bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation of the
MIG1
Gene from
Candida albicans
and Effects of Its Disruption on Catabolite Repression
Authors: Zaragoza Oscar , Rodríguez Cristina , Gancedo Carlos
Published: July 2002

We have cloned a
Candida albicans
gene (
CaMIG1
) that encodes a protein homologous to the DNA-binding protein Mig1 from
Saccharomyces cerevisiae
(
Sc
Mig1). The
C. albicans
Mig1 protein (
Ca
Mig1) differs from
Sc
Mig1, in that, among other things, it lacks a putative phosphorylation site for Snf1 and presents several long stretches rich in glutamine or in asparagine, serine, and threonine and has the effector domain located at some distance (50 amino acids) from the carboxy terminus. Expression of
CaMIG1
was low and was similar in glucose-, sucrose-, or ethanol-containing media. Disruption of the two
CaMIG1
genomic copies had no effect in filamentation or infectivity. Levels of a glucose-repressible α-glucosidase, implicated in both sucrose and maltose utilization, were similar in wild-type or
mig1/mig1
cells. Disruption of
CaMIG1
had also no effect on the expression of the glucose-repressed gene
CaGAL1. CaMIG1
was functional in
S. cerevisiae
, as judged by its ability to suppress the phenotypes produced by
mig1
or
tps1
mutations. In addition,
Ca
Mig1 formed specific complexes with the URS1 region of the
S. cerevisiae FBP1
gene. The existence of a possible functional analogue of
CaMIG1
in
C. albicans
was suggested by the results of band shift experiments.
Cloning, Expression, and Purification of a Thermostable Nonhomodimeric Restriction Enzyme,
Bsl
I
Authors: Hsieh Pei-chung , Xiao Jian-ping , O'loane Diana , Xu Shuang-yong
Published: July 2002

Bsl
I is a thermostable type II restriction endonuclease with interrupted recognition sequence CCNNNNN/NNGG (/, cleavage position). The
Bsl
I restriction-modification system from
Bacillus
species was cloned and expressed in
Escherichia coli
. The system is encoded by three genes: the 2,739-bp
Bsl
I methylase gene (
bslIM
), the
bslIR
α gene, and the
bslIR
β gene. The α and β subunits of
Bsl
I can be expressed independently in
E. coli
in the absence of
Bsl
I methylase (M.
Bsl
I) protection.
Bsl
I endonuclease activity can be reconstituted in vitro by mixing the two subunits together. Gel filtration chromatography and native polyacrylamide gel electrophoresis indicated that
Bsl
I forms heterodimers (αβ), heterotetramers (α
2
β
2
), and possibly oligomers in solution. Two β subunits can be cross-linked by a chemical cross-linking agent, indicating formation of heterotetramer
Bsl
I complex (α
2
β
2
). In DNA mobility shift assays, neither subunit alone can bind DNA. DNA mobility shift activity was detected after mixing the two subunits together. Because of the symmetric recognition sequence of the
Bsl
I endonuclease, we propose that its active form is α
2
β
2
. M.
Bsl
I contains nine conserved motifs of N-4 cytosine DNA methylases within the β group of aminomethyltransferase. Synthetic duplex deoxyoligonucleotides containing cytosine hemimethylated or fully methylated at N-4 in
Bsl
I sites in the first or second cytosine are resistant to
Bsl
I digestion. C-5 methylation of the second cytosine on both strands within the recognition sequence also renders the site refractory to
Bsl
I digestion. Two putative zinc fingers are found in the α subunit of
Bsl
I endonuclease.
Dimers of π Protein Bind the A+T-Rich Region of the R6K γ Origin near the Leading-Strand Synthesis Start Sites: Regulatory Implications
Authors: Krüger Ricardo , Filutowicz Marcin
Published: July 2002

The replication of γ origin, a minimal replicon derived from plasmid R6K, is controlled by the Rep protein π. At low intracellular concentrations, π activates the γ origin, while it inhibits replication at elevated concentrations. Additionally, π acts as a transcription factor (auto)repressing its own synthesis. These varied regulatory functions depend on π binding to reiterated DNA sequences bearing a TGAGNG motif. However, π also binds to a “non-iteron” site (i.e., not TGAGNG) that resides in the A+T-rich region adjacent to the iterons. This positioning places the non-iteron site near the start sites for leading-strand synthesis that also occur in the A+T-rich region of γ origin. We have hypothesized that origin activation (at low π levels) would require the binding of π monomers to iterons, while the binding of π dimers to the non-iteron site (at high π levels) would be required to inhibit priming. Although monomers as well as dimers can bind to an iteron, we demonstrate that only dimers bind to the non-iteron site. Two additional pieces of data support the hypothesis of negative replication control by π binding to the non-iteron site. First, π binds to the non-iteron site about eight times less well than it binds to a single iteron. Second, hyperactive variants of π protein (called copy-up) either do not bind to the non-iteron site or bind to it less well than wild-type π. We propose a replication control mechanism whereby π would directly inhibit primer formation.
Proteome Analysis of Metabolically Engineered
Escherichia coli
Producing Poly(3-Hydroxybutyrate)
Authors: Han Mee-Jung , Yoon Sang Sun , Lee Sang Yup
Published: July 2002

