bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mutations in Topoisomerase Genes of Fluoroquinolone-Resistant Salmonellae in Hong Kong
Authors: Ling J. M. , Chan E. W. , Lam A. W. , Cheng A. F. 
Published: October 2003

ABSTRACT

A total of 88 salmonella isolates (72 clinical isolates for which the ciprofloxacin MIC was >0.06 μg/ml, 15 isolates for which the ciprofloxacin MIC was ≤0.06 μg/ml, and
Salmonella enterica
serotype Typhimurium ATCC 13311) were studied for the presence of genetic alterations in four quinolone resistance genes,
gyrA
,
gyrB
,
parC
, and
parE
, by multiplex PCR amplimer conformation analysis. The genetic alterations were confirmed by direct nucleotide sequencing. A considerable number of strains had a mutation in
parC
, the first to be reported in salmonellae. Seven of the isolates sensitive to 0.06 μg of ciprofloxacin per ml had a novel mutation at codon 57 of
parC
(Tyr57→Ser) which was also found in 29 isolates for which ciprofloxacin MICs were >0.06 μg/ml. Thirty-two isolates had a single
gyrA
mutation (Ser83→Phe, Ser83→Tyr, Asp87→Asn, Asp87→Tyr, or Asp87→Gly), 34 had both a
gyrA
mutation and a
parC
mutation (29 isolates with a
parC
mutation of Tyr57→Ser and 5 isolates with a
parC
mutation of Ser80→Arg). Six isolates which were isolated recently (from 1998 to 2001) were resistant to 4 μg of ciprofloxacin per ml. Two of these isolates had double
gyrA
mutations (Ser83→Phe and Asp87→Asn) and a
parC
mutation (Ser80→Arg) (MICs, 8 to 32 μg/ml), and four of these isolates had double
gyrA
mutations (Ser83→Phe and Asp87→Gly), one
parC
mutation (Ser80→Arg), and one
parE
mutation (Ser458→Pro) (MICs, 16 to 64 μg/ml). All six of these isolates and those with a Ser80→Arg
parC
mutation were
S. enterica
serotype Typhimurium. One
S. enterica
serotype Typhi isolate harbored a single
gyrA
mutation (Ser83→Phe), and an
S. enterica
serotype Paratyphi A isolate harbored a
gyrA
mutation (Ser83→Tyr) and a
parC
mutation (Tyr57→Ser); both of these isolates had decreased susceptibilities to the fluoroquinolones. The MICs of ciprofloxacin, levofloxacin, and sparfloxacin were in general the lowest of those of the six fluoroquinolones tested. Isolates with a single
gyrA
mutation were less resistant to fluoroquinolones than those with an additional
parC
mutation (Tyr57→Ser or Ser80→Arg), while those with double
gyrA
mutations were more resistant.
Attenuation of Itraconazole Fungicidal Activity following Preexposure of
Aspergillus fumigatus
to Fluconazole
Authors: Liu Wei , Lionakis Michail S. , Lewis Russell E. , Wiederhold Nathan , May Gregory S. , Kontoyiannis Dimitrios P. 
Published: October 2003

