bluuebunny/crossref_metadata_2025_split huggingface dataset:
Small-Colony Mutants of
Staphylococcus aureus
Allow Selection of Gyrase-Mediated Resistance to Dual-Target Fluoroquinolones
Authors: Pan Xiao-Su , Hamlyn Penelope J. , Talens-Visconti Raquel , Alovero Fabiana L. , Manzo Ruben H. , Fisher L. Mark 
Published: July 2002

ABSTRACT

Fluoroquinolones acting equally through DNA gyrase and topoisomerase IV in vivo are considered desirable in requiring two target mutations for emergence of resistant bacteria. To investigate this idea, we have studied the response of
Staphylococcus aureus
RN4220 to stepwise challenge with sparfloxacin, a known dual-target agent, and with NSFQ-105, a more potent sulfanilyl fluoroquinolone that behaves similarly. First-step mutants were obtained with both drugs but only at the MIC. These mutants exhibited distinctive small-colony phenotypes and two- to fourfold increases in MICs of NSFQ-105, sparfloxacin, and ciprofloxacin. No changes were detected in the quinolone resistance-determining regions of the
gyrA
,
gyrB
,
grlA
, or
grlB
gene. Quinolone-induced small-colony mutants shared the delayed coagulase response but not the requirement for menadione, hemin, or thymidine characteristic of small-colony variants, a subpopulation of
S. aureus
that is often defective in electron transport. Second-step mutants selected with NSFQ-105 had
gyrA
(S84L) alterations; those obtained with sparfloxacin carried a
gyrA
(D83A) mutation or a novel
gyrB
deletion (ΔRKSAL, residues 405 to 409) affecting a trypsin-sensitive region linking functional domains of
S. aureus
GyrB. Each mutation was associated with four- to eightfold increases in MICs of NSFQ-105 and sparfloxacin, but not of ciprofloxacin, which we confirm targets topoisomerase IV. The presence of wild-type
grlB
-
grlA
gene sequences in second-step mutants excluded involvement of topoisomerase IV in the small-colony phenotype. Growth revertants retaining mutant
gyrA
or
gyrB
alleles were quinolone susceptible, indicating that resistance to NSFQ-105 and sparfloxacin was contingent on the small-colony mutation. We propose that small-colony mutations unbalance target sensitivities, perhaps through altered ATP or topoisomerase levels, such that gyrase becomes the primary drug target. Breaking of target parity by genetic or physiological means eliminates the need for two target mutations and provides a novel mechanism for stepwise selection of quinolone resistance.
Novel Nucleoside Analogue MCC-478 (LY582563) Is Effective against Wild-Type or Lamivudine-Resistant Hepatitis B Virus
Authors: Ono-Nita Suzane Kioko , Kato Naoya , Shiratori Yasushi , Carrilho Flair José , Omata Masao 
Published: July 2002

ABSTRACTThe emergence of resistant hepatitis B virus (HBV) with the L528M mutation and/or the M552V and M552I mutations in the polymerase gene following long-term lamivudine treatment is becoming an important clinical problem. The aim of this study was to investigate the susceptibility of wild-type and lamivudine-resistant HBV to MCC-478 (LY582563), a novel nucleoside analogue derivative of phosphonomethoxyethyl purine. The susceptibility of wild-type HBV and lamivudine-resistant mutants (M552I, M552V, and L528M/M552V) to MCC-478 was examined by transient transfection of full-length HBV DNA into human hepatoma cells. HBV DNA replication was monitored by Southern blot hybridization, and the effective concentration required to reduce replication by 50% (EC50) was determined. The replicative intermediates of wild-type and lamivudine-resistant mutants were progressively diminished by treatment with increasing doses of MCC-478. The MCC-478 EC50s were 0.027 μM for wild-type HBV (about 20 times more efficient than lamivudine), 2.6 μM for M552I, 3.3 μM for M552V, and 2.0 μM for L528M/M552V. Wild-type HBV and lamivudine-resistant mutants are susceptible to MCC-478. MCC-478 appears to be a candidate for the treatment of HBV infection and exhibits potent activity against lamivudine-resistant HBV.
In Vitro Activities of Quinupristin-Dalfopristin and Cefepime, Alone and in Combination with Various Antimicrobials, against Multidrug-Resistant Staphylococci and Enterococci in an In Vitro Pharmacodynamic Model
Authors: Allen George P. , Cha Raymond , Rybak Michael J. 
Published: July 2002

