bluuebunny/crossref_metadata_2025_split huggingface dataset:
Hypoxic Response ofMycobacterium tuberculosisStudied by Metabolic Labeling and Proteome Analysis of Cellular and Extracellular Proteins
Authors: Rosenkrands Ida , Slayden Richard A. , Crawford Janne , Aagaard Claus , Barry Clifton E. , Andersen Peter 
Published: July 2002

ABSTRACTThe events involved in the establishment of a latent infection withMycobacterium tuberculosisare not fully understood, but hypoxic conditions are generally believed to be the environment encountered by the pathogen in the central part of the granuloma. The present study was undertaken to provide insight intoM. tuberculosisprotein expression in in vitro latency models where oxygen is depleted. The response ofM. tuberculosisto low-oxygen conditions was investigated in both cellular and extracellular proteins by metabolic labeling, two-dimensional electrophoresis, and protein signature peptide analysis by liquid chromatography-mass spectrometry. By peptide mass fingerprinting and immunodetection, five proteins more abundant under low-oxygen conditions were identified from several lysates ofM. tuberculosis: Rv0569, Rv2031c (HspX), Rv2623, Rv2626c, and Rv3841 (BfrB). InM. tuberculosisculture filtrates, two additional proteins, Rv0363c (Fba) and Rv2780 (Ald), were found in increased amounts under oxygen limitation. These results extend our understanding of the hypoxic response inM. tuberculosisand potentially provide important insights into the physiology of the latent bacilli.
Bacillus subtilis
Tolerance of Moderate Concentrations of Rifampin Involves the σ
B
-Dependent General and Multiple Stress Response
Authors: Bandow Julia Elisabeth , Brötz Heike , Hecker Michael 
Published: July 2002

ABSTRACT

Low concentrations of the RNA polymerase inhibitor rifampin added to an exponentially growing culture of
Bacillus subtilis
led to an instant inhibition of growth. Survival experiments revealed that during the growth arrest the cells became tolerant to the antibiotic and the culture was able to resume growth some time after rifampin treatment.
l
-[
35
S]methionine pulse-labeled protein extracts were separated by two-dimensional polyacrylamide gel electrophoresis to investigate the change in the protein synthesis pattern in response to rifampin. The σ
B
-dependent general stress proteins were found to be induced after treatment with the antibiotic. Part of the oxidative stress signature was induced as indicated by the catalase KatA and MrgA. The target protein of rifampin, the β subunit (RpoB) of the DNA-dependent RNA polymerase, and the flagellin protein Hag belonging to the σ
D
regulon were also induced. The rifampin-triggered growth arrest was extended in a
sigB
mutant in comparison to the wild-type strain, and the higher the concentration, the more pronounced this effect was. Activity of the RsbP energy-signaling phosphatase in the σ
B
signal transduction network was also important for this protection against rifampin, but the RsbU environmental signaling phosphatase was not required. The
sigB
mutant strain was less capable of growing on rifampin-containing agar plates. When plated from a culture that had already reached stationary phase without previous exposure to the antibiotic during growth, the survival rate of the wild type exceeded that of the
sigB
mutant by a factor of 100. We conclude that the general stress response of
B. subtilis
is induced by rifampin depending on RsbP activity and that loss of SigB function causes increased sensitivity to the antibiotic.
Localization of UvrA and Effect of DNA Damage on the Chromosome of
Bacillus subtilis
Authors: Smith Bradley T. , Grossman Alan D. , Walker Graham C. 
Published: July 2002

ABSTRACT

We found that the nucleotide excision repair protein UvrA, which is involved in DNA damage recognition, localizes to the entire chromosome both before and after damage in living
Bacillus subtilis
cells. We suggest that the UvrA
2
B damage recognition complex is constantly scanning the genome, searching for lesions in the DNA. We also found that DNA damage induces a dramatic reconfiguration of the chromosome such that it no longer fills the entire cell as it does during normal growth. This reconfiguration is reversible after low doses of damage and is dependent on the damage-induced SOS response. We suggest that this reconfiguration of the chromosome after damage may be either a reflection of ongoing DNA repair or an active mechanism to protect the cell’s genome. Similar observations have been made in
Escherichia coli
, indicating that the alteration of chromosome structure after DNA damage may be a widespread phenomenon.
Construction and Synchronization of
dnaA
Temperature-Sensitive Mutants of
Streptomyces
Authors: Lee Li-Fong , Yeh Shun-Hua , Chen Carton W. 
Published: July 2002