ABSTRACT

Fluconazole (FLC), a triazole with limited activity against
Aspergillus
species, is frequently used as prophylaxis in leukemia patients and bone marrow transplant recipients. Prior FLC use has been associated with an increasing incidence of invasive aspergillosis in these patients. We hypothesized that prior exposure of
Aspergillus fumigatus
to FLC could result in altered in vitro susceptibility of this fungus to other, more active triazoles. Thus, we performed serial passages of conidia of 10 clinical isolates of
A. fumigatus
(all itraconazole [ITC] susceptible) on FLC-containing yeast agar glucose plates. The MICs and minimal fungicidal concentrations (MFCs) of amphotericin B, FLC, ITC, and voriconazole (VRC) for
A. fumigatus
conidia were measured following four passages on FLC-containing medium according to the National Committee for Clinical Laboratory Standards microdilution method. Serial passages on FLC-containing plates resulted in a fourfold increase in the MFCs (but not the MICs) of ITC for nine isolates. The attenuated ITC fungicidal activity against
A. fumigatus
following FLC preexposure was medium independent and was also observed against FLC-preexposed
A. fumigatus
hyphae with the viability staining FUN-1 dye. Moreover, FLC preexposure of
A. fumigatus
conidia resulted in an analogous increase in the MFCs (but not the MICs) of VRC. Our findings suggest that preexposure of
A. fumigatus
to FLC attenuates the in vitro fungicidal activity of subsequent ITC use against it. This phenotypic adaptation is not captured by a routine MIC determination but requires MFC measurement. The in vivo significance of this in vitro phenomenon requires further investigation.
Flow Cytometry Antifungal Susceptibility Testing of
Aspergillus fumigatus
and Comparison of Mode of Action of Voriconazole vis-à-vis Amphotericin B and Itraconazole
Authors: Ramani Rama , Gangwar Madhurama , Chaturvedi Vishnu 
Published: October 2003

ABSTRACT

Aspergillus fumigatus
isolates were tested with three antifungals by flow cytometry (FC) and fluorescence-activated cell sorting. FC results after 4 h correlated well with MICs obtained by the NCCLS M38-A method; voriconazole exhibited fungicidal activity, albeit to a lesser extent than amphotericin B, but to a greater extent than itraconazole.
Simulation of Human Gentamicin Pharmacokinetics in an Experimental
Enterococcus faecalis
Endocarditis Model
Authors: Dubé Laurent , Caillon Jocelyne , Gras-Le Guen Christèle , Jacqueline Cédric , Kergueris Marie-France , Granry Jean-Claude , Potel Gilles , Bugnon Denis 
Published: October 2003

ABSTRACT

Significant differences between animal and human pharmacokinetics may be responsible for the conflicting results of experimental studies. This study determined the impact of human pharmacokinetic simulation (HPS) on gentamicin activity in an
Enterococcus faecalis
endocarditis model. The decrease in bacterial counts was greater with HPS than with a dose-equivalent regimen without HPS.
PPE Protein (Rv3873) from DNA Segment RD1 ofMycobacterium tuberculosis: Strong Recognition of Both Specific T-Cell Epitopes and Epitopes Conserved within the PPE Family
Authors: Okkels Limei Meng , Brock Inger , Follmann Frank , Agger Else Marie , Arend Sandra M. , Ottenhoff Tom H. M. , Oftung Fredrik , Rosenkrands Ida , Andersen Peter 
Published: October 2003

ABSTRACTProteins encoded by DNA segment RD1 ofMycobacterium tuberculosishave recently been demonstrated to play important roles in bacterial virulence, vaccine development, and diagnostic reagent design. Previously, we characterized two immunodominant T-cell antigens, the early secreted antigen target (ESAT-6) and the 10-kDa culture filtrate protein (CFP10), which are encoded by theesx-lhpoperon in this region. In the present study we characterized a third putative open reading frame in this region,rv3873, which encodes a PPE protein. We found that therv3873gene is expressed inM. tuberculosisH37Rv and that the native protein, Rv3873, is predominantly associated with the mycobacterial cell or wall. When tested as a His-tagged recombinant protein, Rv3873 stimulated high levels of gamma interferon secretion in peripheral blood mononuclear cells isolated from tuberculosis (TB) patients, as well as from healthy tuberculin purified protein derivative-positive donors. In contrast to other RD1-encoded antigens, Rv3873 was also found to be recognized by a significant proportion ofMycobacterium bovisBCG-vaccinated donors. Epitope mapping performed with overlapping peptides revealed a broad pattern of T-cell recognition comprising both TB-specific epitopes and epitopes also recognized by BCG-vaccinated donors. The immunodominant epitope (residues 118 to 135) for both TB patients and BCG-vaccinated individuals was found to be highly conserved among a large number of PPE family members.
Ribonucleotide Reduction in
Mycobacterium tuberculosis
: Function and Expression of Genes Encoding Class Ib and Class II Ribonucleotide Reductases
Authors: Dawes Stephanie S. , Warner Digby F. , Tsenova Liana , Timm Juliano , McKinney John D. , Kaplan Gilla , Rubin Harvey , Mizrahi Valerie 
Published: October 2003

