bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mechanisms of Azole Resistance in a Clinical Isolate of
Candida tropicalis
Authors: Vandeputte Patrick , Larcher Gérald , Bergès Thierry , Renier Gilles , Chabasse Dominique , Bouchara Jean-Philippe 
Published: October 2005

ABSTRACT

Azole resistance has been insufficiently investigated in the yeast
Candida tropicalis
. Here we determined the molecular mechanisms responsible for azole resistance in a clinical isolate of this pathogenic yeast. Antifungal susceptibility testing performed by a disk diffusion method showed resistance or markedly decreased susceptibility to azoles, which was confirmed by determination of MICs. Considering the relationship between azole susceptibility and the respiration reported for other yeast species, the respiratory activity of this isolate was investigated. Flow cytometry using rhodamine 123 and oxygraphy demonstrated an increased respiratory activity, which was not linked to an overexpression or increased number of copies of the mitochondrial genome. Among previously described resistance mechanisms, an increased activity of efflux pumps was investigated by flow cytometry using rhodamine 6G. However, the efflux of rhodamine 6G was lower in the resistant isolate than in susceptible ones. Likewise, real-time reverse transcription-PCR quantification of the expression of
C. tropicalis MDR1
(Ct
MDR1
), which encodes an efflux protein belonging to the major facilitator superfamily, did not show overexpression of this gene. In contrast, the resistant isolate overexpressed the Ct
ERG11
gene coding for lanosterol 14α-demethylase. This was in agreement with the larger amount of ergosterol found in this isolate. Moreover, sequencing of Ct
ERG11
showed a point mutation leading to a tyrosine substitution in the protein sequence, which might lead to decreased binding affinity for azoles. In conclusion, overexpression of Ct
ERG11
associated with a missense mutation in this gene seemed to be responsible for the acquired azole resistance of this clinical isolate.
Amino Acid Mutations Essential to Production of an Altered PBP 2X Conferring High-Level β-Lactam Resistance in a Clinical Isolate of
Streptococcus pneumoniae
Authors: Smith Anthony M. , Klugman Keith P. 
Published: October 2005

ABSTRACT

Altered penicillin-binding protein 2X (PBP 2X) is a primary β-lactam antibiotic resistance determinant and is essential to the development of penicillin and cephalosporin resistance in the pneumococcus. We have studied the importance for resistance of 23 amino acid substitutions located in the transpeptidase domain (TD) of PBP 2X from an isolate with high-level resistance, isolate 3191 (penicillin MIC, 16 μg/ml; cefotaxime MIC, 4 μg/ml). Strain R6
2X/2B/1A/mur
(for which the MICs are as described for isolate 3191) was constructed by transforming laboratory strain R6 with all the necessary resistance determinants (altered PBPs 2X, 2B, and 1A and altered MurM) from isolate 3191. Site-directed mutagenesis was used to reverse amino acid substitutions in altered PBP 2X, followed by investigation of the impact of these reversions on resistance levels in R6
2X/2B/1A/mur
. Of the 23 substitutions located in the TD of PBP 2X, reversals at six positions decreased the resistance levels in R6
2X/2B/1A/mur
. Reversal of the Thr338Pro and Ile371Thr substitutions individually decreased the penicillin and cefotaxime MICs to 2 and 1 μg/ml, respectively, and individually displayed the greatest impact on resistance. To a lesser extent, reversal of the Leu364Phe, Ala369Val, Arg384Gly, and Tyr595Phe substitutions individually also decreased the penicillin and cefotaxime MICs. Reversal at all six positions collectively decreased both the penicillin and the cefotaxime MICs of R6
2X/2B/1A/mur
to 0.06 μg/ml. This study confirms the essential role of altered PBP 2X as a resistance determinant. Our data reveal that, for isolate 3191, the six amino acid substitutions described above are collectively essential to the production of an altered PBP 2X required for high-level resistance to penicillin and cefotaxime.
Virulence Factors of
Escherichia coli
Isolates That Produce CTX-M-Type Extended-Spectrum β-Lactamases
Authors: Pitout Johann D. D. , Laupland Kevin B. , Church Deirdre L. , Menard Megan L. , Johnson James R. 
Published: October 2005

