lactamase
bla
CTX-M
genes in
Enterobacteriaceae
. Thus, the ability of this insertion sequence to mobilize the
bla
CTX-M-2
gene was tested from its progenitor,
Kluyvera ascorbata
. Insertions of IS
Ecp1B
upstream of the
bla
CTX-M-2
gene in
K. ascorbata
reference strain CIP7953 were first selected with cefotaxime (0.5 and 2 μg/ml). In those cases, IS
Ecp1B
brought promoter sequences enhancing
bla
CTX-M-2
expression in
K. ascorbata
. Then, IS
Ecp1B
-mediated mobilization of the
bla
CTX-M-2
gene from
K. ascorbata
to
Escherichia coli
J53 was attempted. The transposition frequency of IS
Ecp1B-bla
CTX-M-2
occurred at (6.4 ± 0.5) × 10
−7
in
E. coli
. Cefotaxime, ceftazidime, and piperacillin enhanced transposition, whereas amoxicillin, cefuroxime, and nalidixic acid did not. Transposition was also enhanced when studied at 40°C.
Evaluation of Amphotericin B Interpretive Breakpoints for
Candida
Bloodstream Isolates by Correlation with Therapeutic Outcome
Authors: Park Benjamin J. , Arthington-Skaggs Beth A. , Hajjeh Rana A. , Iqbal Naureen , Ciblak Meral A. , Lee-Yang Wendy , Hairston Mario D. , Phelan Maureen , Plikaytis Brian D. , Sofair Andre N. , Harrison Lee H. , Fridkin Scott K. , Warnock David W.
Published: March 2006

One hundred seven
Candida
bloodstream isolates (51
C. albicans
, 24
C. glabrata
, 13
C. parapsilosis
, 13
C. tropicalis
, 2
C. dubliniensis
, 2
C. krusei
, and 2
C. lusitaniae
strains) from patients treated with amphotericin B alone underwent in vitro susceptibility testing against amphotericin B using five different methods. Fifty-four isolates were from patients who failed treatment, defined as death 7 to 14 days after the incident candidemia episode, having persistent fever of ≥5 days' duration after the date of the incident candidemia, or the recurrence of fever after two consecutive afebrile days while on antifungal treatment. MICs were determined by using the Clinical Laboratory Standards Institute (formally National Committee for Clinical Laboratory Standards) broth microdilution procedure with two media and by using Etest. Minimum fungicidal concentrations (MFCs) were also measured in two media. Broth microdilution tests with RPMI 1640 medium generated a restricted range of MICs (0.125 to 1 μg/ml); the corresponding MFC values ranged from 0.5 to 4 μg/ml. Broth microdilution tests with antibiotic medium 3 produced a broader distribution of MIC and MFC results (0.015 to 0.25 μg/ml and 0.06 to 2 μg/ml, respectively). Etest produced the widest distribution of MICs (0.094 to 2 μg/ml). However, none of the test formats studied generated results that significantly correlated with therapeutic success or failure.
Assessment of the Paradoxical Effect of Caspofungin in Therapy of Candidiasis
Authors: Clemons Karl V. , Espiritu Marife , Parmar Rachana , Stevens David A.
Published: March 2006

Paradoxical growth of some
Candida albicans
isolates in the presence of caspofungin (CAS) in vitro has been demonstrated previously. We sought to determine whether a similar phenomenon occurred in vivo. A systemic model of candidiasis was studied in CD-1 mice by intravenous inoculation of different isolates of
C. albicans.
Infected animals were treated with CAS at various dosages (0.01 to 20 mg/kg) and CFU remaining in the kidneys determined. Four clinical isolates that showed paradoxical growth in vitro and one that did not were tested. Recovery of CFU from the kidneys showed that dosages of CAS at 0.1 mg/kg and above were efficacious in the reduction of
C. albicans
, but were not curative. Against isolates that show paradoxical growth in vitro, CAS was efficacious, but lacked dose responsiveness above 0.5 mg/kg against three of the four. One isolate, 95-68, showed paradoxical growth in vivo with significantly higher CFU recovered from mice given CAS at 20 mg/kg than those given CAS at 5 mg/kg, but the effect was not reproducible in a subsequent experiment. When CAS was given prophylactically and therapeutically, improved efficacy and cure rate were observed. Overall, these data indicate that CAS is highly efficacious against systemic murine candidiasis and a paradoxical effect was not reproducibly demonstrated in vivo.
Pharmacokinetics of Tenofovir Disoproxil Fumarate and Ritonavir-Boosted Saquinavir Mesylate Administered Alone or in Combination at Steady State
Authors: Chittick Gregory E. , Zong Jian , Blum M. Robert , Sorbel Jeffrey J. , Begley John A. , Adda Nathalie , Kearney Brian P.
Published: March 2006

A phase I study was conducted to formally evaluate the steady-state pharmacokinetics (PK) of tenofovir disoproxil fumarate (TDF) and ritonavir (RTV)-boosted saquinavir mesylate (SQV) when coadministered in healthy volunteers. Forty subjects received multiple doses of TDF (300 mg, once daily) and SQV/RTV (1,000 mg/100 mg, twice daily) alone and together under steady-state conditions in an open-label, fixed sequence design. Blood samples for tenofovir (TFV) and SQV/RTV PK were drawn over respective 24- and 12-h dosing intervals, and drug concentrations were measured by liquid chromatography-tandem mass spectrometry. Safety was assessed periodically by clinical and laboratory monitoring. Thirty-two subjects completed the study and were fully evaluable; three subjects discontinued participation in the study due to adverse events, three subjects withdrew for personal reasons, and two subjects withdrew because of inadequate venous access for blood sampling. Steady-state TFV PK were not significantly altered upon coadministration with SQV/RTV. Steady-state SQV (administered as SQV/RTV) AUC
tau
,
C
max
, and
C
tau
increased 29, 22, and 47%, respectively, upon coadministration with TDF, and all subjects achieved a
C
tau
of >100 ng/ml. These modestly increased SQV exposures are not clinically meaningful given its clinical use with RTV already results in >10-fold-higher SQV levels. Steady-state RTV AUC
tau
and
C
max
levels were not significantly altered, whereas
C
tau
was 23% higher upon coadministration of SQV/RTV and TDF. Thus, no clinically relevant interactions between TDF and RTV-boosted SQV were observed under conditions simulating clinical practice.
Time Course of Microbiologic Outcome and Gene Expression in
Candida albicans
during and following In Vitro and In Vivo Exposure to Fluconazole
Authors: Lepak A. , Nett J. , Lincoln L. , Marchillo K. , Andes D.
Published: March 2006