ABSTRACT

Mycobacterium tuberculosis
, the causative agent of tuberculosis, possesses a class Ib ribonucleotide reductase (RNR), encoded by the
nrdE
and
nrdF2
genes, in addition to a putative class II RNR, encoded by
nrdZ
. In this study we probed the relative contributions of these RNRs to the growth and persistence of
M. tuberculosis
. We found that targeted knockout of the
nrdF2
gene could be achieved only in the presence of a complementing allele, confirming that this gene is essential under normal, in vitro growth conditions. This observation also implied that the alternate class Ib small subunit encoded by the
nrdF1
gene is unable to substitute for
nrdF2
and that the class II RNR, NrdZ, cannot substitute for the class Ib enzyme, NrdEF2. Conversely, a Δ
nrdZ
null mutant of
M. tuberculosis
was readily obtained by allelic exchange mutagenesis. Quantification of levels of
nrdE
,
nrdF2
,
nrdF1
, and
nrdZ
gene expression by real-time, quantitative reverse transcription-PCR with molecular beacons by using mRNA from aerobic and O
2
-limited cultures showed that
nrdZ
was significantly induced under microaerophilic conditions, in contrast to the other genes, whose expression was reduced by O
2
restriction. However, survival of the Δ
nrdZ
mutant strain was not impaired under hypoxic conditions in vitro. Moreover, the lungs of B6D2/F
1
mice infected with the Δ
nrdZ
mutant had bacterial loads comparable to those of lungs infected with the parental wild-type strain, which argues against the hypothesis that
nrdZ
plays a significant role in the virulence of
M. tuberculosis
in this mouse model.
Absence of the Macrophage Mannose Receptor in Mice Does Not Increase Susceptibility toPneumocystis cariniiInfection In Vivo
Authors: Swain Steve D. , Lee Sena J. , Nussenzweig Michel C. , Harmsen Allen G. 
Published: October 2003

ABSTRACTHost defense against the opportunistic pathogenPneumocystis cariniirequires functional interactions of many cell types. Alveolar macrophages are presumed to be a vital host cell in the clearance ofP. carinii, and the mechanisms of this interaction have come under scrutiny. The macrophage mannose receptor is believed to play an important role as a receptor involved in the binding and phagocytosis ofP. carinii. Although there is in vitro evidence for this interaction, the in vivo role of this receptor inP. cariniiclearance in unclear. Using a mouse model in which the mannose receptor has been deleted, we found that the absence of this receptor is not sufficient to allow infection byP. cariniiin otherwise immunocompetent mice. Furthermore, when mice were rendered susceptible toP. cariniiby CD4+depletion, mannose receptor knockout mice (MR-KO) had pathogen loads equal to those of wild-type mice. However, the MR-KO mice exhibited a greater influx of phagocytes into the alveoli during infection. This was accompanied by increased pulmonary pathology in the MR-KO mice, as well as greater accumulation of glycoproteins in the alveoli (glycoproteins, including harmful hydrolytic enzymes, are normally cleared by the mannose receptor). We also found that the surface expression of the mannose receptor is not downregulated duringP. cariniiinfection in wild-type mice. Our findings suggest that while the macrophage mannose receptor may be important in the recognition ofP. carinii, in vivo, this mechanism may be redundant, and the absence of this receptor may be compensated for.
Pathogenic
Acanthamoeba
spp. Secrete a Mannose-Induced Cytolytic Protein That Correlates with the Ability To Cause Disease
Authors: Hurt Michael , Neelam Sudha , Niederkorn Jerry , Alizadeh Hassan 
Published: October 2003

