bluuebunny/crossref_metadata_2025_split huggingface dataset:
Conservation of Plasmid Maintenance Functions between Linear and Circular Plasmids inBorrelia burgdorferi
Authors: Stewart Philip E. , Chaconas George , Rosa Patricia 
Published: May 2003

ABSTRACTThe Lyme disease agentBorrelia burgdorferimaintains both linear and circular plasmids that appear to be essential for mammalian infection. Recent studies have characterized the circular plasmid regions that confer autonomous replication, but the genetic elements necessary for linear plasmid maintenance have not been experimentally identified. Two vectors derived from linear plasmids lp25 and lp28-1 were constructed and shown to replicate autonomously inB. burgdorferi. These vectors identify internal regions of linear plasmids necessary for autonomous replication inB. burgdorferi. Although derived from linear plasmids, the vectors are maintained in circular form inB. burgdorferi, indicating that plasmid maintenance functions are conserved, regardless of DNA form. Finally, derivatives of these vectors indicate that paralogous gene family 49 is apparently not required for either circular or linear plasmid replication.
Characterization of the Chlorate Reductase from
Pseudomonas chloritidismutans
Authors: Wolterink Arthur F. W. M. , Schiltz Emile , Hagedoorn Peter-Leon , Hagen Wilfred R. , Kengen Servé W. M. , Stams Alfons J. M. 
Published: May 2003

ABSTRACT

A chlorate reductase has been purified from the chlorate-reducing strain
Pseudomonas chloritidismutans
. Comparison with the periplasmic (per)chlorate reductase of strain GR-1 showed that the cytoplasmic chlorate reductase of
P. chloritidismutans
reduced only chlorate and bromate. Differences were also found in N-terminal sequences, molecular weight, and subunit composition. Metal analysis and electron paramagnetic resonance measurements showed the presence of iron and molybdenum, which are also found in other dissimilatory oxyanion reductases.
Alpha-Toxin Is Required for Biofilm Formation by
Staphylococcus aureus
Authors: Caiazza Nicky C. , O'Toole G. A. 
Published: May 2003

ABSTRACT

Staphylococcus aureus
is a common pathogen associated with nosocomial infections. It can persist in clinical settings and gain increased resistance to antimicrobial agents through biofilm formation. We have found that alpha-toxin, a secreted, multimeric, hemolytic toxin encoded by the
hla
gene, plays an integral role in biofilm formation. The
hla
mutant was unable to fully colonize plastic surfaces under both static and flow conditions. Based on microscopy studies, we propose that alpha-hemolysin is required for cell-to-cell interactions during biofilm formation.
The Single Superoxide Dismutase of
Rhodobacter capsulatus
Is a Cambialistic, Manganese-Containing Enzyme
Authors: Tabares Leandro C. , Bittel Cristian , Carrillo Néstor , Bortolotti Ana , Cortez Néstor 
Published: May 2003

ABSTRACT

The phototrophic bacterium
Rhodobacter capsulatus
contains a single, oxygen-responsive superoxide dismutase (SOD
Rc
) homologous to iron-containing superoxide dismutase enzymes. Recombinant SOD
Rc
, however, displayed higher activity after refolding with Mn
2+
, especially when the pH of the assay mixture was raised. SOD
Rc
isolated from
Rhodobacter
cells also preferentially contains manganese, but metal discrimination depends on the culture conditions, with iron fractions increasing from 7% in aerobic cultures up to 40% in photosynthetic cultures. Therefore, SOD
Rc
behaves as a Mn-containing dismutase with cambialistic properties.
The
Bacillus subtilis
Acyl Lipid Desaturase Is a Δ5 Desaturase
Authors: Altabe Silvia G. , Aguilar Pablo , Caballero Gerardo M. , de Mendoza Diego 
Published: May 2003