ABSTRACTUse of combinations of antimicrobials that together achieve synergistic activities against targeted microorganisms is one potential strategy for overcoming bacterial resistance. As the incidence of infections caused by multidrug-resistant staphylococci and enterococci increases, the importance of devising additional synergistic drug combinations for these bacteria is magnified. We evaluated a number of antimicrobial combinations, with a focus on quinupristin-dalfopristin (Q-D), cefepime, and linezolid, using a previously described in vitro pharmacodynamic model. The combination of Q-D with either linezolid or vancomycin, as well as the combination of cefepime-vancomycin, resulted in enhanced killing (≥2-log10increase in killing versus the most-active single agent) against methicillin-resistantStaphylococcus aureus(MRSA) 494. An improved effect (<2 log10kill increase in kill) against MRSA 494 was noted for cefepime plus either Q-D or linezolid, as well as linezolid-vancomycin. Similar relationships were observed for a methicillin-susceptibleS. aureusisolate (isolate 1199). Against methicillin-resistantS. epidermidisR444, enhanced killing was achieved with the combination of cefepime-linezolid, while improvement was noted for vancomycin with either cefepime or linezolid. The combination of cefepime and vancomycin also achieved enhanced killing against a glycopeptide-intermediate-susceptibleS. aureusisolate (isolate 992). The combination of linezolid and doxycycline achieved an enhanced effect against vancomycin-resistantEnterococcus faecalis(VREFc) andE. faecium. Q-D plus ampicillin or linezolid resulted in similar enhancement of activity against the VREFc isolate. The results of this study suggest a number of novel antimicrobial combinations that may be useful against staphylococci and enterococci. Combination regimens including cefepime, Q-D, and/or linezolid warrant further investigation for the treatment of refractive infections due to multidrug-resistant gram-positive pathogens.
Therapeutic Efficacies of GW471552 and GW471558, Two New Azasordarin Derivatives, against Pneumocystosis in Two Immunosuppressed-Rat Models
Authors: Jimenez Elena , Martínez Antonio , Aliouat El Moukhtar , Caballero Jesus , Dei-Cas Eduardo , Gargallo-Viola Domingo 
Published: July 2002

ABSTRACT

Two new azasordarins, GW471552 and GW471558, were studied in vivo for treatment of
Pneumocystis carinii
pneumonia. In the Wistar rat spontaneous pneumonia model, both azasordarins significantly reduced the number of
P. carinii
cysts per gram of lung homogenate when administered at 1 mg/kg of body weight twice a day for 10 days. In a nude rat inoculation model, both compounds showed therapeutic efficacy at 0.25 mg/kg twice a day for 10 days.
Longitudinal Assessment of Antipneumococcal Susceptibility in the United States
Authors: Jones Mark E. , Karlowsky James A. , Blosser-Middleton Renée , Critchley Ian A. , Karginova Elena , Thornsberry Clyde , Sahm Daniel F. 
Published: July 2002

ABSTRACT

The prevalence of antimicrobial resistance among 4,940 U.S. pneumococcal isolates collected during 1999 was as follows: penicillin, 16.2%; amoxicillin-clavulanate, 12.2%; cefuroxime, 28.1%; ceftriaxone, 3.6%; trimethoprim-sulfamethoxazole, 30.3%; azithromycin, 21.4%; levofloxacin, 0.6%; and moxifloxacin, 0.1%. Compared to the previous 1997-1998 study (Jones et al., Antimicrob. Agents Chemother.
44:
2645-2652, 2000), increases were noted for resistance to penicillin (3.7%;
P
< 0.001), amoxicillin-clavulanate (3.9%;
P
< 0.001), cefuroxime (5.7%;
P
< 0.001), azithromycin (2.4%;
P
= 0.014), trimethoprim-sulfamethoxazole (15.4%;
P
< 0.001), and levofloxacin (0.3%;
P
= 0.017). Resistance to ceftriaxone (0.1%;
P
= 0.809) and moxifloxacin (0.03%;
P
= 0.570) decreased. Concurrently, multidrug resistance increased (
P
< 0.001) from 6.3% to 11.3%.
Incidence, Epidemiology, and Characteristics of Quinolone- Nonsusceptible
Streptococcus pneumoniae
in Croatia
Authors: Pankuch Glenn A. , Bozdogan Bülent , Nagai Kensuke , Tambić-Andrašević Arjana , Schoenwald Slavko , Tambić Tera , Kalenić Smilja , Pleško Sanja , Tepeš Nastja K. , Kotarski Zdenka , Payerl-Pal Marina , Appelbaum Peter C. 
Published: July 2002