ABSTRACT

This study determined the phylogenetic groups and virulence factors of 37
Escherichia coli
isolates producing types of CTX-M compared with those of 19 isolates producing different types of extended-spectrum β-lactamases (ESBLs) in a well-defined North American population. Most CTX-M-14 producers (97%) were from phylogenic group D; 67% of the CTX-M-15 producers were from group B2. A single CTX-M-14-producing strain belonged to clonal group A. There were significant prevalence differences for individual virulence factors among CTX-M producers and nonproducers; however, aggregate virulence factor scores were similar. CTX-M producers more commonly caused repeat urinary tract infections. Our results indicate that CTX-M type predicts phylogenetic background, and the virulence potential of ESBL-producing
E. coli
isolates is a complex issue, requiring further study and ongoing surveillance.
Organotypic Epithelial Raft Cultures as a Model for Evaluating Compounds against Alphaherpesviruses
Authors: Andrei Graciela , van den Oord Joost , Fiten Pierre , Opdenakker Ghislain , De Wolf-Peeters Chris , De Clercq Erik , Snoeck Robert 
Published: October 2005

ABSTRACT

The course of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) and varicella-zoster virus (VZV) infections in squamous epithelial cells cultured in a three-dimensional organotypic raft culture was tested. In these raft cultures, normal human keratinocytes isolated from neonatal foreskins grown at the air-liquid interface stratified and differentiated, reproducing a fully differentiated epithelium. Typical cytopathic changes identical to those found in the squamous epithelium in vivo, including ballooning and reticular degeneration with the formation of multinucleate cells, were observed throughout the raft following infection with HSV and VZV at different times after lifting the cultures to the air-liquid interface. For VZV, the aspects of the lesions depended on the stage of differentiation of the organotypic cultures. The activity of reference antiviral agents, acyclovir (ACV), penciclovir (PCV), brivudin (BVDU), foscarnet (PFA), and cidofovir (CDV), was evaluated against wild-type and thymidine kinase (TK) mutants of HSV and VZV in the raft cultures. ACV, PCV, and BVDU protected the epithelium against cytopathic effect induced by wild-type viruses in a concentration-dependent manner, while treatment with CDV and PFA proved protective against the cytodestructive effects induced by both TK
+
and TK
−
strains. The quantification of the antiviral effects in the rafts were accomplished by measuring viral titers by plaque assay for HSV and by measuring viral DNA load by real-time PCR for VZV. A correlation between the degree of protection as determined by histological examination and viral quantification could be demonstrated The three-dimensional epithelial raft culture represents a novel model for the study of antiviral agents active against HSV and VZV. Since no animal model is available for the evaluation of antiviral agents against VZV, the organotypic cultures may be considered a model to evaluate the efficacy of new anti-VZV antivirals before clinical trials.
DNA Sequence and Comparative Genomics of pAPEC-O2-R, an Avian Pathogenic
Escherichia coli
Transmissible R Plasmid
Authors: Johnson Timothy J. , Siek Kylie E. , Johnson Sara J. , Nolan Lisa K. 
Published: October 2005