ABSTRACT

Bacillus subtilis
was recently reported to synthesize unsaturated fatty acids (UFAs) with a double bond at positions Δ5, Δ7, and Δ9 (M. H. Weber, W. Klein, L. Muller, U. M. Niess, and M. A. Marahiel, Mol. Microbiol. 39:1321-1329, 2001). Since this finding would have considerable importance in the double-bond positional specificity displayed by the
B
.
subtilis
acyl lipid desaturase, we have attempted to confirm this observation. We report that the double bond of UFAs synthesized by
B
.
subtilis
is located exclusively at the Δ5 position, regardless of the growth temperature and the length chain of the fatty acids.
Development of a LightCycler PCR Assay for Detection and Quantification ofAspergillus fumigatusDNA in Clinical Samples from Neutropenic Patients
Authors: Spiess Birgit , Buchheidt Dieter , Baust Corinna , Skladny Heyko , Seifarth Wolfgang , Zeilfelder Udo , Leib-Mösch Christine , Mörz Handan , Hehlmann Rüdiger 
Published: May 2003

ABSTRACTThe increasing incidence of invasive aspergillosis, a life-threatening infection in immunocompromised patients, emphasizes the need to improve the diagnostic tools for this disease. We established a LightCycler-based real-time PCR assay to detect and quantify rapidly, specifically, and sensitivelyAspergillus fumigatusDNA in both bronchoalveolar lavage (BAL) and blood samples from high-risk patients. The primers and hybridization probes were derived from anA. fumigatus-specific sequence of the mitochondrial cytochromebgene. The assay is linear in the range between 13.2 fg and 1.3 ng ofA. fumigatusDNA, corresponding to 3 to 300,000 CFU per ml of BAL fluid or blood. No cross-amplification was observed with human DNA or with the DNA of fungal or bacterial pathogens. For clinical evaluation we investigated 10 BAL samples from nine neutropenic patients with malignant hematological diseases and 12 blood samples from seven neutropenic patients with malignant hematological diseases. Additionally, we tested one blood sample and one BAL sample from each of two neutropenic patients. In order to characterize the validity of the novel PCR assay, only samples that had shown positive results by a previously described sensitive and specific nested PCR assay were tested. Twelve of 12 BAL samples and 6 of 14 blood samples gave positive results by the LightCycler PCR assay. Eight of 14 blood samples gave negative results by the novel method. The LightCycler PCR-mediated quantification of the fungal burden showed 15 to 269,018 CFU per ml of BAL sample and 298 to 104,114 CFU per ml of blood sample. Twenty of 20 BAL samples and 50 of 50 blood samples from subjects without evidence of invasive pulmonary aspergillosis (IPA) were PCR negative. Compared to a previously described nested PCR assay, these preliminary data for the novel real-time PCR assay indicate a less sensitive rate of detection of IPA in high-risk patients, but the assay may be valuable for quantification of the fungal burden in individual clinical samples.
Characterization of a Tandem Repeat Polymorphism inLegionella pneumophilaand Its Use for Genotyping
Authors: Pourcel C. , Vidgop Y. , Ramisse F. , Vergnaud G. , Tram C. 
Published: May 2003