Pharmacodynamics (PD) considers the relationship between drug exposure and effect. The two factors that have been used to distinguish the PD behaviors of antimicrobials are the impact of concentration on the extent of organism killing and the duration of persistent microbiologic suppression (postantibiotic effect). The goals of these studies were (i) to examine the relationship between antimicrobial PD and gene expression and (ii) to gain insight into the mechanism of fluconazole effects persisting following exposure. Microarrays were used to estimate the transcriptional response of
Candida albicans
to a supra-MIC F exposure over time in vitro. Fluconazole at four times the MIC was added to a log-phase
C. albicans
culture, and cells were collected to determine viable growth and for microarray analyses. We identified differential expression of 18% of all genes for at least one of the time points. More genes were upregulated (
n
= 1,053 [16%]) than downregulated (174 [3%]). Of genes with known function that were upregulated during exposure, most were related to plasma membrane/cell wall synthesis (18%), stress responses (7%), and metabolism (6%). The categories of downregulated genes during exposure included protein synthesis (15%), DNA synthesis/repair (7%), and transport (7%) genes. The majority of genes identified at the postexposure time points were from the protein (17%) and DNA (7%) synthesis categories. In subsequent studies, three genes (
CDR1
,
CDR2
, and
ERG11
) were examined in greater detail (more concentration and time points) following fluconazole exposure in vitro and in vivo. Expression levels from the in vitro and in vivo studies were congruent.
CDR1
and
CDR2
transcripts were reduced during in vitro fluconazole exposure and during supra-MIC exposure in vivo. However, in the postexposure period, the mRNA abundance of both pumps increased.
ERG11
expression increased during exposure and fell in the postexposure period. The expression of the three genes responded in a dose-dependent manner. In sum, the microarray data obtained during and following fluconazole exposure identified genes both known and unknown to be affected by this drug class. The expanded in vitro and in vivo expression data set underscores the importance of considering the time course of exposure in pharmacogenomic investigations.
Novel Carbapenem-Hydrolyzing Oxacillinase OXA-62 from
Pandoraea pnomenusa
Authors: Schneider Ines , Queenan Anne Marie , Bauernfeind Adolf
Published: March 2006

Pandoraea
spp. are gram-negative, glucose nonfermenting rods detectable in blood cultures and sputa of cystic fibrosis patients. They are resistant to various antibiotic groups, with imipenem being the only active β-lactam. We isolated an imipenem-resistant (MIC, 64 μg/ml)
Pandoraea pnomenusa
strain from a cystic fibrosis patient. Cloning and sequencing identified two β-lactamases of Bush group 2d, namely, the known OXA-33, located on an integron, and the novel carbapenem-hydrolyzing oxacillinase OXA-62. OXA-62 is only distantly related to other oxacillinases (OXA-50 being closest with 43% amino acid identity). It hydrolyzes penicillins, oxacillin, imipenem, and meropenem but not expanded-spectrum cephalosporins. The
bla
OXA-62
gene is chromosome located. No transposable elements were found in its genetic neighborhood. With OXA-62-specific primers,
bla
OXA-62
could be identified in all
P. pnomenusa
strains and appears to be species specific. This additional mechanism of carbapenem resistance further complicates the treatment of infections caused by
P. pnomenusa
.
Involvement of the MexXY-OprM Efflux System in Emergence of Cefepime Resistance in Clinical Strains of
Pseudomonas aeruginosa
Authors: Hocquet Didier , Nordmann Patrice , El Garch Farid , Cabanne Ludovic , Plésiat Patrick
Published: March 2006

Cefepime (FEP) and ceftazidime (CAZ) are potent β-lactam antibiotics with similar MICs (1 to 2 μg/ml) for wild-type strains of
Pseudomonas aeruginosa
. However, recent epidemiological studies have highlighted the occurrence of isolates more resistant to FEP than to CAZ (FEP
r
/CAZ
s
profile). We thus investigated the mechanisms conferring such a phenotype in 38 clonally unrelated strains collected in two French teaching hospitals. Most of the bacteria (
n
= 32; 84%) appeared to stably overexpress the
mexY
gene, which codes for the RND transporter of the multidrug efflux system MexXY-OprM. MexXY up-regulation was the sole FEP resistance mechanism identified (
n
= 12) or was associated with increased levels of pump MexAB-OprM (
n
= 5) or MexJK (
n
= 2), synthesis of secondary β-lactamase PSE-1 (
n
= 10), derepression of cephalosporinase AmpC (
n
= 1), coexpression of both OXA-35 and MexJK (
n
= 1), or production of both PSE-1 and MexAB-OprM (
n
= 1). Down-regulation of the
mexXY
operon in seven selected strains by the plasmid-borne repressor gene
mexZ
decreased FEP resistance from two- to eightfold, thereby demonstrating the significant contribution of MexXY-OprM to the FEP
r
/CAZ
s
phenotype. The six isolates of this series that exhibited wild-type levels of the
mexY
gene were found to produce β-lactamase PSE-1 (
n
= 1), OXA-35 (
n
= 4), or both PSE-1 and OXA-35 (
n
= 1). Altogether, these data provide evidence that MexXY-OprM plays a major role in the development of FEP resistance among clinical strains of
P. aeruginosa
.
Antitrypanosomal and Antileishmanial Activities of Flavonoids and Their Analogues: In Vitro, In Vivo, Structure-Activity Relationship, and Quantitative Structure-Activity Relationship Studies
Authors: Tasdemir Deniz , Kaiser Marcel , Brun Reto , Yardley Vanessa , Schmidt Thomas J. , Tosun Fatma , Rüedi Peter
Published: March 2006