ABSTRACT

In this study, a 101-kb IncF plasmid from an avian pathogenic
Escherichia coli
(APEC) strain (APEC O2) was sequenced and analyzed, providing the first completed APEC plasmid sequence. This plasmid, pAPEC-O2-R, has functional transfer and antimicrobial resistance-encoding regions. The resistance-encoding region encodes resistance to eight groups of antimicrobial agents, including silver and other heavy metals, quaternary ammonium compounds, tetracycline, sulfonamides, aminoglycosides, trimethoprim, and beta-lactam antimicrobial agents. This region of the plasmid is unique among previously described IncF plasmids in that it possesses a class 1 integron that harbors three gene cassettes and a heavy metal resistance operon. This region spans 33 kb and is flanked by the RepFII plasmid replicon and an assortment of plasmid maintenance genes. pAPEC-O2-R also contains a 32-kb transfer region that is nearly identical to that found in the
E. coli
F plasmid, rendering it transferable by conjugation to plasmid-less strains of bacteria, including an APEC strain, a fecal
E. coli
strain from an apparently healthy bird, a
Salmonella enterica
serovar Typhimurium strain, and a uropathogenic
E. coli
strain from humans. Differences in the G+C contents of individual open reading frames suggest that various regions of pAPEC-O2-R had dissimilar origins. The presence of pAPEC-O2-R-like plasmids that encode resistance to multiple antimicrobial agents and that are readily transmissible from APEC to other bacteria suggests the possibility that such plasmids may serve as a reservoir of resistance genes for other bacteria of animal and human health significance.
Mouse Adenovirus Type 1 Infection in SCID Mice: an Experimental Model for Antiviral Therapy of Systemic Adenovirus Infections
Authors: Lenaerts L. , Verbeken E. , De Clercq E. , Naesens L. 
Published: October 2005

ABSTRACTThe importance of human adenovirus infections in immunocompromised patients urges for new and adequate antiadenovirus compounds. Since human adenoviruses are species specific, animal models for systemic adenovirus infections rely on a nonhuman adenovirus. We established mouse adenovirus type 1 (MAV-1) infection of BALB/c SCID mice as a model for the evaluation of antiadenovirus therapy. In vitro studies with mouse embryonic fibroblasts pointed to the acyclic nucleoside phosphonate cidofovir and the N-7-substituted acyclic derivative 2-amino-7-(1,3-dihydroxy-2-propoxymethyl)purine (S-2242) as markedly active compounds against MAV-1. SCID mice, infected intranasally with MAV-1, developed a fatal disseminated infection after approximately 19 days, characterized by hemorrhagic enteritis. Several techniques were optimized to monitor viral, immunological, and pathological aspects of MAV-1 infection. Real-time PCR quantification of viral DNA revealed that after replication in the lungs, virus disseminated to several organs, including the brain, liver, spleen, intestine, heart, and kidneys (resulting in viruria). Immunohistochemical staining showed that MAV-1 was localized in the endothelial cells of the affected organs. Using reverse transcription-PCR, tissue levels of proinflammatory cytokines (i.e., interleukin-1β and tumor necrosis factor alpha) were found to be markedly increased. The MAV-1/SCID model appears to be an appropriate model for in vivo evaluation of antiadenovirus agents. Treatment with cidofovir or S-2242 at a dose of 100 mg per kg of body weight resulted in a significant delay in MAV-1-related death, although these antivirals were unable to completely suppress virus replication despite continued drug treatment. These findings suggest that complete virus clearance during antiviral therapy for disseminated adenovirus infection may require an efficient adaptive immune response from the host.
Comparative Effects of Two Neutralizing Anti-Respiratory Syncytial Virus (RSV) Monoclonal Antibodies in the RSV Murine Model: Time versus Potency
Authors: Mejías Asunción , Chávez-Bueno Susana , Ríos Ana María , Aten Mónica Fonseca , Raynor Brett , Peromingo Estrella , Soni Perla , Olsen Kurt D. , Kiener Peter A. , Gómez Ana María , Jafri Hasan S. , Ramilo Octavio 
Published: October 2005