ABSTRACTWe have analyzed the variability of minisatellite sequences (also called variable-number tandem repeats [VNTRs]) in the genome ofLegionella pneumophila. Based upon the genome sequence of the Philadelphia-1 strain (serogroup 1), 25 minisatellites were selected and their polymorphisms were analyzed by PCR with the DNA of serogroup 1 to 14 reference strains. For 22 markers, a PCR product of the expected size was found with the DNA of the Philadelphia-1 strain. Most of these markers did not amplify the DNA of otherLegionellaspecies or other bacteria used as controls. A polymorphism was observed for seven markers among theL. pneumophilastrains tested. To check whether these markers could be used to compare strains ofL. pneumophila, we analyzed two groups of isolates from clinical and environmental samples which had been independently genotyped by other methods. The results showed that, for the isolates in these two sets of samples, VNTR typing is as informative as pulsed-field gel electrophoresis for comparison of strains. Sequencing of one minisatellite from 14 reference strains was performed. Comparison of the sequences allowed a classification and confirmed the existence of subspecies ofL. pneumophila. We also tested the usefulness of one very polymorphic marker as a tool for the rapid screening of colonies grown from water samples. This allowed the rapid identification of theL. pneumophilacolonies and gave a first hint as to the presence of several strains in a single sample.
Virulence Markers of Enteroaggregative
Escherichia coli
Isolated from Children and Adults with Diarrhea in Brasília, Brazil
Authors: Piva Iriane C. , Pereira Alex L. , Ferraz Lúcia R. , Silva Rejane S. N. , Vieira Ataíza C. , Blanco Jesus E. , Blanco Miguel , Blanco Jorge , Giugliano Loreny G. 
Published: May 2003

ABSTRACT

Escherichia coli
strains isolated from sporadic cases of acute diarrhea in children and adults and from children without diarrhea were investigated for the presence of the pAA plasmid. Strains harboring the pAA plasmid were isolated at similar frequencies from children with (19.6%) and without (10.8%) diarrhea and from adults with diarrhea (11.8%). The genotypic and phenotypic virulence markers of these strains were further analyzed. Most of the strains were positive for EAST1 (73%), and this toxin was detected significantly more frequently in strains from children with diarrhea than in strains from adults with diarrhea (
P
< 0.05). Likewise,
pic
sequences were detected significantly more frequently in strains from children with diarrhea than in strains from adults with diarrhea (
P
< 0.005) and controls (
P
< 0.025). Furthermore, the association of pAA positivity (pAA
+
) and
pic
positivity (
pic
+
) was more frequently found for strains from children with diarrhea than for strains from controls, indicating that pAA
+
pic
+
strains may represent a subset of pAA
+
strains associated with disease in children. Most of the strains (82.5%) adhered to cells presenting the typical aggregative pattern. The frequency of occurrence of enteropathogenic
E
.
coli
(EPEC) serogroups in the strains from children with diarrhea was very high (56%), while none of the strains from adults with diarrhea belonged to EPEC serogroups. Extraintestinal virulence markers were very commonly found in strains from adults with diarrhea. The frequencies of occurrence of the adhesins AFA and SFA were significantly higher in strains from adults with diarrhea than in strains from children with diarrhea. More than one extraintestinal virulence marker was found in 58% of the strains from adults with diarrhea but in only 7.7% of the strains from children with diarrhea. Our results show that pAA
+
strains isolated from children and adults with diarrhea present very different profiles when enteroaggregative
E. coli
virulence markers, serotypes, and extraintestinal virulence markers are considered.
Persistence of Oropharyngeal
Candida albicans
Strains with Reduced Susceptibilities to Fluconazole among Human Immunodeficiency Virus-Seropositive Children and Adults in a Long-Term Care Facility
Authors: Makarova Natalya U. , Pokrowsky V. V. , Kravchenko A. V. , Serebrovskaya L. V. , James Michael J. , McNeil Michael M. , Lasker Brent A. , Warnock David W. , Reiss Errol 
Published: May 2003

ABSTRACT

Nineteen oropharyngeal
Candida albicans
isolates from six children and seven adults living with AIDS at the Russia AIDS Centre, Moscow, from 1990 to 1998 were selected for molecular typing. Two fluconazole-resistant
C. albicans
genotypes were identified from a child who contracted human immunodeficiency virus infection during the Elista Hospital outbreak in the Kalmyk Republic in 1989. Highly related strains were observed 4 years later in the oral lesions and colonization of two patients and a health care worker. There may be a tendency for persons who are living with AIDS in a long-term care facility and who receive fluconazole therapy for oropharyngeal candidiasis to harbor and spread fluconazole-resistant
C. albicans
strains.
Racial Distribution of
Candida dubliniensis
Colonization among South Africans
Authors: Blignaut Elaine , Pujol Claude , Joly Sophie , Soll David R. 
Published: May 2003