ABSTRACT

The pathogenesis of
Acanthamoeba
keratitis begins when
Acanthamoeba
trophozoites bind specifically to mannosylated glycoproteins upregulated on the surfaces of traumatized corneal epithelial cells. When
Acanthamoeba castellanii
trophozoites are grown in
methyl
-α-
d
-mannopyranoside, they are induced to secrete a novel 133-kDa protein that is cytolytic to corneal epithelial cells. Clinical isolates of
Acanthamoeba
spp., and not the soil isolates, were proficient at producing a mannose-induced protein (MIP-133) and generating disease in Chinese hamsters. The purified protein was efficient at killing corneal epithelial cells, the first mechanistic barrier, by inducing apoptosis in a caspase 3-dependent pathway. Subsequent steps in pathogenesis require the amoebae to penetrate and degrade collagen. Only the clinical isolates tested were efficient at migrating through a collagenous matrix in vitro, presumably by MIP-133 degradation of both human type I and human type IV collagen. A chicken anti-MIP-133 antiserum effectively bound to the protein and blocked collagenolytic activity, migration, and cytopathic effects (CPE) against corneal cells in vitro. Chinese hamsters orally immunized with MIP-133 displayed a >30% reduction in disease. Immunoglobulin A isolated from immunized animals bound MIP-133 and blocked CPE on corneal cells in vitro. Animals induced to generate severe chronic infections displayed significant reductions in disease symptoms upon oral immunization postinfection. These data suggest that MIP-133 production might be necessary to initiate corneal disease and that it may play an important role in the subsequent steps of the pathogenic cascade of
Acanthamoeba
keratitis. Furthermore, as antibodies produced both prior to and after infection reduced clinical symptoms of disease, the protein may represent an important immunotherapeutic target for
Acanthamoeba
keratitis.
Evaluation ofBrucella abortusPhosphoglucomutase (pgm) Mutant as a New Live Rough-Phenotype Vaccine
Authors: Ugalde Juan Esteban , Comerci Diego José , Leguizamón M. Susana , Ugalde Rodolfo Augusto 
Published: October 2003

ABSTRACTBrucella abortusS19 is the vaccine most frequently used against bovine brucellosis. Although it induces good protection levels, it cannot be administered to pregnant cattle, revaccination is not advised due to interference in the discrimination between infected and vaccinated animals during immune-screening procedures, and the vaccine is virulent for humans. Due to these reasons, there is a continuous search for new bovine vaccine candidates that may confer protection levels comparable to those conferred by S19 but without its disadvantages. A previous study characterized the phenotype associated with the phosphoglucomutase (pgm) gene disruption inBrucella abortusS2308, as well as the possible role for the smooth lipopolysaccharide (LPS) in virulence and intracellular multiplication in HeLa cells (J. E. Ugalde, C. Czibener, M. F. Feldman, and R. A. Ugalde, Infect. Immun.68:5716-5723, 2000). In this report, we analyze the protection, proliferative response, and cytokine production induced in BALB/c mice by a Δpgmdeletion strain. We show that this strain synthesizes O antigen with a size of approximately 45 kDa but is rough. This is due to the fact that the Δpgmstrain is unable to assemble the O side chain in the complete LPS. Vaccination with the Δpgmstrain induced protection levels comparable to those induced by S19 and generated a proliferative splenocyte response and a cytokine profile typical of a Th1 response. On the other hand, we were unable to detect a specific anti-O-antigen antibody response by using the fluorescence polarization assay. In view of these results, the possibility that the Δpgmmutant could be used as a vaccination strain is discussed.
Transfer Region of pO113 from EnterohemorrhagicEscherichia coli: Similarity with R64 and Identification of a Novel Plasmid-Encoded Autotransporter, EpeA
Authors: Leyton Denisse L. , Sloan Joan , Hill Rebecca E. , Doughty Steven , Hartland Elizabeth L. 
Published: October 2003