ABSTRACTRespiratory syncytial virus (RSV) is the leading viral pathogen responsible for bronchiolitis and pneumonia in infants and young children worldwide. We have previously shown in the mouse model that treatment with an anti-RSV neutralizing monoclonal antibody (MAb) against the F glycoprotein of RSV, palivizumab, decreased lung inflammation, airway obstruction, and postmethacholine airway hyperresponsiveness. MEDI-524, or Numax, is a new MAb derived from palivizumab with enhanced neutralizing activity against RSV. We compared the effects of these two MAbs on different markers of disease severity using the murine model of RSV infection. BALB/c mice were intranasally inoculated with RSV A2. Palivizumab or MEDI-524 was administered once at either 24 h before or 48 h after RSV inoculation. Regardless of the time of administration, all treated mice showed significantly decreased RSV loads in bronchoalveolar lavage samples measured by plaque assay. Only MEDI-524 given at −24 h significantly decreased lung RSV RNA loads on days 5 and 28 after RSV inoculation. Pulmonary histopathologic scores, airway obstruction, and postmethacholine airway hyperresponsiveness were significantly reduced in mice treated with MEDI-524 at 24 h before inoculation, compared with untreated controls and the other regimens evaluated. MEDI-524 was superior to palivizumab on several outcome variables of RSV disease assessed in the mouse model: viral replication, inflammatory and clinical markers of acute disease severity, and long-term pulmonary abnormalities.
Molecular Analysis of the Simultaneous Production of Two SHV-Type Extended-Spectrum Beta-Lactamases in a Clinical Isolate of
Enterobacter cloacae
by Using Single-Nucleotide Polymorphism Genotyping
Authors: Szabó Dóra , Melan Melissa A. , Hujer Andrea M. , Bonomo Robert A. , Hujer Kristine M. , Bethel Christopher R. , Kristóf Katalin , Paterson David L. 
Published: October 2005

ABSTRACT

Bacteria that simultaneously produce multiple extended-spectrum beta-lactamases are frequently isolated. We report an
Enterobacter cloacae
isolate, ES24, producing four different beta-lactamases (AmpC type beta-lactamase, TEM-1, SHV-7, and a novel extended-spectrum beta-lactamase, SHV-30). Direct sequencing of
bla
SHV
gene products gave a “double peak” at position 703, suggesting the presence of more than one allele. Using fluorescence resonance energy transfer real-time PCR to detect single-nucleotide polymorphisms, we were able to distinguish two different
bla
SHV
genes in a single isolate. This may prove to be a useful technique in surveys of beta-lactamase production in contemporary clinical isolates.
Maraviroc (UK-427,857), a Potent, Orally Bioavailable, and Selective Small-Molecule Inhibitor of Chemokine Receptor CCR5 with Broad-Spectrum Anti-Human Immunodeficiency Virus Type 1 Activity
Authors: Dorr Patrick , Westby Mike , Dobbs Susan , Griffin Paul , Irvine Becky , Macartney Malcolm , Mori Julie , Rickett Graham , Smith-Burchnell Caroline , Napier Carolyn , Webster Rob , Armour Duncan , Price David , Stammen Blanda , Wood Anthony , Perros Manos 
Published: October 2005

ABSTRACTMaraviroc (UK-427,857) is a selective CCR5 antagonist with potent anti-human immunodeficiency virus type 1 (HIV-1) activity and favorable pharmacological properties. Maraviroc is the product of a medicinal chemistry effort initiated following identification of an imidazopyridine CCR5 ligand from a high-throughput screen of the Pfizer compound file. Maraviroc demonstrated potent antiviral activity against all CCR5-tropic HIV-1 viruses tested, including 43 primary isolates from various clades and diverse geographic origin (geometric mean 90% inhibitory concentration of 2.0 nM). Maraviroc was active against 200 clinically derived HIV-1 envelope-recombinant pseudoviruses, 100 of which were derived from viruses resistant to existing drug classes. There was little difference in the sensitivity of the 200 viruses to maraviroc, as illustrated by the biological cutoff in this assay (= geometric mean plus two standard deviations [SD] of 1.7-fold). The mechanism of action of maraviroc was established using cell-based assays, where it blocked binding of viral envelope, gp120, to CCR5 to prevent the membrane fusion events necessary for viral entry. Maraviroc did not affect CCR5 cell surface levels or associated intracellular signaling, confirming it as a functional antagonist of CCR5. Maraviroc has no detectable in vitro cytotoxicity and is highly selective for CCR5, as confirmed against a wide range of receptors and enzymes, including the hERG ion channel (50% inhibitory concentration, >10 μM), indicating potential for an excellent clinical safety profile. Studies in preclinical in vitro and in vivo models predicted maraviroc to have human pharmacokinetics consistent with once- or twice-daily dosing following oral administration. Clinical trials are ongoing to further investigate the potential of using maraviroc for the treatment of HIV-1 infection and AIDS.
Molecular Mechanisms of β-Lactam Resistance Mediated by AmpC Hyperproduction inPseudomonas aeruginosaClinical Strains
Authors: Juan Carlos , Maciá María D. , Gutiérrez Olivia , Vidal Carmen , Pérez José L. , Oliver Antonio 
Published: October 2005