ABSTRACT

Candida dubliniensis
is a yeast species that has only recently been differentiated from
Candida albicans. C. dubliniensis
colonization was initially associated with human immunodeficiency virus (HIV)-positive individuals. Because of the large proportion of AIDS patients in South Africa, we tested the generality of this association by assessing the prevalence of
C. dubliniensis
colonization among 253 black HIV-positive individuals, 66 healthy black individuals, 22 white HIV-positive individuals, and 55 healthy white individuals in South Africa carrying germ tube-positive yeasts in their oral cavities. Molecular fingerprinting with Ca3, a complex DNA fingerprinting probe specific for
C. albicans
, and Cd25, a complex DNA fingerprinting probe specific for
C. dubliniensis
, provides the first conclusive evidence of the existence of
C. dubliniensis
among South African clinical yeast isolates and reveals a higher relative prevalence of this species among white healthy individuals (16%) than among HIV-positive white individuals (9%), black healthy individuals (0%), and black HIV-positive individuals (1.5%). A cluster analysis separated South African
C. dubliniensis
isolates into two previously described groups, groups I and II, with the majority of isolates clustering in group I. Isolates from white healthy individuals exhibited a higher level of relatedness. A comparison of the
C. dubliniensis
isolates from South Africa with a general collection of
C. dubliniensis
isolates collected worldwide revealed no South Africa-specific clade, as has been demonstrated for
C. albicans
. These results suggest that in South Africa,
C. dubliniensis
carriage is influenced more by race than by HIV infection status.
Evaluation of Pulsed-Field Gel Electrophoresis as a Tool for Determining the Degree of Genetic Relatedness between Strains of
Escherichia coli
O157:H7
Authors: Davis Margaret A. , Hancock Dale D. , Besser Thomas E. , Call Douglas R. 
Published: May 2003

ABSTRACT

Pulsed-field gel electrophoresis (PFGE) has been used extensively to investigate the epidemiology of
Escherichia coli
O157:H7, although it has not been evaluated as a tool for establishing genetic relationships. This is a critical issue when molecular genetic data are used to make inferences about pathogen dissemination. To evaluate this further, genomic DNAs from 62 isolates of
E. coli
O157:H7 from different cattle herds were digested with
Xba
I and
Bln
I and subjected to PFGE. The correlation between the similarity coefficients for these two enzymes was only 0.53. Four additional restriction enzymes (
Nhe
I,
Pac
I,
Sfi
I, and
Spe
I) were used with DNAs from a subset of 14 isolates. The average correlations between similarity coefficients using sets of one, two, and three enzymes were 0.405, 0.568, and 0.648, respectively. Probing with lambda DNA demonstrated that some DNA fragments migrated equal distances in the gel but were composed of nonhomologous genetic material. Genome sequence data from EDL933 indicated that 40 PFGE fragments would be expected from complete
Xba
I digestion, yet only 19 distinguishable fragments were visible. Two reasons that similarity coefficients from single-enzyme PFGE are poor measures of relatedness (and hence are poorly correlated with other enzymes) are evident from this study: (i) matching bands do not always represent homologous genetic material and (ii) there are limitations to the power of PFGE to resolve bands of nearly identical size. The findings of the present study indicate that if genetic relationships must be inferred in the absence of epidemiologic data, six or more restriction enzymes would be needed to provide a reasonable estimate using PFGE.
Rapid Detection of
Mycoplasma genitalium
,
Mycoplasma hominis
,
Ureaplasma parvum
, and
Ureaplasma urealyticum
Organisms in Genitourinary Samples by PCR-Microtiter Plate Hybridization Assay
Authors: Yoshida Takashi , Maeda Shin-Ichi , Deguchi Takashi , Miyazawa Takamaro , Ishiko Hiroaki 
Published: May 2003