Recombinant
Escherichia coli
strains harboring heterologous polyhydroxyalkanoate (PHA) biosynthesis genes were shown to accumulate unusually large amounts of PHA. In the present study, integrated cellular responses of metabolically engineered
E. coli
to the accumulation of poly(3-hydroxybutyrate) (PHB) in the early stationary phase were analyzed at the protein level by two-dimensional gel electrophoresis. Out of 20 proteins showing altered expression levels with the accumulation of PHB, 13 proteins were identified with the aid of mass spectrometry. Three heat shock proteins, GroEL, GroES, and DnaK, were significantly up-regulated in PHB-accumulating cells. Proteins which play essential roles in protein biosynthesis were unfavorably influenced by the accumulation of PHB. Cellular demand for the large amount of acetyl coenzyme A and NADPH for the PHB biosynthesis resulted in the increased synthesis of two enzymes of the glycolytic pathway and one enzyme of the Entner-Doudoroff pathway. The expression of the
yfiD
gene encoding a 14.3-kDa protein, which is known to be produced at low pH, was greatly induced with the accumulation of PHB. Therefore, it could be concluded that the accumulation of PHB in
E. coli
acted as a stress on the cells, which reduced the cells' ability to synthesize proteins and induced the expression of various protective proteins.
Identification of Two
prpDBC
Gene Clusters in
Corynebacterium glutamicum
and Their Involvement in Propionate Degradation via the 2-Methylcitrate Cycle
Authors: Claes Wilfried A. , Pühler Alfred , Kalinowski Jörn
Published: July 2002

Genome sequencing revealed that the
Corynebacterium glutamicum
genome contained, besides
gltA
, two additional citrate synthase homologous genes (
prpC
) located in two different
prpDBC
gene clusters, which were designated
prpD1B1C1
and
prpD2B2C2
. The coding regions of the two gene clusters as well as the predicted gene products showed sequence identities of about 70 to 80%. Significant sequence similarities were found also to the
prpBCDE
operons of
Escherichia coli
and
Salmonella enterica
, which are known to encode enzymes of the propionate-degrading 2-methylcitrate pathway. Homologous and heterologous overexpression of the
C. glutamicum prpC1
and
prpC2
genes revealed that their gene products were active as citrate synthases and 2-methylcitrate synthases. Growth tests showed that
C. glutamicum
used propionate as a single or partial carbon source, although the beginning of the exponential growth phase was strongly delayed by propionate for up to 7 days. Compared to growth on acetate, the specific 2-methylcitrate synthase activity increased about 50-fold when propionate was provided as the sole carbon source, suggesting that in
C. glutamicum
the oxidation of propionate to pyruvate occurred via the 2-methylcitrate pathway. Additionally, two-dimensional gel electrophoresis experiments combined with mass spectrometry showed strong induction of the expression of the
C. glutamicum prpD2B2C2
genes by propionate as an additional carbon source. Mutational analyses revealed that only the
prpD2B2C2
genes were essential for the growth of
C. glutamicum
on propionate as a sole carbon source, while the function of the
prpD1B1C1
genes remains obscure.
Effects of
rodA
and
pbp2b
Disruption on Cell Morphology and Oxidative Stress Response of
Streptococcus thermophilus
CNRZ368
Authors: Thibessard Annabelle , Fernandez Annabelle , Gintz Brigitte , Leblond-Bourget Nathalie , Decaris Bernard
Published: July 2002

Insertional mutagenesis was used to isolate clones from
Streptococcus thermophilus
CNRZ368 that were modified in their abilities to tolerate oxidative stress. During this process, two menadione-sensitive clones (6G4 and 18C3) were found to display abnormal cell morphologies and distorted chain topologies and were further studied. Molecular characterization of both 6G4 and 18C3 mutants indicated that they were disrupted in open reading frames homologous to
rodA
and
pbp2b
, respectively. Both genes encoded proteins in
Escherichia coli
that were described as being implicated in peptidoglycan synthesis during the process of cell elongation and to function in determining the rod shape of the cell. This work reports a possible connection between peptidoglycan biosynthesis and oxidative stress defense in
S. thermophilus
CNRZ368.
Oxidative-Stress Resistance Mutants ofHelicobacter pylori
Authors: Olczak Adriana A. , Olson Jonathan W. , Maier Robert J.
Published: July 2002