Trypanosomiasis and leishmaniasis are important parasitic diseases affecting millions of people in Africa, Asia, and South America. In a previous study, we identified several flavonoid glycosides as antiprotozoal principles from a Turkish plant. Here we surveyed a large set of flavonoid aglycones and glycosides, as well as a panel of other related compounds of phenolic and phenylpropanoid nature, for their in vitro activities against
Trypanosoma brucei rhodesiense
,
Trypanosoma cruzi
, and
Leishmania donovani
. The cytotoxicities of more than 100 compounds for mammalian L6 cells were also assessed and compared to their antiparasitic activities. Several compounds were investigated in vivo for their antileishmanial and antitrypanosomal efficacies in mouse models. Overall, the best in vitro trypanocidal activity for
T. brucei rhodesiense
was exerted by 7,8-dihydroxyflavone (50% inhibitory concentration [IC
50
], 68 ng/ml), followed by 3-hydroxyflavone, rhamnetin, and 7,8,3′,4′-tetrahydroxyflavone (IC
50
s, 0.5 μg/ml) and catechol (IC
50
, 0.8 μg/ml). The activity against
T. cruzi
was moderate, and only chrysin dimethylether and 3-hydroxydaidzein had IC
50
s less than 5.0 μg/ml. The majority of the metabolites tested possessed remarkable leishmanicidal potential. Fisetin, 3-hydroxyflavone, luteolin, and quercetin were the most potent, giving IC
50
s of 0.6, 0.7, 0.8, and 1.0 μg/ml, respectively. 7,8-Dihydroxyflavone and quercetin appeared to ameliorate parasitic infections in mouse models. Generally, the test compounds lacked cytotoxicity in vitro and in vivo. By screening a large number of flavonoids and analogues, we were able to establish some general trends with respect to the structure-activity relationship, but it was not possible to draw clear and detailed quantitative structure-activity relationships for any of the bioactivities by two different approaches. However, our results can help in directing the rational design of 7,8-dihydroxyflavone and quercetin derivatives as potent and effective antiprotozoal agents.
Overexpression of the
MDR1
Gene Is Sufficient To Confer Increased Resistance to Toxic Compounds in
Candida albicans
Authors: Hiller Davina , Sanglard Dominique , Morschhäuser Joachim
Published: March 2006

Overexpression of
MDR1
, which encodes a membrane transport protein of the major facilitator superfamily, is one mechanism by which the human fungal pathogen
Candida albicans
can develop increased resistance to the antifungal drug fluconazole and other toxic compounds. In clinical
C. albicans
isolates, constitutive
MDR1
overexpression is accompanied by the upregulation of other genes, but it is not known if these additional alterations are required for Mdr1p function and drug resistance. To investigate whether
MDR1
overexpression is sufficient to confer a drug-resistant phenotype in
C. albicans
, we expressed the
MDR1
gene from the strong
ADH1
promoter in
C. albicans
laboratory strains that did not express the endogenous
MDR1
gene as well as in a fluconazole-resistant clinical
C. albicans
isolate in which the endogenous
MDR1
alleles had been deleted and in a matched fluconazole-susceptible isolate from the same patient. Forced
MDR1
overexpression resulted in increased resistance to the putative Mdr1p substrates cerulenin and brefeldin A, and this resistance did not depend on the additional alterations which occurred during drug resistance development in the clinical isolates. In contrast, artificial expression of the
MDR1
gene from the
ADH1
promoter did not enhance or only slightly enhanced fluconazole resistance, presumably because Mdr1p expression levels in the transformants were considerably lower than those observed in the fluconazole-resistant clinical isolate. These results demonstrate that
MDR1
overexpression in
C. albicans
is sufficient to confer resistance to some toxic compounds that are substrates of this efflux pump but that the degree of resistance depends on the Mdr1p expression level.
Impaired Target Site Penetration of Vancomycin in Diabetic Patients following Cardiac Surgery
Authors: Skhirtladze Keso , Hutschala Doris , Fleck Tatjana , Thalhammer Florian , Ehrlich Marek , Vukovich Thomas , Müller Markus , Tschernko Edda M.
Published: March 2006

Soft tissue infections constitute a serious complication following surgery in diabetic patients and frequently require the administration of vancomycin. However, despite antibiotic treatment, mortality of patients with postoperative infections remains high and might be related to an impaired penetration of anti-infective agents to target tissues. Therefore, the present study was designed to measure vancomycin tissue concentrations in six diabetic and six nondiabetic patients after cardiac surgery. Vancomycin was administered as a continuous intravenous infusion at an infusion rate of 80 to 120 mg/h. Vancomycin concentrations in soft tissues and plasma were measured in all patients during steady state as “therapeutic window” concentrations in plasma by microdialysis on day 8 ± 4 after initiation of vancomycin treatment. Vancomycin tissue concentrations in diabetic patients were significantly lower than in nondiabetics (3.7 mg/liter versus 11.9 mg/liter;
P
= 0.002). The median vancomycin
tissue
/vancomycin
plasma
concentration ratio was 0.1 in diabetic patients and 0.3 in nondiabetics (
P
= 0.002). Our study demonstrated that vancomycin penetration into target tissues is substantially impaired in diabetic patients versus nondiabetics. Insufficient tissue concentrations could therefore possibly contribute to failure of antibiotic treatment and the development of antimicrobial resistance in diabetic patients.
Pharmacodynamics of a New Cephalosporin, PPI-0903 (TAK-599), Active against Methicillin-Resistant
Staphylococcus aureus
in Murine Thigh and Lung Infection Models: Identification of an In Vivo Pharmacokinetic-Pharmacodynamic Target
Authors: Andes D. , Craig W. A.
Published: March 2006