ABSTRACT

Among 585
Streptococcus pneumoniae
strains isolated in 22 Croatian hospitals 21 strains (3.6%) were quinolone nonsusceptible. MICs of all quinolones were high for seven strains tested with the same serotype (23F) and mutations in
gyrA
,
parC
, and
parE
. The remaining 14 strains were more heterogeneous and had mutations only in
parC
and/or
parE
, and the MICs of quinolones were lower for these strains.
Biguanide-Atovaquone Synergy against
Plasmodium falciparum
In Vitro
Authors: Jones K. , Ward S. A. 
Published: July 2002

ABSTRACT

The synergistic potential of a range of biguanides, their triazine metabolites, tetracyclines, and pyrimethamine in combination with atovaquone has been assessed. All five biguanides tested interacted synergistically with atovaquone against
Plasmodium falciparum
in vitro. All of the other compounds tested were either additive or antagonistic.
Noninvasive Quantitative Measurement of Bacterial Growth in Porous Media under Unsaturated-Flow Conditions
Authors: Yarwood R. R. , Rockhold M. L. , Niemet M. R. , Selker J. S. , Bottomley P. J. 
Published: July 2002

ABSTRACT

Glucose-dependent growth of the
luxCDABE
reporter bacterium
Pseudomonas fluorescens
HK44 was monitored noninvasively in quartz sand under unsaturated-flow conditions within a 45- by 56- by 1-cm two-dimensional light transmission chamber. The spatial and temporal development of growth were mapped daily over 7 days by quantifying salicylate-induced bioluminescence. A nonlinear model relating the rate of increase in light emission after salicylate exposure to microbial density successfully predicted growth over 4 orders of magnitude (
r
2
= 0.95). Total model-predicted growth agreed with growth calculated from the mass balance of the system by using previously established growth parameters of HK44 (predicted, 1.2 × 10
12
cells; calculated, 1.7 × 10
12
cells). Colonization expanded in all directions from the inoculation region, including upward migration against the liquid flow. Both the daily rate of expansion of the colonized zone and the population density of the first day's growth in each newly colonized region remained relatively constant throughout the experiment. Nonetheless, substantial growth continued to occur on subsequent days in the older regions of the colonized zone. The proportion of daily potential growth that remained within the chamber declined progressively between days 2 and 7 (from 97 to 13%). A densely populated, anoxic region developed in the interior of the colonized zone even though the sand was unsaturated and fresh growth medium continued to flow through the colonized zone. These data illustrate the potential of a light transmission chamber, bioluminescent bacteria, and sensitive digital camera technology to noninvasively study real-time hydrology-microbiology interactions associated with unsaturated flow in porous media.
Comparison of a Multiplex Flow Cytometric Assay with Enzyme-Linked Immunosorbent Assay for Quantitation of Antibodies to Tetanus, Diphtheria, and
Haemophilus influenzae
Type b
Authors: Pickering Jerry W. , Martins Thomas B. , Schroder M. Carl , Hill Harry R. 
Published: July 2002

ABSTRACT

We developed a multiplexed indirect immunofluorescence assay for antibodies to
Haemophilus influenza
type b (Hib) polysaccharide and the toxoids of
Clostridium tetani
(Tet) and
Corynebacterium diphtheriae
(Dip) based on the Luminex multiple-analyte profiling system. A pooled serum standard was calibrated against World Health Organization standards for Dip and Tet and an international standard for Hib. The multiplexed Luminex assay was compared to individual enzyme-linked immunosorbent assays (ELISAs) for the same analytes. By both methods, 75 (92.6%) of 81 of random serum samples had protective levels of antibody to Tet (≥0.1 IU/ml). For Dip, 81.5% of the samples had protective antibody levels (≥0.1 IU/ml) by ELISA and 80.2% had protective antibody levels by Luminex. Protective levels (≥1.0 μg/ml) of antibody to Hib were found in 45.0% of the samples tested by ELISA and in 39.0% of the samples tested by Luminex. The correlations (
R
2
) between ELISA and Luminex of the 81 samples were 0.96, 0.96, and 0.91 for Tet, Dip, and Hib, respectively. There was also similar agreement between Luminex and ELISA for sera collected before and 1 month after Tet, Dip, and Hib vaccine administration. Both methods detected strong postvaccination responses. The Luminex method is an attractive alternative to ELISA since it reduces labor and reagent costs, as well as assay time.
Widal Test in Diagnosis of Typhoid Fever in Turkey
Authors: Willke Ayse , Ergonul Onder , Bayar Banu 
Published: July 2002