ABSTRACT

We present a method for detecting the presence of
Mycoplasma genitalium
,
Mycoplasma hominis
,
Ureaplasma parvum
, and
Ureaplasma urealyticum
organisms, which are thought to be associated with nongonococcal urethritis (NGU) and other genitourinary infections, in clinical samples. This method consists of PCR amplification of a part of the 16S rRNA gene followed by 96-well microtiter plate hybridization assay using four species-specific capture probes to detect the targets. To test the efficacy of this method, we applied it to the detection of the four species in the urine of patients with NGU. There were no cross-reactions with other human mycoplasmas or ureaplasmas, and the PCR-microtiter plate hybridization assay detected as few as 10 copies of the 16S rRNA gene of each of the four species. Based on these results, this PCR-microtiter plate hybridization assay can be considered an effective tool for the diagnosis of genitourinary infections with mycoplasmas or ureaplasmas.
Specific Delayed-Type Hypersensitivity Responses to ESAT-6 Identify Tuberculosis-Infected Cattle
Authors: Pollock J. M. , McNair J. , Bassett H. , Cassidy J. P. , Costello E. , Aggerbeck H. , Rosenkrands I. , Andersen P. 
Published: May 2003

ABSTRACT

Human and bovine tuberculosis have long been detected by skin testing with purified protein derivative (PPD), a complex mix of partly denatured mycobacterial antigens with suboptimal specificity. In the present study, skin tests based on ESAT-6, a recombinantly produced antigen highly specific for tuberculosis infection, were investigated. Although ESAT-6 was strongly recognized in vitro and induced high levels of gamma interferon, initial investigations demonstrated that higher doses of ESAT-6 than of PPD were needed to induce substantial delayed-type hypersensitivity reactions. Also, the kinetics of the skin test response differed for the two reagents; PPD showed maximal response at 72 h, but the response to ESAT-6 often peaked later at 96 h. Tests based on an optimized strategy (400 μg of ESAT-6 measured between 72 and 96 h), in cattle infected with
Mycobacterium bovis
(
n
= 22) and animals sensitized by exposure to environmental mycobacteria showed ESAT-6 to have a promising diagnostic potential (sensitivity, 82%; specificity, 100%; optimal cutoff, 3 mm), compared with PPD (sensitivity, 86%; specificity, 90%; optimal cutoff, 4 mm). Larger investigations are required to refine cutoff points for any new diagnostic test, but the present results indicate great potential for skin tests based on specific antigens for accurate in vivo diagnosis of tuberculosis.
Evaluation and Utilization as a Public Health Tool of a National Molecular Epidemiological Tuberculosis Outbreak Database within the United Kingdom from 1997 to 2001
Authors: Drobniewski F. A. , Gibson A. , Ruddy M. , Yates M. D. 
Published: May 2003

ABSTRACTThe aim of this study was to develop a national model and analyze the value of a molecular epidemiologicalMycobacterium tuberculosisDNA fingerprint-outbreak database. Incidents were investigated by the United Kingdom PHLS Mycobacterium Reference Unit (MRU) from June 1997 to December 2001, inclusive. A total of 124 incidents involving 972 tuberculosis cases, including 520 patient cultures from referred incidents and 452 patient cultures related to two population studies, were examined by using restriction fragment length polymorphism IS6110fingerprinting and rapid epidemiological typing. Investigations were divided into the following three categories, reflecting different operational strategies: retrospective passive analysis, retrospective active analysis, and retrospective prospective analysis. The majority of incidents were in the retrospective passive analysis category, i.e., the individual submitting isolates has a suspicion they may be linked. Outbreaks were examined in schools, hospitals, farms, prisons, and public houses, and laboratory cross-contamination events and unusual clinical presentations were investigated. Retrospective active analysis involved a major outbreak centered on a high school. Contact tracing of a teenager with smear-positive pulmonary tuberculosis matched 14 individuals, including members of his class, and another 60 cases were identified in schools clinically and radiologically and by skin testing. Retrospective prospective analysis involved an outbreak of 94 isoniazid-resistant tuberculosis cases in London, United Kingdom, that began after cases were identified at one hospital in January 2000. Contact tracing and comparison with MRU databases indicated that the earliest matched case had occurred in 1995. Subsequently, the MRU changed to an active prospective analysis targeting linked isoniazid-monoresistant isolates for follow up. The patients were multiethnic, born mainly in the United Kingdom, and included professionals, individuals from the music industry, intravenous drug abusers, and prisoners.
Evaluation ofCoxiella burnetiiAntibiotic Susceptibilities by Real-Time PCR Assay
Authors: Brennan Robert E. , Samuel James E. 
Published: May 2003