Within a large family of peroxidases, one member that catalyzes the reduction of organic peroxides to alcohols is known as alkyl hydroperoxide reductase, or AhpC. Gene disruption mutations in the gene encoding AhpC ofHelicobacter pylori(ahpC) were generated by screening transformants under low-oxygen conditions. Two classes of mutants were obtained. Both types lack AhpC protein, but the major class (type I) isolated was found to synthesize increased levels (five times more than the wild type) of another proposed antioxidant protein, an iron-binding, neutrophil-activating protein (NapA). The other class of mutants, the minor class (type II), produced wild-type levels of NapA. The two types of AhpC mutants differed in their frequencies of spontaneous mutation to rifampin resistance and in their sensitivities to oxidative-stress chemicals, with the type I mutants exhibiting less sensitivity to organic hydroperoxides as well as having a lower mutation frequency. ThenapApromoter regions of the two types of AhpC mutants were identical, and primer extension analysis revealed their transcription start site to be the same as for the wild type. Gene disruption mutations were obtained innapAalone, and a double mutant strain (ahpC napA) was also created. All four of the oxidative-stress resistance mutants could be distinguished from the wild type in oxygen sensitivity or in some other oxidative-stress resistance phenotype (i.e., in sensitivity to stress-related chemicals and spontaneous mutation frequency). For example, growth of the NapA mutant was more sensitive to oxygen than that of the wild-type strain and both of the AhpC-type mutants were highly sensitive to paraquat and to cumene hydroperoxide. Of the four types of mutants, the double mutant was the most sensitive to growth inhibition by oxygen and by organic peroxides and it had the highest spontaneous mutation frequency. Notably, two-dimensional gel electrophoresis combined with protein sequence analysis identified another possible oxidative-stress resistance protein (HP0630) that was up-regulated in the double mutant. However, the transcription start site of the HP0630 gene was the same for the double mutant as for the wild type. It appears thatH. pylorican readily modulate the expression of other resistance factors as a compensatory response to loss of a major oxidative-stress resistance component.
Mycobacterium microti
Llama-Type Infection Presenting as Pulmonary Tuberculosis in a Human Immunodeficiency Virus-Positive Patient
Authors: Horstkotte Matthias A. , Sobottka Ingo , Schewe Carl K. , Schäfer Peter , Laufs Rainer , Rüsch-Gerdes Sabine , Niemann Stefan
Published: July 2002

A rare case of
Mycobacterium microti
infection in a human immunodeficiency virus-positive patient is described. Because of unusual morphological and cultural features, the pathogen was analyzed by spoligotyping and identified as the
Mycobacterium microti
llama type. Although culture of
M. microti
is difficult, drug susceptibility testing could be performed, which correlated with the clinical outcome.
Evaluation of a Rapid Immunochromatographic Test for Detection of
Streptococcus pneumoniae
Antigen in Urine Samples from Adults with Community-Acquired Pneumonia
Authors: Murdoch David R. , Laing Richard T. R. , Mills Graham D. , Karalus Noel C. , Town G. Ian , Mirrett Stanley , Reller L. Barth
Published: July 2002

Streptococcus pneumoniae
is the most common cause of community-acquired pneumonia but is undoubtedly underdiagnosed. Isolation of
S. pneumoniae
from blood is specific but lacks sensitivity, while isolation of
S. pneumoniae
from sputum may represent colonization. We evaluated a new immunochromatographic test (NOW
S. pneumoniae
urinary antigen test; Binax, Portland, Maine) that is simple to perform and that can detect
S. pneumoniae
antigen in urine within 15 min. Urine samples from 420 adults with community-acquired pneumonia and 169 control patients who did not have pneumonia were tested. Urine from 315 (75%) of the pneumonia patients and all controls was tested both before and after 25-fold concentration, while the remaining 105 samples were only tested without concentration.
S. pneumoniae
urinary antigen tests were positive for 120 (29%) patients with pneumonia and for none of the controls. Of the urine samples tested with and without concentration, 96 were positive, of which 6 were positive only after concentration.
S. pneumoniae
antigen was detected in the urine from 16 of the 20 (80%) patients with blood cultures positive for
S. pneumoniae
and from 28 of the 54 (52%) patients with sputum cultures positive for
S. pneumoniae
. The absence of
S. pneumoniae
antigen in the urine from controls suggests that the specificity is high. Concentration of urine prior to testing resulted in a small increase in yield. The NOW
S. pneumoniae
urinary antigen test should be a useful adjunct to culture for determining the etiology of community-acquired pneumonia in adults.
Plasmid-BornesmrGene Causes Resistance to Quaternary Ammonium Compounds in BovineStaphylococcus aureus
Authors: Bjorland Jostein , Sunde Marianne , Waage Steinar
Published: July 2002