PPI-0903 is a new cephalosporin with broad-spectrum activity, including beta-lactam-resistant
Streptococcus pneumoniae
and
Staphylococcus aureus
. We used the neutropenic murine thigh and lung infection models to examine the pharmacodynamic characteristics of PPI-0903. Serum drug levels following four fourfold-escalating single doses of PPI-0903 were measured by microbiologic assay. In vivo postantibiotic effects (PAEs) were determined after doses of 1.56, 6.25, 25, and 100 mg/kg of body weight in mice infected with
S. pneumoniae
ATCC 10813,
S. aureus
ATCC 29213, or
Escherichia coli
ATCC 25922. Dose fractionation studies over a 24-h dose range of 0.39 to 1,600 mg/kg were administered every 3, 6, 12, or 24 hours. Nonlinear regression analysis was used to determine which pharmacokinetic-pharmacodynamic (PK-PD) index (total and free 65% drug) best correlated with CFU/thigh at 24 h. Similar to other beta-lactam antibiotics, PPI-0903 produced short to modest in vivo PAEs with either
S. pneumoniae
or
E. coli
. The percent time that serum concentrations were above the MIC (%T>MIC) was the PK-PD index that best correlated with efficacy (
R
2
= 84 to 88% for the three organisms, compared with 9 to 41% for peak/MIC and 30 to 82% for the area under the concentration-time curve/MIC). In subsequent studies we used the neutropenic murine thigh infection model to determine if the magnitude of the free-drug %T>MIC needed for efficacy of PPI-0903 varied among pathogens (including resistant strains). Mice infected with one of five isolates of
S. pneumoniae
, four isolates of
S. aureus
, or four gram-negative bacilli were treated for 24 h with 0.10 to 400 mg/kg of PPI-0903 every 6 h. A sigmoid dose-response model was used to estimate the doses (mg/kg/24 h) required to achieve a net bacteriostatic affect over 24 h and to produce a reduction in the burden of organisms from the start of therapy by 1 and 2 log
10
CFU/thigh. MICs ranged from 0.008 to 1 μg/ml. Mean free-drug %T>MICs ± the standard deviation associated with the static effect endpoint for
S. pneumoniae
,
S. aureus
, and gram-negative isolates were 39 ± 9, 26 ± 8, and 32 ± 6, respectively. Methicillin and penicillin resistance did not alter the magnitude of free-drug %T>MIC required for efficacy. The free-drug %T>MIC necessary for efficacy was slightly reduced in animals with normal neutrophil counts. Treatment effect was similar in both the thigh and lung infection models. The pharmacodynamic characteristics of PPI-0903 are similar to those of other compounds within the cephalosporin class.
Phosphorothioate Oligonucleotides Inhibit Human Immunodeficiency Virus Type 1 Fusion by Blocking gp41 Core Formation
Authors: Vaillant Andrew , Juteau Jean-Marc , Lu Hong , Liu Shuwen , Lackman-Smith Carol , Ptak Roger , Jiang Shibo
Published: March 2006

Several studies have shown that phosphorothioate oligodeoxynucleotides (PS-ONs) have a sequence-independent antiviral activity against human immunodeficiency virus type 1 (HIV-1). It has also been suggested that PS-ONs inhibit HIV-1 by acting as attachment inhibitors that bind to the V3 loop of gp120 and prevent the gp120-CD4 interaction. Here we show that PS-ONs (and their fully 2′-O-methylated derivatives) are potent inhibitors of HIV-1-mediated membrane fusion and HIV-1 replication in a size-dependent, phosphorothioation-dependent manner. PS-ONs interact with a peptide derived from the N-terminal heptad repeat region of gp41, and the HIV-1 fusion-inhibitory activity of PS-ONs is closely correlated with their ability to block gp41 six-helix bundle formation, a critical step during the process of HIV-1 fusion with the target cell. These results suggest that the increased hydrophobicity of PS-ONs may contribute to their inhibitory activity against HIV-1 fusion and entry, because longer PS-ONs (≥30 bases) which have a greater hydrophobicity are more potent in blocking the hydrophobic interactions involved in the gp41 six-helix bundle formation and inhibiting the HIV-1-mediated cell-cell fusion than shorter PS-ONs (<30 bases). This novel antiviral mechanism of action of long PS-ONs has implications for therapy against infection by HIV-1 and other enveloped viruses with type I fusion proteins.
Protease-Stable Polycationic Photosensitizer Conjugates between Polyethyleneimine and Chlorin(e6) for Broad-Spectrum Antimicrobial Photoinactivation
Authors: Tegos George P. , Anbe Masahiro , Yang Changming , Demidova Tatiana N. , Satti Minahil , Mroz Pawel , Janjua Sumbul , Gad Faten , Hamblin Michael R.
Published: March 2006