ABSTRACTEnterohemorrhagicEscherichia coli(EHEC) is a prominent, food-borne cause of diarrhea, bloody diarrhea, and the hemolytic uremic syndrome in industrialized countries. Most strains of EHEC carry the locus for enterocyte effacement (LEE) pathogenicity island, but a proportion of isolates from patients with severe disease do not carry LEE and very little is known about virulence factors in these organisms. LEE-negative strains of EHEC typically express Shiga toxin 2 and carry a large plasmid that encodes the production of EHEC hemolysin. In this study, we determined the nucleotide sequence of the transfer region of pO113, the large hemolysin plasmid from LEE-negative EHEC O113:H21 (EH41). This 63.9-kb region showed a high degree of similarity with the transfer region of R64, and pO113 was capable of self-transmission at low frequencies. Unlike R64 and the relateddot/icmsystem ofLegionella pneumophila, however, pO113 was unable to mobilize RSF1010. In addition, the pO113 transfer region encoded a novel high-molecular-weight serine protease autotransporter ofEnterobacteriaceae(SPATE) protein, termed EpeA. Like other SPATEs, EpeA exhibited protease activity and mucinase activity, but expression was not associated with a cytopathic effect on epithelial cells. Analysis of a second high-molecular-weight secreted protein revealed that pO113 also encodes EspP, a cytopathic SPATE identified previously in EHEC O157:H7. The nucleotide sequences encoding the predicted β-domains ofespPandepeAwere identical and also shared significant homology with a third SPATE protein, EspI. BothespPandepeAwere detected in several LEE-negative clinical isolates of EHEC and thus may contribute to the pathogenesis of this subset of EHEC.
Relative Immunogenicity of PorA Subtypes in a Multivalent
Neisseria meningitidis
Vaccine Is Not Dependent on Presentation Form
Authors: Luijkx Thomas A. , van Dijken Harry , Hamstra Hendrik-Jan , Kuipers Betsy , van der Ley Peter , van Alphen Loek , van den Dobbelsteen Germie 
Published: October 2003

ABSTRACT
The hexavalent meningococcal vaccine HexaMen, containing six PorAs on two vesicles, was tested in clinical studies. Although fourfold increases in serum bactericidal activity (SBA) titers against all of the PorAs were observed, there were significant differences between PorA-specific SBA titers. SBA titers were mainly directed against one PorA from each vesicle, P1.5-2,10 and P1.5-1,2-2, and were lower against the other PorAs, especially P1.7-2,4 and P1.19,15-1. We investigated whether these differences were due to immunological interference that resulted in competition between the three PorAs on the same vesicle or whether they were caused by a difference in the immunogenicities of the separate PorAs. Therefore, mice were immunized either with HexaMen, with six monovalent outer membrane vesicles (OMVs) representing the same six PorAs simultaneously (HexaMix), or with only one of the monovalent OMVs. The immunoglobulin G and SBA titers after HexaMen immunization in mice resembled the results obtained in clinical studies. Although immunization with HexaMix gave higher titers than immunization with HexaMen for some PorAs, the pattern of high and low titers was the same. Similar differences in immunogenicity between subtypes were seen after monovalent immunization when interference was eliminated as a cause of the differences. Monovalent immunization resulted in higher titers for P1.5-1,2-2 and P1.7,16 than immunization with HexaMen. However, no significant differences were found for the weakly immunogenic PorAs, P1.7-2,4 and P1.19,15-1. Since immunization with the six PorAs in the trivalent presentation form (HexaMen) and in the mixture of monovalent vesicles (HexaMix) resulted in the same pattern of high and low titers, we concluded that the differences between the PorA-specific responses are due to differences in the immunogenicities of the various PorAs and not due to interference that results in competition between different PorAs.
Revaccination of Neonatal Calves withMycobacterium bovisBCG Reduces the Level of Protection against Bovine Tuberculosis Induced by a Single Vaccination
Authors: Buddle B. M. , Wedlock D. N. , Parlane N. A. , Corner L. A. L. , de Lisle G. W. , Skinner M. A. 
Published: October 2003