ABSTRACTThe molecular mechanisms of β-lactam resistance mediated by AmpC hyperproduction in natural strains ofPseudomonas aeruginosawere investigated in a collection of 10 isogenic, ceftazidime-susceptible and -resistant pairs of isolates, each sequentially recovered from a different intensive care unit patient treated with β-lactams. All 10 ceftazidime-resistant mutants hyperproduced AmpC (β-lactamase activities were 12- to 657-fold higher than those of the parent strains), but none of them harbored mutations inampRor theampC-ampRintergenic region. On the other hand, six of them harbored inactivating mutations inampD: four contained frameshift mutations, one had a C→T mutation, creating a premature stop codon, and finally, one had a large deletion, including the completeampDEregion. Complementation studies revealed that only three of the sixampDmutants could be fully transcomplemented with eitherampD- orampDE-harboring plasmids, whereas one of them could be transcomplemented only withampDEand two of them (including the mutant with the deletion of theampDEregion and one with anampDframeshift mutation leading to anampDE-fused open reading frame) could not be fully transcomplemented with any of the plasmids. Finally, one of the four mutants with no mutations inampDcould be transcomplemented, but only withampDE. Although the inactivation of AmpD is found to be the most frequent mechanism of AmpC hyperproduction in clinical strains, our findings suggest that for certain types of mutations, AmpE plays an indirect role in resistance and that there are other unknown genes involved in AmpC hyperproduction, with at least one of them apparently located close to theampDEoperon.
Spontaneous Conversion to Quinolone and Fluoroquinolone Resistance among Wild-Type
Escherichia coli
Isolates in Relation to Phylogenetic Background and Virulence Genotype
Authors: Johnson James R. , Johnston Brian , Kuskowski Michael A. , Colodner Raul , Raz Raul 
Published: October 2005

ABSTRACT

Human clinical isolates of
Escherichia coli
that are resistant to quinolone or fluoroquinolone agents typically exhibit fewer extraintestinal virulence factors (VFs) than susceptible isolates, along with a different phylogenetic background. To experimentally assess the basis for this as-yet-unexplained phenomenon, 40
E. coli
strains (20
E. coli
Reference collection members and 20 Israeli cystitis isolates) were subjected to serial selective passaging to obtain derivatives resistant to nalidixic acid (NA) and ciprofloxacin (C). PCR-based VF profiling and phylotyping were performed on the parents and their respective resistant derivatives. All 40 susceptible parent strains yielded NA- and C-resistant derivatives after a median of 6 (range, 4 to 12) serial selective passages on agar plates containing increasing concentrations of NA and C. The numbers of passages required for resistance did not differ by collection origin, phylogenetic group, basal VF profile, source (urine versus fecal), or host group (human versus animal). With the development of C resistance, only one VF was lost in a single strain. Resistant derivatives exhibited the same phylotype as their susceptible parents. These findings suggest that the sparse VF profiles and the low-virulence phylogenetic background of NA- and C-resistant
E. coli
clinical isolates probably are not attributable to the loss of VFs from intrinsically high-virulence strains during conversion to resistance or to enhanced emergence of drug resistance among intrinsically low-virulence strains. A more likely explanation is the importation of resistant strains from an as-yet-undefined low-virulence external selection reservoir.
CTX-M-2 and a New CTX-M-39 Enzyme Are the Major Extended-Spectrum Beta-Lactamases in Multiple
Escherichia coli
Clones Isolated in Tel Aviv, Israel
Authors: Chmelnitsky Inna , Carmeli Yehuda , Leavitt Azita , Schwaber Mitchell J. , Navon-Venezia Shiri 
Published: October 2005