ABSTRACT

Temperature-sensitive mutants of
Streptomyces
defective in initiation of chromosome replication were created by in vitro site-directed mutagenesis in the
dnaA
gene followed by gene replacement. When they were shifted to 39°C replication in the mutants ceased in about 90 min but resumed on return to 30°C. This allowed manipulations to achieve replication synchronization.
Evaluation of 5′ Nuclease Assay for Detection of
Actinobacillus pleuropneumoniae
Authors: Angen Øystein , Jensen Jannie , Lavritsen Dorte T. 
Published: July 2002

ABSTRACT

Sequence detection by the 5′ nuclease TaqMan assay uses online detection of internal fluorogenic probes in closed PCR tubes. Primers and probe were chosen from a part of the
omlA
gene common to all serotypes of
Actinobacillus pleuropneumoniae
, which gave an amplicon of 92 bp. The test was evaluated with 73 lung isolates and 120 tonsil isolates of
A. pleuropneumoniae
as well as with a collection of reference strains. By using a

C
t

value (cycle number in which the fluorescence exceeds the threshold defined by the software) of 30 as the cutoff limit, the 5′ nuclease assay represents a test with 100% sensitivity and 100% specificity. A high degree of reproducibility of the test was demonstrated. If samples with

C
t

values of ≤30 are considered positive, the detection limit of the assay was 1 CFU/reaction tube, corresponding to a 10-fold higher number of DNA templates. After cycle 30, nonspecific reactions appeared when testing dilutions of DNA templates or pure cultures of
A. pleuropneumoniae
, as well as when testing tonsil scrapings from specific-pathogen-free herds. The diagnostic sensitivity, as evaluated with 586 tonsil scrapings from animals infected with
A. pleuropneumoniae
, is the same level as that of a PCR test based on the
omlA
gene described previously. The 5′ nuclease assay represents a fast method for species-specific detection and identification of
A. pleuropneumoniae
in pure and mixed cultures. The evaluation shows, however, that a

C
t

value cutoff limit of ≤30 must be chosen in order to obtain reliable results. The investigation emphasizes that a thorough evaluation of the criteria used to define a positive test result is necessary.
Great Diversity of Group A Rotavirus Strains and High Prevalence of Mixed Rotavirus Infections in India
Authors: Jain Vivek , Das Bimal K. , Bhan Maharaj K. , Glass Roger I. , Gentsch Jon R. ,   the Indian Strain Surveillance Collaborating Laboratories
Published: July 2002

ABSTRACT

We previously observed a marked diversity of rotavirus strains and a high prevalence of the uncommon serotype G9 in a small survey of rotavirus strains collected from six centers in India. In the present study, we characterized a larger collection of strains from children hospitalized with severe diarrhea in seven Indian cities between 1996 and 1998. A total of 287 strains were G and P genotyped by reverse transcription-PCR, and some were further characterized by electropherotyping and subgrouping. Of the four strains common globally, three were found in only 43% of samples (P[8], G1, 15%; P[4], G2, 22%; P[8], G4, 6%), whereas G9 strains made up 17% of the total. Three different G9 strains were present: a P[8], G9 strain, which displayed the long electropherotype and subgroup II VP6 specificity, and two P[6], G9 strains, one with the long electropherotype and subgroup II specificity and the other with the short electropherotype and subgroup I specificity. Marked diversity was observed among strains collected from different cities and collected over time. Of the 253 strains that were fully typed, 54 (21%) had a mixed G or P genotype. Serotype G2 strains were detected more often in infections caused by single strains than in mixed infections (
P
< 0.05), whereas serotype G1 strains were found more often in mixed infections than in infections caused by single strains (
P
< 0.05). The diversity of rotavirus strains and the high prevalence of mixed infections confirm trends reported earlier and help to better characterize the strains of rotavirus circulating in India. Vaccines under development should clearly target G9 strains, and G9 should be included as one of the common global serotypes.
Mannosidase Production by Viridans Group Streptococci
Authors: Homer K. A. , Roberts G. , Byers H. L. , Tarelli E. , Whiley R. A. , Philpott-Howard J. , Beighton D. 
Published: July 2002