ABSTRACTCattle may provide a suitable model for testing ways of improving tuberculosis vaccine efficacy in human infants. A vaccination and challenge study was undertaken in calves to determine the optimal time to vaccinate neonatal animals withMycobacterium bovisbacillus Calmette-Guérin (BCG) for protection against tuberculosis and to determine whether revaccination with BCG was beneficial. Calves (10 per group) were vaccinated with BCG within 8 h of birth or at 6 weeks of age, when immune responses to antigens of environmental mycobacteria were detectable, or vaccinated at birth and revaccinated at 6 weeks. A control group was not vaccinated. BCG vaccination at birth induced strong antigen-specific gamma interferon (IFN-γ) and interleukin-2 (IL-2) responses and antigen-specific activation in CD4+, CD8+, and WC1+γδ T-cell subsets from blood. The proportions of animals per group with macroscopic tuberculous lesions after challenge were 0/10 for BCG at birth, 1/9 for BCG at 6 weeks, 4/10 for the revaccinated group, and 10/10 for the nonvaccinated group. There was no significant difference in the levels of protection between groups vaccinated at birth or at 6 weeks, while animals vaccinated both at birth and at 6 weeks had significantly less protection than those vaccinated only at birth. The revaccinated calves that subsequently developed tuberculous lesions had significantly stronger IFN-γ and IL-2 responses to bovine purified protein derivative after the BCG booster than those in the same group that did not develop lesions. The results indicated that BCG vaccination at birth induced a high level of immunity and that the sensitization of very young animals to antigens of environmental mycobacteria by 6 weeks of age did not affect the effectiveness of BCG. However, BCG revaccination of these young animals was contraindicated.
CapG
−/−
Mice Have Specific Host Defense Defects That Render Them More Susceptible than CapG
+/+
Mice to
Listeria monocytogenes
Infection but Not to
Salmonella enterica
Serovar Typhimurium Infection
Authors: Parikh Shefal S. , Litherland Sally A. , Clare-Salzler Michael J. , Li Wei , Gulig Paul A. , Southwick Frederick S. 
Published: October 2003

ABSTRACT

Loss of the actin filament capping protein CapG has no apparent effect on the phenotype of mice maintained under sterile conditions; however, bone marrow-derived macrophages from CapG
−/−
mice exhibited distinct motility defects. We examined the ability of CapG
−/−
mice to clear two intracellular bacteria,
Listeria monocytogenes
and
Salmonella enterica
serovar Typhimurium. The 50% lethal dose of
Listeria
was 10-fold lower for CapG
−/−
mice than for CapG
+/+
mice (6 × 10
3
CFU for CapG
−/−
mice and 6 × 10
4
CFU for CapG
+/+
mice), while no difference was observed for
Salmonella
. The numbers of
Listeria
cells in the spleens and livers were significantly higher in CapG
−/−
mice than in CapG
+/+
mice at days 5 to 9, while the bacterial counts were identical on day 5 for
Salmonella-
infected mice. Microscopic analysis revealed qualitatively similar inflammatory responses in the spleens and livers of the two types of mice. Specific immunofluorescence staining analyzed by fluorescence-activated cell sorting revealed similar numbers of macrophages and dendritic cells in infected CapG
−/−
and CapG
+/+
spleens. However, analysis of bone marrow-derived macrophages revealed a 50% reduction in the rate of phagocytosis of
Listeria
in CapG
−/−
cells but a normal rate of phagocytosis of
Salmonella
. Stimulation of bone marrow-derived dendritic cells with granulocyte-macrophage colony-stimulating factor resulted in a reduction in the ruffling response of CapG
−/−
cells compared to the response of CapG
+/+
cells, and CapG
−/−
bone-marrowed derived neutrophils migrated at a mean speed that was nearly 50% lower than the mean speed of CapG
+/+
neutrophils. Our findings suggest that specific motility deficits in macrophages, dendritic cells, and neutrophils render CapG
−/−
mice more susceptible than CapG
+/+
mice to
Listeria
infection.