We previously showed that covalent conjugates between poly-
l
-lysine and chlorin(e6) were efficient photosensitizers (PS) of both gram-positive and gram-negative bacteria. The polycationic molecular constructs increased binding and penetration of the PS into impermeable gram-negative cells. We have now prepared a novel set of second-generation polycationic conjugates between chlorin(e6) and three molecular forms of polyethyleneimine (PEI): a small linear, a small cross-linked, and a large cross-linked molecule. The conjugates were characterized by high-pressure liquid chromatography and tested for their ability to kill a panel of pathogenic microorganisms, the gram-positive
Staphylococcus aureus
and
Streptococcus pyogenes
, the gram-negative
Escherichia coli
and
Pseudomonas aeruginosa
, and the yeast
Candida albicans
, after exposure to low levels of red light. The large cross-linked molecule efficiently killed all organisms, while the linear conjugate killed gram-positive bacteria and
C. albicans
. The small cross-linked conjugate was the least efficient antimicrobial PS and its remarkably low activity could not be explained by reduced photochemical quantum yield or reduced cellular uptake. In contrast to polylysine conjugates, the PEI conjugates were resistant to degradation by proteases such as trypsin that hydrolyze lysine-lysine peptide bonds, The advantage of protease stability combined with the ready availability of PEI suggests these molecules may be superior to polylysine-PS conjugates for photodynamic therapy of localized infections.
Comparative Analysis of Chromosome-Encoded Microcins
Authors: Poey María Eloisa , Azpiroz María F. , Laviña Magela
Published: March 2006

Microcins are ribosomally synthesized peptide antibiotics that are produced by enterobacterial strains. Although the first studies concentrated on plasmid-encoded activities, in the last years three chromosome-encoded microcins have been described: H47, E492, and M. Here, a new microcin, I47, is presented as a fourth member of this group. Common features exhibited by chromosome-encoded microcins were searched for. The comparison of the genetic clusters responsible for microcin production revealed a preserved general scheme. The clusters essentially comprise a pair of activity-immunity genes which determine antibiotic specificity and a set of microcin maturation and secretion genes which are invariably present and whose protein products are highly homologous among the different producing strains. A strict functional relationship between the maturation and secretion pathways of microcins H47, I47, and E492 was demonstrated through genetic analyses, which included heterologous complementation assays. The peptide precursors of these microcins share a maturation process which implies the addition of a catecholate siderophore of the salmochelin type. Microcins thus acquire the ability to enter gram-negative cells through the catechol receptors. In addition, they employ a common mode of secretion to reach the external milieu by means of a type I export apparatus. The results presented herein lead us to propose that chromosome-encoded microcins constitute a defined subgroup of peptide antibiotics which are strictly related by their modes of synthesis, secretion, and uptake.
Efficacy of Hospital Germicides against Adenovirus 8, a Common Cause of Epidemic Keratoconjunctivitis in Health Care Facilities
Authors: Rutala William A. , Peacock Jeffrey E. , Gergen Maria F. , Sobsey Mark D. , Weber David J.
Published: March 2006

The inactivation of virus-contaminated nonporous inanimate surfaces was investigated using adenovirus type 8, a common cause of epidemic keratoconjunctivitis. A 10-μl inoculum of adenovirus was placed onto each stainless steel disk (1-cm diameter), and the inoculum was allowed to air dry for 40 min. Twenty-one different germicides (including disinfectants and antiseptics) were selected for this study based on their current uses in health care. After a 1- or 5-minute exposure to 50 μl of the germicide, the virus-germicide test mixture was neutralized and assayed for infectivity. Using an efficacy criterion of a 3-log
10
reduction in the titer of virus infectivity and regardless of the virus suspending medium (i.e., hard water, sterile water, and hard water with 5% fetal calf serum), only five disinfectants proved to be effective against the test virus at 1 min: 0.55%
ortho
-phthalaldehyde, 2.4% glutaraldehyde, 2.65% glutaraldehyde, ∼6,000 ppm chlorine, and ∼1,900 ppm chlorine. Four other disinfectants showed effectiveness under four of the five testing conditions: 70% ethanol, 65% ethanol with 0.63% quaternary ammonium compound, 79.6% ethanol with 0.1% quaternary ammonium compound, and 0.2% peracetic acid. Of the germicides suitable for use as an antiseptic, 70% ethanol achieved a 3-log
10
reduction under four of the five test conditions. These results emphasize the need for proper selection of germicides for use in disinfecting noncritical surfaces and semicritical medical devices, such as applanation tonometers, in order to prevent outbreaks of epidemic keratoconjunctivitis.
Screen for Inhibitors of the Coupled Transglycosylase-Transpeptidase of Peptidoglycan Biosynthesis in
Escherichia coli
Authors: Ramachandran Vasanthi , Chandrakala B. , Kumar Vidya P. , Usha Veeraraghavan , Solapure Suresh M. , de Sousa Sunita M.
Published: March 2006

Class A high-molecular-weight penicillin-binding protein 1a (PBP1a) and PBP1b of
Escherichia coli
have both transglycosylase (TG) and transpeptidase (TP) activity. These enzymes are difficult to assay, since their substrates are difficult to prepare. We show the activity of PBP1a or PBP1b can be measured in membranes by cloning the PBP into an
E. coli ponB
::Spc
r
strain. Using this assay, we show that PBP1a is ∼10-fold more sensitive to penicillin than PBP1b and that the 50% inhibitory concentration (IC
50
) of moenomycin, a TG inhibitor, is ∼10-fold higher in the PBP transformants than in wild-type membranes; this increase in IC
50
in transformants can be used to test the specificity of test compounds for inhibition of the TG. Alternatively, the coupled TG-TP activity of PBP1b can be directly measured in a two-step microplate assay. In the first step, radiolabeled lipid II, the TG substrate, was made in membranes of the
E. coli ponB
::Spc
r
strain by incubation with the peptidoglycan sugar precursors. In the second step, the TG-TP activity was assayed by adding a source of PBP1b to the membranes. The coupled TG-TP activity converts lipid II to cross-linked peptidoglycan, which was specifically captured by wheat germ agglutinin-coated scintillation proximity beads in the presence of 0.2% Sarkosyl (B. Chandrakala et al., Antimicrob. Agents Chemother.
48:
30-40, 2004). The TG-TP assay was inhibited by penicillin and moenomycin as expected. Surprisingly, tunicamycin and nisin also inhibited the assay, and paper chromatography analysis revealed that both inhibited the transglycosylase. The assay can be used to screen for novel antibacterial agents.
Human β-Defensin 2 Induces a Vigorous Cytokine Response in Peripheral Blood Mononuclear Cells
Authors: Boniotto Michele , Jordan William J. , Eskdale Joyce , Tossi Alessandro , Antcheva Nikolinka , Crovella Sergio , Connell Nancy D. , Gallagher Grant
Published: March 2006