Resistance to quaternary ammonium compounds (QAC) in staphylococci is common in hospital environments and has been described in the food industry. Little is known about staphylococcal QAC resistance associated with animal disease, although such disinfectants are widely used in veterinary medicine. In order to investigate the occurrence of QAC resistance in staphylococci isolated from QAC-exposed animals, 32 penicillin- and tetracycline-resistant and 23 penicillin- and tetracycline-susceptibleStaphylococcus aureusisolates collected from milk from cows with mastitis during a 4-year period were selected for QAC susceptibility studies and genetic characterization. The isolates originated from four different herds that used a common pasture with a joint milking parlor in the summer. During the pasture season, a teat cream containing the QAC cetyltrimethylammonium bromide had been used daily for more than 10 years for mastitis control. Three of the penicillin- and tetracycline-resistant isolates, which were recovered from three different cows during a 20-month period, were resistant to QAC. Plasmid analysis, PCR, and DNA sequencing revealed a novel plasmid of 2,239 bp containing thesmrgene. The plasmid, designated pNVH99, has similarities to small,smr-containing staphylococcal plasmids previously found in human and food isolates. pNVH99 is a new member of the pC194 family of rolling-circle replication plasmids. The three QAC-resistant isolates, as well as 28 of the 29 remaining penicillin- and tetracycline-resistant isolates, were indistinguishable by pulsed-field gel electrophoresis. The study indicates that the occurrence and spread of QAC-resistantS. aureusamong dairy cows may be a problem that needs further investigation.
Pulsed-Field Gel Electrophoresis in Differentiation of
Erysipelothrix
Species Strains
Authors: Okatani Alexandre Tomomitsu , Uto Takehiko , Taniguchi Takahide , Horisaka Tomoko , Horikita Tetsuya , Kaneko Ken-Ichi , Hayashidani Hideki
Published: July 2002

We report here the first analysis of
Erysipelothrix
spp. using pulsed-field gel electrophoresis (PFGE). Seventy strains of
Erysipelothrix
spp. were analyzed.
Sma
I,
Asc
I, and
Not
I were tested for the ability to cleave the DNA extracted from those strains, and among them,
Sma
I was the most reliable enzyme. Sixty-three distinct PFGE patterns were produced, and no DNA degradation was observed, allowing the identification of all of the strains. Based on these results and on those of a previous analysis using randomly amplified polymorphic DNA and ribotyping, PFGE with
Sma
I might be considered to be more sensitive than those methods and to be the best method for epidemiological studies of strains of this genus.
Burden of Unidentifiable Mycobacteria in a Reference Laboratory
Authors: Tortoli Enrico , Bartoloni Alessandro , Böttger Erik C. , Emler Stefan , Garzelli Carlo , Magliano Enrico , Mantella Antonia , Rastogi Nalin , Rindi Laura , Scarparo Claudio , Urbano Pasquale
Published: July 2002

Modern identification techniques at the genomic level have greatly improved the taxonomic knowledge of mycobacteria. In adjunct to nucleic acid sequences, mycobacterial identification has been endorsed by investigation of the lipidic patterns of unique mycolic acids in such organisms. In the present investigation, the routine use of high-performance liquid chromatography (HPLC) of mycolic acids, followed by the sequencing of the 16S rRNA, allowed us to select 72 mycobacterial strains, out of 1,035 screened, that do not belong to any of the officially recognized mycobacterial species. Most strains (i.e., 47) were isolated from humans, 13 were from the environment, 3 were from animals, and 9 were from unknown sources. The majority of human isolates were grown from the respiratory tract and were therefore most likely not clinically significant. Some, however, were isolated from sterile sites (blood, pleural biopsy, central venous catheter, or pus). Many isolates, including several clusters of two or more strains, mostly slow growers and scotochromogenic, presented unique genetic and lipidic features. We hope the data reported here, including the results of major conventional identification tests, the HPLC profiles of strains isolated several times, and the whole sequences of the 16S rRNA hypervariable regions of all 72 mycobacteria, may encourage reporting of new cases. The taxonomy of the genus
Mycobacterium
is, in our opinion, still far from being fully elucidated, and the reporting of unusual strains provides the best background for the recognition of new species. Our report also shows the usefulness of the integration of novel technology to routine diagnosis, especially in cases involving slow-growing microorganisms such as mycobacteria.
Pyrazinamide-Monoresistant
Mycobacterium tuberculosis
in the United States
Authors: Hannan Margaret M. , Desmond Edward P. , Morlock Glenn P. , Mazurek Gerald H. , Crawford Jack T.
Published: July 2002