ABSTRACT

The production of mannosidase activity by all currently recognized species of human viridans group streptococci was determined using an assay in which bacterial growth was dependent on the degradation of the high-mannose-type glycans of RNase B and subsequent utilization of released mannose. RNase B is an excellent substrate for the demonstration of mannosidase activity since it is a glycoprotein with a single glycosylation site which is occupied by high-mannose-type glycoforms containing five to nine mannose residues. Mannosidase activity was produced only by some members of the mitis group (
Streptococcus mitis, Streptococcus oralis, Streptococcus gordonii, Streptococcus cristatus, Streptococcus infantis, Streptococcus parasanguinis
, and
Streptococcus pneumoniae
) and
Streptococcus intermedius
of the anginosus group. None of the other species within the salivarius and mutans groups or
Streptococcus peroris
and
Streptococcus sanguinis
produced mannosidase activity. Using matrix-assisted laser desorption ionization time-of-flight mass spectrometry, it was demonstrated that the Man
5
glycan alone was degraded while Man
6
to Man
9
, which contain terminal α(1→2) mannose residues in addition to the α(1→3), α(1→6), and β(1→4) residues present in Man
5
, remained intact. Investigations on mannosidase production using synthetic (4-methylumbelliferone- or
p
-nitrophenol-linked) α- or β-mannosides as substrates indicated that there was no correlation between degradation of these substrates and degradation of the Man
5
glycan of RNase B. No species degraded these α-linked mannosides, while degradation of the β-linked synthetic substrates was restricted to strains within the
Streptococcus anginosus, S. gordonii
, and
S. intermedius
species. The data generated using a native glycoprotein as the substrate demonstrate that mannosidase production within the viridans group streptococci is more widely distributed than had previously been considered.
Toward Standardization of Epstein-Barr Virus DNA Load Monitoring: Unfractionated Whole Blood as Preferred Clinical Specimen
Authors: Stevens Servi J. C. , Pronk Inge , Middeldorp Jaap M. 
Published: July 2002

ABSTRACT

Epstein-Barr virus (EBV) DNA load monitoring in peripheral blood has been shown to be a useful tool for the diagnosis of aberrant EBV infections. In the present study we compared the relative diagnostic values of EBV DNA load monitoring in unfractionated whole blood and simultaneously obtained serum or plasma samples from Burkitt's lymphoma (BL) patients, transplant recipients, human immunodeficiency virus (HIV)-infected individuals, and infectious mononucleosis (IM) patients by a quantitative competitive PCR (Q-PCR). The EBV DNA load in BL patients was mainly situated in the cellular blood compartment (up to 4.5 × 10
6
copies/ml). EBV DNA loads in unfractionated whole blood and parallel serum samples showed no correlation. In transplant recipients, IM patients, and HIV-infected patients, the EBV burden in the circulation was almost exclusively restricted to the cellular blood compartment, because serum or plasma samples from these patients yielded negative results by Q-PCR, despite high viral loads in corresponding whole-blood samples. A 10-fold more sensitive but qualitative
Bam
HI-W-repeat PCR occasionally revealed the presence of EBV at <2,000 copies of EBV DNA per ml of serum. Spiking of 100 copies of EBV DNA in samples with negative Q-PCR results excluded the presence of inhibitory factors in serum or plasma that influenced the Q-PCR result. Serum samples from all populations were often positive for β-globin DNA, indicating cell damage in vivo or during serum preparation. We conclude that serum is an undesirable clinical specimen for EBV DNA load monitoring because it omits the presence of cell-associated virus and uncontrolled cell lysis may give irreproducible results or overestimation of the DNA load. Unfractionated whole blood is strongly preferred since it combines all blood compartments that may harbor EBV and it best reflects the absolute viral burden in the patient's circulation.
Comparison of Variable Number Tandem Repeat and IS
6110
-Restriction Fragment Length Polymorphism Analyses for Discrimination of High- and Low-Copy-Number IS
6110 Mycobacterium tuberculosis
Isolates
Authors: Barlow Rachael E. L. , Gascoyne-Binzi Deborah M. , Gillespie Stephen H. , Dickens Anne , Qamer Shabnam , Hawkey Peter M. 
Published: July 2002