β-Defensins are a family of small cationic peptides involved in the innate response to microbial infection. Although their role in microbial killing is well established, the mechanisms through which this occurs remain largely undefined. Here, using protein array technology, we describe a role for human β-defensins in the induction of an inflammatory cytokine response by human peripheral blood mononuclear cells (PBMCs). Human β-defensins 1, 2, and 3 were examined for induction of an array of cytokines and chemokines. Some cytokines, such as interleukin 8 (IL-8) and monocyte chemoattractant protein 1, were up-regulated by all three defensins, while others, such as IL-6 and IL-10, were induced more selectively. It was notable that each defensin induced a unique pattern of cytokines. This report documents, for the first time, an analysis of the composite cytokine response of human PBMCs to β-defensins. The induction or up-regulation of a number of cytokines involved in the adaptive immune response suggests a possible role for these defensins in linking innate and acquired immunity.
Genetic Structures at the Origin of Acquisition and Expression of the Carbapenem-Hydrolyzing Oxacillinase Gene
bla
OXA-58
in
Acinetobacter baumannii
Authors: Poirel Laurent , Nordmann Patrice
Published: March 2006

Genetic structures surrounding the carbapenem-hydrolyzing oxacillinase gene
bla
OXA-58
were characterized in a series of OXA-58-positive
Acinetobacter baumannii
strains isolated from different countries. We showed that in most of the cases, acquisitions of the
bla
OXA-58
-containing overall structure, including insertion sequence elements, may be likely the results of recombination events. In type strain
A. baumannii
MAD, the genetic structure surrounding the
bla
OXA-58
gene was bracketed by two 27-bp repeated sequences. The isolation of a clonally related OXA-58-negative
A. baumannii
isolate that possessed the same plasmid backbone as
A. baumannii
MAD but lacked this genetic structure indicated that the mechanism of acquisition could be reversible. Parts of the structure identified in
A. baumannii
MAD were conserved in other
bla
OXA-58
-positive isolates from various European countries. Primer extension experiments showed that
bla
OXA-58
expression was related to promoter sequences brought by different insertion sequence elements, such as an IS
Aba3
-like element, IS
Aba1
, IS
Aba2
, and IS
18
. This work identified novel structures at the origin of acquisition and expression of a carbapenem-hydrolyzing β-lactamase identified in non-clonally related
A. baumannii
isolates.
Insights into In Vivo Activities of Lantibiotics from Gallidermin and Epidermin Mode-of-Action Studies
Authors: Bonelli Raquel Regina , Schneider Tanja , Sahl Hans-Georg , Wiedemann Imke
Published: March 2006

The activity of lanthionine-containing peptide antibiotics (lantibiotics) is based on different killing mechanisms which may be combined in one molecule. The prototype lantibiotic nisin inhibits peptidoglycan synthesis and forms pores through specific interaction with the cell wall precursor lipid II. Gallidermin and epidermin possess the same putative lipid II binding motif as nisin; however, both peptides are considerably shorter (22 amino acids, compared to 34 in nisin). We demonstrate that in model membranes, lipid II-mediated pore formation by gallidermin depends on membrane thickness. With intact cells, pore formation was less pronounced than for nisin and occurred only in some strains. InLactococcus lactissubsp.cremorisHP, gallidermin was not able to release K+, and a mutant peptide, [A12L]gallidermin, in which the ability to form pores was disrupted, was as potent as wild-type gallidermin, indicating that pore formation does not contribute to killing. In contrast, nisin rapidly formed pores in theL. lactisstrain; however, it was approximately 10-fold less effective in killing. The superior activity of gallidermin in a cell wall biosynthesis assay may help to explain this high potency. Generally, it appears that the multiple activities of lantibiotics combine differently for individual target strains.
Adding Selectivity to Antimicrobial Peptides: Rational Design of a Multidomain Peptide againstPseudomonasspp
Authors: Eckert Randal , Qi Fengxia , Yarbrough Daniel K. , He Jian , Anderson Maxwell H. , Shi Wenyuan
Published: March 2006

Currently available antimicrobials exhibit broad killing with regard to bacterial genera and species. Indiscriminate killing of microbes by these conventional antibiotics can disrupt the ecological balance of the indigenous microbial flora, often resulting in negative clinical consequences. Species-specific antimicrobials capable of precisely targeting pathogenic bacteria without damaging benign microorganisms provide a means of avoiding this problem. In this communication, we report the successful creation of the first synthetic, target-specific antimicrobial peptide, G10KHc, via addition of a rationally designedPseudomonas-specific targeting moiety (KH) to a generally killing peptide (novispirin G10). The resulting chimeric peptide showed enhanced bactericidal activity and faster killing kinetics againstPseudomonasspp. than G10 alone. The enhanced killing activities are due to increased binding and penetration of the outer membrane ofPseudomonassp. cells. These properties were not observed in tests of untargeted bacterial species, and this specificity allowed G10KHc to selectively eliminatePseudomonasspp. from mixed cultures. This work lays a foundation for generating target-specific “smart” antimicrobials to complement currently available conventional antibiotics.
Preclinical Evaluation of Synthetic −2 RANTES as a Candidate Vaginal Microbicide To Target CCR5
Authors: Kish-Catalone Tina M. , Lu Wuyuan , Gallo Robert C. , DeVico Anthony L.
Published: March 2006