Mycobacterium bovis
is naturally resistant to the antituberculosis drug pyrazinamide (PZA). To determine whether all
Mycobacterium tuberculosis
complex isolates demonstrating PZA monoresistance were truly
M. bovis
, we examined the phenotype and genotype of isolates reported as PZA monoresistant in five counties in California from January 1996 through June 1999. Isolates reported by local laboratories to be PZA monoresistant were sent to the state reference laboratory for repeat susceptibility testing using the BACTEC radiometric method and to the Centers for Disease Control and Prevention for
pncA
sequencing and PCR-restriction fragment length polymorphism (RFLP) analysis of the
oxyR
gene. Of 1,916 isolates, 14 were reported as PZA monoresistant and 11 were available for retesting. On repeat testing, 6 of the 11 isolates were identified as PZA-susceptible
M. tuberculosis
, 1 was identified as PZA-monoresistant
M. bovis
, and 1 was identified as
M. bovis
BCG. The three remaining isolates were identified as PZA-monoresistant
M. tuberculosis
. Sequencing of the
pncA
and
oxyR
genes genotypically confirmed the two
M. bovis
and the six susceptible
M. tuberculosis
species. Each of the three PZA-monoresistant
M. tuberculosis
isolates had different, previously unreported,
pncA
gene mutations: a 24-bp deletion in frame after codon 88, a base substitution at codon 104 (Ser
104
Cys), and a base substitution at codon 90 (Ile
90
Ser). This study demonstrates that PZA monoresistance is not an absolute marker of
M. bovis
species but may also occur in
M. tuberculosis
, associated with a number of different mutational events in the
pncA
gene. It is the first report of PZA-monoresistant
M. tuberculosis
in the United States.
Rotavirus Strain Diversity in Blantyre, Malawi, from 1997 to 1999
Authors: Cunliffe N. A. , Gondwe J. S. , Graham S. M. , Thindwa B. D. M. , Dove W. , Broadhead R. L. , Molyneux M. E. , Hart C. A.
Published: July 2002

In a 2-year study of viral gastroenteritis in children in Blantyre, Malawi, the diversity of rotavirus strains was investigated by using electropherotyping, reverse transcription-PCR amplification of the VP7 and VP4 genes (G and P genotyping), and nucleotide sequencing. Of 414 rotavirus strains characterized, the following strain types were identified: P[8], G1 (
n
= 111; 26.8%); P[6], G8 (
n
= 110; 26.6%); P[8], G3 (
n
= 93; 22.5%); P[4], G8 (
n
= 31; 7.5%); P[8], G4 (
n
= 21; 5.1%); P[6], G3 (
n
= 12; 2.9%); P[6], G1 (
n
= 7; 1.7%); P[6], G9 (
n
= 3; 0.7%); P[6], G4 (
n
= 3; 0.7%); P[4], G3 (
n
= 1; 0.2%); and mixed (
n
= 15; 3.6%). While all strains could be assigned a G type, seven strains (1.7%) remained P nontypeable. The majority of serotype G8 strains and all serotype G9 strains had short electropherotype profiles. All remaining typeable strains had long electropherotypes. Divergent serotype G1 rotaviruses, which contained multiple base substitutions in the 9T-1 primer binding site, were commonly identified in the second year of surveillance. Serotype G2 was not identified. Overall, G8 was the most frequently identified VP7 serotype (
n
= 144; 34.8%) and P[8] was the most frequently detected VP4 genotype (
n
= 227; 54.8%). Partial sequence analysis of the VP4 gene of genotype P[8] rotaviruses identified three distinct clusters, which predominantly (but not exclusively) comprised strains belonging to a distinct VP7 serotype (G1, G3, or G4). As a result of mutations in the 1T-1 primer binding site, strains belonging to each cluster required a separate primer for efficient typing. One cluster, represented by P[8], G4 strain OP354, was highly divergent from the established Wa and F45 VP4 P[8] lineages. As is the case for some other countries, the diversity of rotaviruses in Malawi implies that rotavirus vaccines in development will need to protect against a wider panel of serotypes than originally envisioned.
Comparison of the E-test with the NCCLS M38-P Method for Antifungal Susceptibility Testing of Common and Emerging Pathogenic Filamentous Fungi
Authors: Espinel-Ingroff Ana
Published: July 2002