ABSTRACT

The present study was designed to evaluate the use of variable number tandem repeat (VNTR) and IS
6110
-restriction fragment length polymorphism (RFLP) analyses in combination as a two-step strategy for discrimination (as measured by the Hunter-Gaston Discrimination Index [HGDI]) of both high- and low-copy-number IS
6110 Mycobacterium tuberculosis
isolates compared to IS
6110
-RFLP alone with an unselected collection of isolates. Individually, IS
6110
-RFLP fingerprinting produced six clusters that accounted for 69% of the low-copy-number IS
6110
isolates (five clusters) and 5% of the high-copy-number IS
6110
isolates (one cluster). A total of 39% of all the isolates were clustered (HGDI = 0.97). VNTR analysis generated a total of 35 different VNTR allele profile sets from 93 isolates (HGDI = 0.938). Combining IS
6110
-RFLP analysis with VNTR analysis reduced the overall percentage of clustered isolates to 29% (HGDI = 0.988) and discriminated a further 27% of low-copy-number isolates that would have been clustered by IS
6110
-RFLP alone. The use of VNTR analysis as an initial typing strategy facilitates further analysis by IS
6110
-RFLP, and more importantly, VNTR analysis subdivides some IS
6110
-RFLP-defined clusters containing low- and single-copy IS
6110
isolates.
O-Antigen Diversity among
Acinetobacter baumannii
Strains from the Czech Republic and Northwestern Europe, as Determined by Lipopolysaccharide-Specific Monoclonal Antibodies
Authors: Pantophlet Ralph , Nemec Alexandr , Brade Lore , Brade Helmut , Dijkshoorn Lenie 
Published: July 2002

ABSTRACT

O-antigen-specific monoclonal antibodies (MAbs) are currently being generated to develop an O-serotyping scheme for the genus
Acinetobacter
and to provide potent tools to study the diversity of O-antigens among
Acinetobacter
strains. In this report,
Acinetobacter baumannii
strains from the Czech Republic and from two clonal groups identified in Northwestern Europe (termed clones I and II) were investigated for their reactivity with a panel of O-antigen-specific MAbs generated against
Acinetobacter
strains from various species. The bacteria were characterized for their ribotype, biotype, and antibiotic susceptibility and the presence of the 8.7-kb plasmid pAN1. By using the combination of these typing profiles, the Czech strains could be classified into four previously defined groups (A. Nemec, L. Janda, O. Melter, and L. Dijkshoorn, J. Med. Microbiol. 48:287–296, 1999): two relatively homogeneous groups of multiresistant strains (termed groups A and B), a heterogeneous group of other multiresistant strains, and a group of susceptible strains. O-antigen reactivity was observed primarily with MAbs generated against
Acinetobacter calcoaceticus
and
Acinetobacter baumannii
strains. A comparison of reaction patterns confirmed the previously hypothesized clonal relationship between group A and clone I strains, which are also similar in other properties. The results show that there is limited O-antigen variability among strains with similar geno- and phenotypic characteristics and are suggestive of a high prevalence of certain
A. baumannii
serotypes in the clinical environment. It is also shown that O-antigen-specific MAbs are useful for the follow-up of strains causing outbreaks in hospitals.
Differentiation of Metronidazole-Sensitive and -Resistant Clinical Isolates of
Helicobacter pylori
by Immunoblotting with Antisera to the RdxA Protein
Authors: Latham Stephanie R. , Owen Robert J. , Elviss Nicola C. , Labigne Agnès , Jenks Peter J. 
Published: July 2002

ABSTRACT

Antimicrobial resistance in
Helicobacter pylori
is a serious and increasing problem, and the development of rapid, reliable methods for detecting resistance would greatly improve the selection of antibiotics used to treat gastric infection with this organism. We assessed whether detection of the RdxA protein could provide the basis for determining the susceptibility of
H. pylori
to metronidazole. In order to raise polyclonal antisera to RdxA, we cloned the
rdxA
gene from
H. pylori
strain 26695 into the commercial expression vector pMAL-c2, purified the resultant fusion protein by affinity chromatography, and used this recombinant RdxA preparation to immunize rabbits. We then used this specific anti-RdxA antibody to perform immunoblotting on whole bacterial cell lysates of 17 metronidazole-sensitive and 27 metronidazole-resistant clinical isolates of
H. pylori
. While a 24-kDa immunoreactive band corresponding to the RdxA protein was observed in all metronidazole-sensitive strains, this band was absent in 25 of 27 resistant isolates. Our results indicate that testing for the absence of the RdxA protein would identify the majority of clinical isolates that will respond poorly to metronidazole-containing eradication regimens and have implications for the development of assays capable of detecting metronidazole resistance in
H. pylori
.
Semiautomation of Multilocus Sequence Typing for the Characterization of Clinical Isolates of
Neisseria meningitidis
Authors: Clarke S. C. , Diggle M. A. , Edwards G. F. S. 
Published: July 2002