A potential strategy that can be used to combat the worldwide AIDS epidemic is the development of a vaginal microbicide that prevents the sexual transmission of human immunodeficiency virus type 1 (HIV-1). Certain CC chemokines, including RANTES, MIP-1α, and MIP-1β, might facilitate the development of such microbicides since they potently suppress HIV-1 infection by binding to CCR5, the viral coreceptor used by most sexually transmitted strains of HIV-1 to enter host cells. In this study, we evaluated whether a CCR5-specific fragment of RANTES that lacks two N-terminal residues (−2 RANTES) and possesses especially potent HIV-1 suppressive activity has toxicity profiles conducive to the advancement of testing in candidate microbicide formulations. Analyses were carried out with a synthetic version of the chemokine, which was formulated with either Novasomes 7474, a nonphospholipid liposome, or methylcellulose gel. Dialysis studies demonstrated that the formulated −2 RANTES was released from both vehicles and retained anti-HIV-1 activity. Preclinical toxicity studies carried out with Swiss Webster mouse and New Zealand White rabbit vaginal irritation models demonstrated minimal inflammation and minimal adverse changes in cervicovaginal tissue integrity after short-term (10 min) and long-term (24 h) exposure to formulations containing up to 1 mg/ml of −2 RANTES. Similarly, no toxicity was observed with formulations of bioactive murine RANTES in the Swiss Webster mouse vaginal irritation model. Overall, these preclinical studies suggest that −2 RANTES is suitable for further testing as a candidate anti-HIV-1 microbicide.
Substrate Envelope and Drug Resistance: Crystal Structure of RO1 in Complex with Wild-Type Human Immunodeficiency Virus Type 1 Protease
Authors: Prabu-Jeyabalan Moses , King Nancy M. , Nalivaika Ellen A. , Heilek-Snyder Gabrielle , Cammack Nick , Schiffer Celia A.
Published: March 2006

In our previous crystallographic studies of human immunodeficiency virus type 1 (HIV-1) protease-substrate complexes, we described a conserved “envelope” that appears to be important for substrate recognition and the selection of drug-resistant mutations. In this study, the complex of HIV-1 protease with the inhibitor RO1 was determined and comparison with the substrate envelope provides a rationale for mutational patterns.
The Antimicrobial Peptide Polyphemusin Localizes to the Cytoplasm of
Escherichia coli
following Treatment
Authors: Powers Jon-Paul S. , Martin Morgan M. , Goosney Danika L. , Hancock Robert E. W.
Published: March 2006

The horseshoe crab peptide polyphemusin I possesses high antimicrobial activity, but its mechanism of action is as yet not well defined. Using a biotin-labeled polyphemusin I analogue and confocal fluorescence microscopy, we showed that the peptide accumulates in the cytoplasm of wild-type
Escherichia coli
within 30 min after addition without causing substantial membrane damage.
In Vivo Selection of Fluoroquinolone-Resistant
Escherichia coli
Isolates Expressing Plasmid-Mediated Quinolone Resistance and Expanded-Spectrum β-Lactamase
Authors: Poirel Laurent , Pitout Johann D. D. , Calvo Lucy , Rodriguez-Martinez Jose-Manuel , Church Deirdre , Nordmann Patrice
Published: March 2006

A ciprofloxacin-resistant
Escherichia coli
isolate, isolate 1B, was obtained from a urinary specimen of a Canadian patient treated with norfloxacin for infection due to a ciprofloxacin-susceptible isolate, isolate 1A. Both isolates harbored a plasmid-encoded
sul1
-type integron with
qnrA1
and
bla
VEB-1
genes. Isolate 1B had amino acid substitutions in gyrase and topoisomerase.
Plasmodium falciparum
In Vitro Susceptibility to Antimalarial Drugs in Casamance (Southwestern Senegal) during the First 5 Years of Routine Use of Artesunate-Amodiaquine
Authors: Agnamey P. , Brasseur P. , de Pecoulas P. Eldin , Vaillant Michel , Olliaro P.
Published: March 2006

We have monitored the in vitro sensitivities of
Plasmodium falciparum
isolates predeployment and during the deployment of artesunate plus amodiaquine treatment in Mlomp, Casamance (southwestern Senegal) during 2000 to 2004. Parasites remained susceptible to both drugs. Chloroquine resistance levels were high but stable. Quinine continues to be effective.
Antimalarial Activity of a Synthetic Endoperoxide (RBx-11160/OZ277) against
Plasmodium falciparum
Isolates from Gabon
Authors: Kreidenweiss Andrea , Mordmüller Benjamin , Krishna Sanjeev , Kremsner Peter G.
Published: March 2006

OZ277 is a newly developed, fully synthetic endoperoxide antimalarial that we tested against field isolates from Gabon. A comparison of activities of OZ277 with artesunate, mefloquine, and chloroquine showed OZ277 to be highly active against all parasite isolates. Artesunate and mefloquine also showed potent antiparasitic activity, but all isolates were chloroquine resistant.
Gatifloxacin Resistance and Mutations in
gyrA
after Unsuccessful
Helicobacter pylori
Eradication in Japan
Authors: Nishizawa Toshihiro , Suzuki Hidekazu , Kurabayashi Kumiko , Masaoka Tatsuhiro , Muraoka Hiroe , Mori Mikiji , Iwasaki Eisuke , Kobayashi Intetsu , Hibi Toshifumi
Published: March 2006