The National Committee for Clinical Laboratory Standards (NCCLS) M38-P method describes standard parameters for testing the fungistatic antifungal activities (MICs) of established agents against filamentous fungi (molds). The present study evaluated the in vitro fungistatic activities of itraconazole and amphotericin B by the E-test and the NCCLS M38-P microdilution method against 186 common and emerging pathogenic molds (123 isolates of
Aspergillus
spp. [five species], 16 isolates of
Fusarium
spp. [two species], 4
Paecilomyces lilacinus
isolates, 5
Rhizopus arrhizus
isolates, 15
Scedosporium
spp., 18 dematiaceous fungi, and 5
Trichoderma longibrachiatum
isolates). The agreement between the methods for amphotericin B MICs ranged from 70% for
Fusarium solani
to ≥90% for most of the other species after the first reading; agreement was dependent on both the incubation time and the species being evaluated. Major discrepancies between the amphotericin B MICs determined by the E-test and the NCCLS M38-P method were demonstrated for three of the five species of
Aspergillus
tested and the two species of
Fusarium
tested. This discrepancy was more marked after 48 h of incubation; the geometric mean MICs determined by the E-test increased between 24 and 48 h from between 1.39 and 3.3 μg/ml to between 5.2 and >8 μg/ml for
Aspergillus flavus, Aspergillus fumigatus
, and
Aspergillus nidulans
. The agreement between the itraconazole MICs determined by the E-test and the NCCLS M38-P method ranged from 83.3% for
A. nidulans
to ≥90% for all the other species tested; the overall agreement was higher (92.7%) than that for amphotericin B (87.9%). The agreement was less dependent on the incubation time. Clinical trials need to be conducted to establish the role of the results of either the E-test or the NCCLS M38-P method in vitro for molds with the two agents as predictors of clinical outcome.
Detection of
Aspergillus
Species DNA by PCR in Bronchoalveolar Lavage Fluid
Authors: Hayette Marie-Pierre , Vaira Dolores , Susin Fabrice , Boland Pascal , Christiaens Geneviève , Melin Pierrette , De Mol Patrick
Published: July 2002

The usefulness of a nested PCR assay for detection of
Aspergillus
sp. DNA was evaluated in 177 bronchoalveolar lavage (BAL) fluid specimens. This test was accurate both to diagnose culture-negative BAL fluid specimens from patients with invasive pulmonary aspergillosis and to confirm culture-positive samples. However, it did not differentiate between infection and colonization.
Potential Impact of the VITEK 2 System and the Advanced Expert System on the Clinical Laboratory of a University-Based Hospital
Authors: Sanders Christine C. , Peyret Michel , Moland Ellen Smith , Cavalieri Stephen J. , Shubert Carole , Thomson Kenneth S. , Boeufgras Jean-Marc , Sanders W. Eugene
Published: July 2002

A study was designed to assess the impact of the VITEK 2 automated system and the Advanced Expert System (AES) on the clinical laboratory of a typical university-based hospital. A total of 259 consecutive, nonduplicate isolates of
Enterobacteriaceae
members,
Pseudomonas aeruginosa
, and
Staphylococcus aureus
were collected and tested by the VITEK 2 system for identification and antimicrobial susceptibility testing, and the results were analyzed by the AES. The results were also analyzed by a human expert and compared to the AES analyses. Among the 259 isolates included in this study, 245 (94.6%) were definitively identified by VITEK 2, requiring little input from laboratory staff. For 194 (74.9%) isolates, no inconsistencies between the identification of the strain and the antimicrobial susceptibility determined by VITEK 2 were detected by the AES. Thus, no input from laboratory staff was required for these strains. The AES suggested one or more corrections to results obtained with 65 strains to remove inconsistencies. The human expert thought that most of these corrections were appropriate and that some resulted from a failure of the VITEK 2 system to detect certain forms of resistance. Antimicrobial phenotypes assigned to the strains by the AES for β-lactams, aminoglycosides, quinolones, macrolides, tetracyclines, and glycopeptides were similar to those assigned by the human expert for 95.7 to 100% of strains. These results indicate that the VITEK 2 system and AES can provide accurate information in tests for most of the clinical isolates examined and remove the need for human analysis of results for many. Certain problems were identified in the study that should be remediable with further work on the software supporting the AES.
Prevalence of the Pandemic Genotype of
Vibrio parahaemolyticus
in Dhaka, Bangladesh, and Significance of Its Distribution across Different Serotypes
Authors: Bhuiyan N. A. , Ansaruzzaman M. , Kamruzzaman M. , Alam Khorshed , Chowdhury N. R. , Nishibuchi M. , Faruque Shah M. , Sack David A. , Takeda Yoshifumi , Nair G. Balakrish
Published: July 2002