ABSTRACT

The rate of occurrence of the extended-spectrum beta-lactamase (ESBL)-producing phenotype among
Escherichia coli
isolates in Tel Aviv is 12% (22). The aim of this study was to understand the molecular epidemiology of
E. coli
ESBL producers and to identify the ESBL genes carried by them. We studied 20 single-patient ESBL-producing
E. coli
clinical isolates. They comprised 11 distinct nonrelated pulsed-field gel electrophoresis (PFGE) genotypes: six isolates belonged to the same PFGE clone, four other clones included two isolates each, and six unrelated clones included only one isolate. All isolates produced various beta-lactamases with pIs ranging from 5.2 to 8.2, varying within similar PFGE clones. The most prevalent ESBL gene was
bla
CTX-M
; 16 isolates carried
bla
CTX-M-2
and three carried a new ESBL gene designated
bla
CTX-M-39
. Three strains carried
bla
SHV
(two
bla
SHV-12
and one
bla
SHV-5
), and two strains carried inhibitor-resistant ESBL genes,
bla
TEM-33
and
bla
TEM-30
; 18 strains carried
bla
TEM-1
and eight strains carried
bla
OXA-2
. Plasmid mapping and Southern blot analysis with a CTX-M-2 probe demonstrated that
bla
CTX-M-2
is plasmid borne. The wide dissemination of ESBLs among
E. coli
isolates in our institution is partly related to clonal spread, but more notably to various plasmid-associated ESBL genes, occurring in multiple clones, wherein the CTX-M gene family appears almost uniformly. We report here a new CTX-M gene, designated
bla
CTX-M-39
, which revealed 99% homology with
bla
CTX-M-26
, with a substitution of arginine for glutamine at position 225.
Increased Mutation Frequencies in
Escherichia coli
Isolates Harboring Extended-Spectrum β-Lactamases
Authors: Baquero María-Rosario , Galán Juan Carlos , del Carmen Turrientes María , Cantón Rafael , Coque Teresa M. , Martínez José Luis , Baquero Fernando 
Published: October 2005

ABSTRACT

Hypermutable (mutation frequency [
f
], ≥4 × 10
−8
)
Escherichia coli
strains were more frequently found (43%) in a collection of 89 extended-spectrum β-lactamase (ESBL)-producing isolates from different patients (77 pulsed-field gel electrophoresis clones, 12 ESBL types) than in non-ESBL
E. coli
(26%) strains (
P
= 0.03). Among urinary tract isolates, the frequency of hypermutation was 40% in ESBL versus 26% in non-ESBL isolates (
P
= 0.03).
Kinetics Study of KPC-3, a Plasmid-Encoded Class A Carbapenem-Hydrolyzing β-Lactamase
Authors: Alba Jimena , Ishii Yoshikazu , Thomson Kenneth , Moland Ellen Smith , Yamaguchi Keizo 
Published: October 2005

ABSTRACT

The kinetic activity of KPC-3, a plasmid-encoded class A carbapenemase, was studied. It hydrolyzed penicillins, cephalosporins, carbapenems, and even sulbactam. The best substrate was cephalothin (
k

cat
/K

m
= 3.48 μM
−1
s
−1
). The efficiency of the enzyme was similar for imipenem and meropenem (
k
cat
/

K
m

, 1.4 and 1.94 μM
−1
s
−1
, respectively).