A high resistance rate (47.9%) to gatifloxacin (GAT; 8-methoxy fluoroquinolone) in
Helicobacter pylori
(
H. pylori
) strains from 48 Japanese patients is observed after unsuccessful
H. pylori
eradication. A significant association between MICs for GAT equal to or above 1 μg/ml and mutations of the
gyrA
gene of
H. pylori
was demonstrated.
Autolytic Properties of Glycopeptide-Intermediate
Staphylococcus aureus
Mu50
Authors: Utaida Sugunya , Pfeltz Richard F. , Jayaswal R. K. , Wilkinson B. J.
Published: March 2006

Whole-cell autolytic activity of prototypical glycopeptide-intermediate
Staphylococcus aureus
(GISA) Mu50 was reduced versus that of hetero-GISA Mu3 and glycopeptide-susceptible
S. aureus
, consistent with other GISA strains. In contrast, autolytic activity was relatively high in Mu50 crude cell walls and autolysin extracts against purified cell walls, reflecting the complexities of autolytic activity regulation.
Quadruple Mutations in Dihydrofolate Reductase of
Plasmodium falciparum
Isolates from Car Nicobar Island, India
Authors: Ahmed Anwar , Das Manoj K. , Dev Vas , Saifi Muheet A. , Wajihullah  , Sharma Yagya D.
Published: March 2006

Quadruple mutations in the
Plasmodium falciparum
dihydrofolate reductase (PFDHFR) enzyme give rise to the highest level of pyrimethamine resistance leading to treatment failures. We describe here the presence of these quadruple mutations in a majority of
P. falciparum
isolates from Car Nicobar (Andaman and Nicobar) Island, India. Isolates from the mainland, however, continue to show a prevalence of double PFDHFR mutations and some with triple but none with quadruple mutations. In conclusion, the antifolate drug pressure is very high in the island, which should be a cause of concern for the malaria control program in the country.
In Vivo Efficacy of Aerosolized Nanostructured ItraconazoleFormulations for Prevention of Invasive Pulmonary Aspergillosis
Authors: Hoeben Barbara J. , Burgess David S. , McConville Jason T. , Najvar Laura K. , Talbert Robert L. , Peters Jay I. , Wiederhold Nathan P. , Frei Bradi L. , Graybill John R. , Bocanegra Rosie , Overhoff Kirk A. , Sinswat Prapasri , Johnston Keith P. , Williams Robert O.
Published: March 2006

Aerosolized
evaporative precipitation into aqueous solution and spray freezing into
liquid nanostructured formulations of itraconazole as prophylaxis
significantly improved survival relative to commercial itraconazole
oral solution and the control in a murine model of invasive pulmonary
aspergillosis. Aerosolized administration of nanostructured
formulations also achieved high lung tissue concentrations while
limiting systemic
exposure.
In Vitro Activity of Tigecycline against
Burkholderia pseudomallei
and
Burkholderia thailandensis
Authors: Thamlikitkul Visanu , Trakulsomboon Suwanna
Published: March 2006

Investigation of the in vitro activity of tigecycline against
Burkholderia pseudomallei
and
Burkholderia thailandensis
revealed that the inhibition zone diameters of tigecycline against all isolates were ≥20 mm and that the MIC
50
values were 0.5 and 1 μg/ml and the MIC
90
values were 2 and 1.5 μg/ml for
B. pseudomallei
and
B. thailandensis
, respectively.
Bactericidal Activities of R207910 and Other Newer Antimicrobial Agents against
Mycobacterium leprae
in Mice
Authors: Ji Baohong , Chauffour Aurélie , Andries Koen , Jarlier Vincent
Published: March 2006

As measured by a proportional bactericidal technique in the mouse footpad system, the bactericidal activity against
Mycobacterium leprae
of R207910 was equal to that of rifapentine, rifampin, or moxifloxacin and significantly greater than those of minocycline, PA-824, and linezolid. These data suggest that R207910 may play an important role in treatment of leprosy.
Prevalence of First-Step Mutants among Levofloxacin-Susceptible Invasive Isolates of
Streptococcus pneumoniae
in the United States
Authors: Pletz Mathias W. R. , Shergill Ardaman P. , McGee Lesley , Beall Bernard , Whitney Cynthia G. , Klugman Keith P.
Published: March 2006

By use of a PCR-restriction fragment length polymorphism assay, we screened 496 levofloxacin-susceptible invasive pneumococcal strains (MIC ≤ 2 mg/liter) for quinolone resistance-determining region mutations known to confer fluoroquinolone resistance. Among those with a levofloxacin MIC of 2 mg/liter, 16.2% of isolates recovered from nursing home residents and 6.4% from non-nursing home residents had first-step mutations.
Ether Lipid Ester Derivatives of Cidofovir Inhibit Polyomavirus BK Replication In Vitro
Authors: Randhawa Parmjeet , Farasati Noush Afarin , Shapiro Ron , Hostetler Karl Y.
Published: March 2006

Polyomavirus BK is a significant pathogen in transplant recipients, but no effective antiviral therapy is available. We show that cidofovir can inhibit BK virus replication in vitro. Esterification of cidofovir with hexadecyloxypropyl, octadecyloxyethyl, and oleyloxyethyl groups results in up to a 3-log lowering of the 50% effective concentration and an increased selectivity index.
Sequential or Combination Antifungal Therapy with Voriconazole and Liposomal Amphotericin B in a Guinea Pig Model of Invasive Aspergillosis
Authors: Kirkpatrick William R. , Coco Brent J. , Patterson Thomas F.
Published: March 2006

We evaluated combinations of voriconazole (VRC) and liposomal amphotericin B (L-AMB) in a guinea pig invasive aspergillosis model. Simultaneous VRC and L-AMB was most effective, although VRC monotherapy was also effective. These regimens as well as sequential L-AMB followed by VRC were more effective than L-AMB alone or VRC followed by L-AMB.