Sixty-six strains of
Vibrio parahaemolyticus
belonging to 14 serotypes were isolated from hospitalized patients in Dhaka, Bangladesh, from January 1998 to December 2000. Among these, 48 strains belonging to four serotypes had the pandemic genotype and possessed the
tdh
gene. A marker (open reading frame ORF8) for a filamentous phage previously thought to correspond to the pandemic genotype was found to have a poor correlation with the pandemic genotype.
Primary Meningoencephalitis by
Naegleria fowleri
: First Reported Case from Mangalore, South India
Authors: Shenoy Shalini , Wilson Godwin , Prashanth H. V. , Vidyalakshmi K. , Dhanashree B. , Bharath R.
Published: July 2002

A fatal case of primary amebic meningoencephalitis (PAM) in a 5-month-old infant is described. The disease may have been contracted during bathing. The source of water was from an artificial well. The clinical presentation, the isolation of the ameba from the cerebrospinal fluid, the poor response to amphotericin B, and the ultimate fatal outcome are all consistent with the diagnosis of PAM. On the basis of its ability to grow at temperatures above 30°C, the morphology of the trophozoite, and the presence of flagellate forms, the ameba was identified as
Naegleria fowleri
. Pathogenic
N. fowleri
amebae were recovered from samples of water from the well. To our knowledge this case represents the second case of PAM in an infant in the absence of the history of swimming.
rpoB
Genotypes of
Mycobacterium tuberculosis
Beijing Family Isolates from East Asian Countries
Authors: Qian Lishi , Abe Chiyoji , Lin Tao-Ping , Yu Ming-Chih , Cho Sang-Nae , Wang Sumin , Douglas James T.
Published: July 2002

The 81-bp region of the
rpoB
gene in 66 Rif
r
Mycobacterium tuberculosis
isolates from China, Japan, Korea, and Taiwan was analyzed. Twelve single-nucleotide substitutions in the
rpoB
gene were detected. The most prevalent mutations were at Ser-531 (52%), Asp-516 (17%), and His-526 (11%). Mutations were not found in seven (11%) of the isolates. Higher mutation rates in 50 Beijing family isolates were found than in other isolates for mutations at Asp-516 (18 and 12.5%, respectively) and His-526 (12 and 6.3%, respectively). The different rates of mutation may reflect the choice of rifamycin analogs.
Incidence of and Risk Factors for Symptomatic Visceral Leishmaniasis among Human Immunodeficiency Virus Type 1-Infected Patients from Spain in the Era of Highly Active Antiretroviral Therapy
Authors: de la Rosa Rafael , Pineda Juan A , Delgado Juan , Macías Juan , Morillas Francisco , Mira José A. , Sánchez-Quijano Armando , Leal Manuel , Lissen Eduardo
Published: July 2002

The way in which the extensive use of highly active antiretroviral therapy (HAART) has influenced the incidence of visceral leishmaniasis (VL) among human immunodeficiency type 1 (HIV-1)-infected patients is not yet understood. The present study assessed whether the incidence of symptomatic VL in HIV-infected patients has decreased since the introduction of HAART. Likewise, the role of other potential risk factors for VL was also analyzed. Therefore, 479 HIV-1-infected patients receiving antiretroviral treatment, according to the available drugs at each moment, were prospectively followed from April 1989 to June 2000 in two university hospitals in southern Spain. A bone marrow aspiration was performed when patients showed symptoms suggestive of kala-azar. A diagnosis of VL was made whenLeishmaniaamastigotes were seen in Giemsa-stained samples or promastigotes were cultured in specific media. The median follow-up time was 1,380 [8 to 4,536] days. Twenty-one patients were diagnosed with symptomatic VL. The density of incidence of VL has decreased 64.8% as of January 1997, when HAART began to be used extensively in our area. The use of HAART was the main independent factor associated with VL; this therapy was a protective factor (adjusted hazard ratio [HR], 0.05; 95% confidence interval [CI], 0.02 to 0.15). CDC clinical category C at entry in the cohort (HR, 4.08; 95% CI, 1.46 to 11.35) and CD4+cell counts below 300 cells/mm3during the follow-up (HR, 3.96; 95% CI, 1.56 to 10.01) were also independently associated with kala-azar. A VL diagnosis prior to follow-up and low compliance with antiretroviral therapy were not independently associated with symptomatic VL, although statistical significance was almost reached (P= 0.1 andP= 0.08, respectively). In summary, the use of HAART has led to a fall in the incidence of symptomatic VL in HIV-infected patients. The main risk factor associated with kala-azar emergence in patients infected with HIV is deep immunosuppression.
Cloning of a Truncated
Babesia equi
Gene Encoding an 82-Kilodalton Protein and Its Potential Use in an Enzyme-Linked Immunosorbent Assay
Authors: Hirata Haruyuki , Ikadai Hiromi , Yokoyama Naoaki , Xuan Xuenan , Fujisaki Kozo , Suzuki Naoyoshi , Mikami Takeshi , Igarashi Ikuo
Published: July 2002