ABSTRACT

The Scottish Meningococcus and Pneumococcus Reference Laboratory (SMPRL) provides a national service for the laboratory confirmation of meningococcal and pneumococcal disease in Scotland. Part of this service includes the serogrouping of meningococcal isolates followed by typing and subtyping. The procedures for this are labor-intensive but important for the identification of linked cases and the surveillance of disease so that effective public health measures can be taken. However, different strains of meningococci, such as those within the electrophoretic type 37 complex, occurring during case clusters of disease are now indistinguishable by current methods. The SMPRL has started using multilocus sequence typing (MLST) as a routine method for the characterization of isolates of
Neisseria meningitidis
. MLST produces nucleotide sequence data of seven housekeeping genes providing results that are useful for public health management. However, the method is laborious and time-consuming and therefore lends itself towards automation. The SMPRL therefore developed a semiautomated method for MLST using a 96-well format liquid handler and an automated DNA sequencer. Semiautomated MLST is now provided as a reference service for Scotland. This work describes the methodology required for the characterization of
N. meningitidis
and highlights its usefulness for public health intervention.
Molecular Evidence of
Helicobacter cinaedi
Organisms in Human Gastric Biopsy Specimens
Authors: Peña J. A. , McNeil K. , Fox J. G. , Versalovic J. 
Published: July 2002

ABSTRACT

One hundred twenty-six urease-negative gastric biopsy specimens were evaluated for the presence of
Helicobacter
genus-specific 16S ribosomal DNA (rDNA) and
H. pylori
-specific
glmM
DNA sequences by PCR. The species specificity of the
glmM
PCR assay was demonstrated, as
H. pylori
was the only
Helicobacter
species that yielded the expected
glmM
amplicon. Most urease-negative specimens (118 of 126 specimens) lacked
Helicobacter
DNA. However, 8 of 126 urease-negative specimens contained
Helicobacter
16S rDNA. In order to identify the
Helicobacter
species present in urease-negative gastric biopsy specimens, 16S rDNA amplicons were cloned and sequenced. Sequence comparisons were performed by analyses of the sequences in public sequence databases. Two samples contained 16S rDNA that was identified as
H. cinaedi
with 100% identity and that spanned approximately 400 bp (398 and 398 bp, respectively). In contrast, multiple differences (97% identity; 390 of 398 bp) were observed with
H. pylori
16S rDNA in this region. This finding was verified by sequencing an overlapping 537-bp fragment within the 5′ portion of 16S rDNA. Although the clinical findings were consistent with
H. pylori
infection (e.g., duodenal ulcer disease), rapid urease testing and DNA sequence analyses suggested the presence of
H. cinaedi
organisms and the absence of
H. pylori
in two human antral biopsy specimens. This study represents the first report of an enteric urease-negative helicobacter in the human stomach. Although these organisms were previously associated with extragastric infections, the roles of these organisms in the pathogenesis of chronic gastritis or peptic ulcer disease remain unclear.
Comparative Analysis of Titers of Antibody against Measles Virus in Sera of Vaccinated and Naturally Infected Japanese Individuals of Different Age Groups
Authors: Itoh Masae , Okuno Yoshinobu , Hotta Hak 
Published: July 2002

ABSTRACT

The anti-measles virus (MV) antibody titers in the sera of vaccinees and naturally infected individuals of different age groups were measured to help assess the efficacy of the current MV vaccination in Japan. Neutralizing (NT) antibody titers induced by vaccination were 2
3.2
times lower than those induced by natural infection and declined significantly by age 20. The once-decreased NT antibody titers of the vaccinees increased 2
3.6
times during their twenties to titers comparable to those of naturally infected individuals of the same age, implying the possible occurrence of natural infection in vaccinees with decreased anti-MV immunity. Although the current field strains in Japan, types D3 and D5, were reported to differ antigenically from each other and from vaccine strains (type A) to some extent, as demonstrated by different reactivities to monoclonal antibodies, the sera of vaccinees neutralized the two types of field strains and the vaccine strain with the same efficiency. This result suggests that the current vaccine strain would be suitable to elicit protection against types D3 and D5, as long as viral antigenicity is concerned. However, when compared at given hemagglutination inhibition titers, NT antibody titers of vaccinees were 2
1.1
to 2
3.2
times lower than those of naturally infected individuals, suggesting a qualitative difference(s) of anti-MV antibodies between the two groups. It should be emphasized that protective immunity induced by the one-dose vaccination currently implemented in Japan may not be strong enough to ensure lifelong immunity. A two-dose vaccination program with higher vaccination coverage needs to be considered in order to effectively control measles in Japan.