ABSTRACT

We studied the value of the Widal tube agglutination test for the diagnosis of typhoid fever. The subjects were all adults >18 years of age and were divided into four groups: (i) 317 healthy blood donor controls, (ii) 31 bacteriologically confirmed patients with
Salmonella enterica
serotype Typhi, (iii) 21 patients with a clinical diagnosis of typhoid fever, and (iv) 41 febrile nontyphoid patients. Blood donor controls were screened with a slide agglutination test for the
Salmonella enterica
serotype Typhi O and H antigens, and positives were then tested with the Widal test. Acute- and convalescent-phase sera from patients in groups 2, 3, and 4 were obtained 7 to 10 days apart and tested by the Widal test. Using a cutoff of ≥1/200 for the O antigen test performed on acute-phase serum gave a sensitivity of 52% and a specificity of 88% with a positive predictive value (PPV) of 76% and a negative predictive value (NPV) of 71%. This increased to 90% sensitivity and specificity with a PPV of 88% and an NPV of 93% when the convalescent-phase serum was tested. We concluded that O and H agglutinin titers of ≥1/200 are of diagnostic significance. The Widal test is easy, inexpensive, and relatively noninvasive. It can be of diagnostic value when blood cultures are not available or practical. The results must be interpreted cautiously because of the low sensitivity of the test. The Widal test done on convalescent-phase serum gave more-reliable results with higher specificity and sensitivity.
In Vitro Cultivation of
Cryptosporidium
Species
Authors: Arrowood Michael J. 
Published: July 2002

SUMMARY

The in vitro cultivation of protozoan parasites of the genus
Cryptosporidium
has advanced significantly in recent years. These obligate, intracellular parasites colonize the epithelium of the digestive and respiratory tracts, are often difficult to obtain in significant numbers, produce durable oocysts that defy conventional chemical disinfection methods, and are persistently infectious when stored at refrigerated temperatures (4 to 8°C). While continuous culture and efficient life cycle completion (oocyst production) have not yet been achieved in vitro, routine methods for parasite preparation and cell culture infection and assays for parasite life cycle development have been established. Parasite yields may be limited, but in vitro growth is sufficient to support a variety of research studies, including assessing potential drug therapies, evaluating oocyst disinfection methods, and characterizing life cycle stage development and differentiation.
Human Parvovirus B19
Authors: Heegaard Erik D. , Brown Kevin E. 
Published: July 2002

SUMMARYParvovirus B19(B19) was discovered in 1974 and is the only member of the familyParvoviridaeknown to be pathogenic in humans. Despite the inability to propagate the virus in cell cultures, much has been learned about the pathophysiology of this virus, including the identification of the cellular receptor (P antigen), and the control of the virus by the immune system. B19 is widespread, and manifestations of infection vary with the immunologic and hematologic status of the host. In healthy immunocompetent individuals B19 is the cause of erythema infectiosum and, particularly in adults, acute symmetric polyarthropathy. Due to the tropism of B19 to erythroid progenitor cells, infection in individuals with an underlying hemolytic disorder causes transient aplastic crisis. In the immunocompromised host persistent B19 infection is manifested as pure red cell aplasia and chronic anemia. Likewise, the immature immune response of the fetus may render it susceptible to infection, leading to fetal death in utero, hydrops fetalis, or development of congenital anemia. B19 has also been suggested as the causative agent in a variety of clinical syndromes, but given the common nature, causality is often difficult to infer. Diagnosis is primarily based on detection of specific antibodies by enzyme-linked immunosorbent assay or detection of viral DNA by dot blot hybridization or PCR. Treatment of persistent infection with immunoglobulin reduces the viral load and results in a marked resolution of anemia. Vaccine phase I trials show promising results.
Recurrent Variable Region Gene Usage and Somatic Mutation in the Human Antibody Response to the Capsular Polysaccharide ofStreptococcus pneumoniaeType 23F
Authors: Zhou Jianhui , Lottenbach Kathleen R. , Barenkamp Stephen J. , Lucas Alexander H. , Reason Donald C. 
Published: July 2002