ABSTRACTCoxiella burnetiiis an obligate intracellular bacterium. The inability to cultivate this organism on axenic medium has made calculation of infectious units challenging and prevents the use of conventional antibiotic susceptibility assays. A rapid and reliable real-time PCR assay was developed to quantifyC. burnetiicells from J774.16 mouse macrophage cells and was applied to antibiotic susceptibility testing ofC. burnetiiNine Mile, phase I. For calculation of bacterial replication, real-time PCR performed equally as well as immunofluorescent-antibody (IFA) assay when J774.16 cells were infected with 10-fold serial dilutions ofC. burnetiiand was significantly (P< 0.05) more repeatable than IFA when 2-fold dilutions were used. Newly infected murine macrophage-like J774.16 cells were treated with 8 μg of chloramphenicol per ml, 4 μg of tetracycline per ml, 4 μg of rifampin per ml, 4 μg of ampicillin per ml, or 1 μg of ciprofloxacin per ml. After 6 days of treatment, tetracycline, rifampin, and ampicillin significantly (P< 0.01) inhibited the replication ofC. burnetii, while chloramphenicol and ciprofloxacin did not. In general, these results are consistent with those from prior reports on the efficacy of these antibiotics againstC. burnetiiNine Mile, phase I, and indicate that a real-time PCR-based assay is an appropriate alternative to the present methodology for evaluation of the antibiotic susceptibilities ofC. burnetii.
Evaluation of the Etest and Disk Diffusion Methods for Determining Susceptibilities of 235 Bloodstream Isolates of
Candida glabrata
to Fluconazole and Voriconazole
Authors: Pfaller M. A. , Diekema D. J. , Boyken L. , Messer S. A. , Tendolkar S. , Hollis R. J. 
Published: May 2003

ABSTRACT

The performances of the Etest and the disk diffusion methods for testing of the susceptibilities of 235
Candida glabrata
isolates to fluconazole and voriconazole were compared with that of the National Committee for Clinical Laboratory Standards (NCCLS) approved standard broth microdilution (BMD) method. The NCCLS method used RPMI 1640 broth medium, and MICs were read after incubation for 48 h at 35°C. Etest MICs were determined with RPMI 1640 agar containing 2% glucose (RPG agar) and with Mueller-Hinton agar containing 2% glucose and 0.5 μg of methylene blue per ml (MBE agar) and were read after incubation for 48 h at 35°C. Disk diffusion testing was performed with MBE agar, 25-μg fluconazole disks, and 1-μg voriconazole disks and by incubation at 35°C for 24 h. Overall agreements between the Etest and the BMD MICs obtained with RPG and MBE agars were 91 and 96%, respectively, for fluconazole and 93 and 95%, respectively, for voriconazole. Categorical agreements between the agar-based methods and BMD were 52.3 to 64.7% with fluconazole and 94.8 to 97.4% with voriconazole. The vast majority of the discrepancies by the disk diffusion and Etest methods with fluconazole were minor errors. The agar-based methods performed well in identifying isolates with resistance to fluconazole and decreased susceptibility to voriconazole.