ABSTRACTCombinatorial cloning and expression library analysis were used to isolate human antibody Fab fragments specific for the capsular polysaccharide ofStreptococcus pneumoniaeserotype 23F. Thirty 23F-specific Fabs were isolated from seven vaccinated donors, and the sequences of the heavy (H)- and light (L)-chain variable regions were determined. All individuals utilized either the Vκ A23 L chain, the Vκ L6 L chain, or both chains in forming the 23F-specific combining site. Vκ A23 L chains paired primarily with VH3-23 H chains. Vκ L6 L chains were more promiscuous in heavy-chain usage between individuals. Both H and L chains were mutated, primarily in the complementarity-determining regions, compared to their closest germ line counterpart, suggesting a recall response that has undergone affinity maturation. H-chain isotypes were reflective of those found in the serum. Shared somatic modifications demonstrated that immunoglobulin G2 (IgG2) and IgA antibodies arose from the same somatically matured B cell. Our results indicate that the response to the serotype 23F pneumococcal capsular polysaccharide is oligoclonal within the individual, with one or two paratope families accounting for the majority of expressed antibody. We also determined that, in spite of the combinatorial diversity available to the immune system, the 23F-specific response is highly restricted at the population level, with the same two L-chain-determined paratope families recurring in all individuals. Lastly, analysis of the isolated Fabs indicate all have undergone extensive somatic mutation, as well as class switch, maturational events that presumably require the participation of T cells.
Disruption of the Gene Homologous to MammalianNramp1inMycobacterium tuberculosisDoes Not Affect Virulence in Mice
Authors: Boechat Neio , Lagier-Roger Béatrice , Petit Stéphanie , Bordat Yann , Rauzier Jean , Hance Allan J. , Gicquel Brigitte , Reyrat Jean-Marc 
Published: July 2002

ABSTRACTNatural-resistance-associated macrophage protein 1 (Nramp1) is a divalent cation transporter belonging to a family of transporter proteins highly conserved in eukaryotes and prokaryotes. Mammalian and bacterial transporters may compete for essential metal ions during mycobacterial infections. The mycobacterialNramphomolog may therefore be involved inMycobacterium tuberculosisvirulence. Here, we investigated this possibility by inactivating theM. tuberculosis Nramp1gene (Mramp) by allelic exchange mutagenesis. Disruption ofMrampdid not affect the extracellular growth of bacteria under standard conditions. However, theMrampmutation was associated with growth impairment under conditions of limited iron availability. TheMrampmutant displayed no impairment of growth or survival in macrophages derived from mouse bone marrow or inNramp1+/+andNramp1−/−congenic murine macrophage cell lines. Following intravenous challenge in BALB/c mice, counts of parental andMrampmutant strains were similar in the lungs and spleens of the animals at all time points studied. These results indicate thatMrampdoes not contribute to the virulence ofM. tuberculosisin mice.
Identification of Chromosomal
Shigella flexneri
Genes Induced by the Eukaryotic Intracellular Environment
Authors: Runyen-Janecky L. J. , Payne S. M. 
Published: July 2002

ABSTRACT

Upon entry into the eukaryotic cytosol, the facultative intracellular bacterium
Shigella flexneri
is exposed to an environment that may necessitate the expression of particular genes for it to survive and grow intracellularly. To identify genes that are induced in response to the intracellular environment, we screened a library containing fragments of the
S. flexneri
chromosome fused to a promoterless green fluorescent protein gene (
gfp
). Bacteria containing promoter fusions that had a higher level of
gfp
expression when
S. flexneri
was intracellular (in Henle cells) than when
S. flexneri
was extracellular (in Luria-Bertani broth) were isolated by using fluorescence-activated cell sorting. Nine different genes with increased expression in Henle cells were identified. Several genes (
uhpT
,
bioA
, and
lysA
) were involved in metabolic processes. The
uhpT
gene, which encoded a sugar phosphate transporter, was the most frequently isolated gene and was induced by glucose-6-phosphate in vitro. Two of the intracellularly induced genes (
pstS
and
phoA
) encode proteins involved in phosphate acquisition and were induced by phosphate limitation in vitro. Additionally, three iron-regulated genes (
sufA
,
sitA
, and
fhuA
) were identified. The
sufA
promoter was derepressed in iron-limiting media and was also induced by oxidative stress. To determine whether intracellularly induced genes are required for survival or growth in the intracellular environment, we constructed mutations in the
S. flexneri uhpT
and
pstS
genes by allelic exchange. The
uhpT
mutant could not use glucose-6-phosphate as a sole carbon source in vitro but exhibited normal plaque formation on Henle cell monolayers. The
pstS
mutant had no apparent growth defect in low-phosphate media in vitro but formed smaller plaques on Henle cell monolayers than the parent strain. Both mutants were as effective as the parent strain in inducing apoptosis in a macrophage cell line.