To isolate
Babesia equi
genes encoding immunodominant proteins, a cDNA expression library prepared from
B. equi
mRNA was immunoscreened with
B. equi
-infected horse serum. Eighteen positive cDNA clones were obtained, and the clone that showed the strongest immunoreactivity, designated Be82, was further characterized. The Be82 gene consisted of 1,953 bp and contained a partial open reading frame lacking the 5′-terminal sequence. As shown by Western blot analyses, immune sera from mice intraperitoneally injected with the Be82 gene product recognized the 82- and 52-kDa proteins of
B. equi
but not those of
Babesia caballi
. The glutathione
S
-transferase fusion protein expressed in
Escherichia coli
that was purified and used as the antigen in the enzyme-linked immunosorbent assay reacted specifically with
B. equi
-infected horse sera. These results suggest that the Be82 gene product is a potential diagnostic antigen candidate in the detection of
B. equi
infection in horses that will be useful both in the performance of epidemiological studies and in the granting of quarantine passes.
Shiga Toxin-Producing
Escherichia coli
Infection and Antibodies against Stx2 and Stx1 in Household Contacts of Children with Enteropathic Hemolytic-Uremic Syndrome
Authors: Ludwig Kerstin , Sarkim Volkan , Bitzan Martin , Karmali Mohamed A. , Bobrowski Christoph , Ruder Hans , Laufs Rainer , Sobottka Ingo , Petric Martin , Karch Helge , Müller-Wiefel Dirk E.
Published: July 2002

Ninety-five household contacts (aged 2 months to 73 years) of patients with enteropathic hemolytic-uremic syndrome (HUS) were investigated for the presence of immunoglobulin (Ig) G antibodies to Shiga toxins Stx2 and Stx1 by Western blot assay. Thirty-one percent of the household contacts and 19% of 327 controls had anti-Stx2 IgG (heavy and light chain [H + L]), 5 and 8%, respectively, had anti-Stx1 IgG (H + L), and 3 and 2%, respectively, had both anti-Stx2 and anti-Stx1 IgG (H + L). The incidence of infections with Stx-producing
Escherichia coli
(STEC) was determined based on the following diagnostic criteria: STEC isolation, detection of
stx
gene sequences, free fecal Stx in stool filtrates, and serum IgM antibodies against
E. coli
O157 lipopolysaccharide. Evidence of STEC infection was observed in 25 household contacts, of whom 18 (72%) were asymptomatic and represented a potential source of infection. Six of 13 (46%) household contacts with Stx2-producing
E. coli
O157:H7 in stool culture developed anti-Stx2 IgG (H + L), compared to 71% of Stx2-associated HUS cases. In individuals showing anti-Stx2 IgG (H + L), the antibody response was directed against the B subunit in 69% of household contacts and 71% of controls, in contrast to 28% of HUS patients. In this investigation controls had a significant increase of the median of IgM antibodies to O157 lipopolysaccharide (LPS) with age, up to the fifth decade. The lack of disease in household contacts with B subunit-specific antibodies, as well as the significantly higher median of anti-O157 LPS IgM antibodies in controls beyond 4.9 years of age, suggests a protective role for anti-Stx and anti-O157 LPS antibodies.
Correlation between the Elastase Activity Index and Invasiveness of Clinical Isolates ofAspergillus fumigatus
Authors: Blanco Jose L. , Hontecillas Raquel , Bouza Emilio , Blanco Isabel , Pelaez Teresa , Muñoz Patricia , Perez Molina Jose , Garcia Marta E.
Published: July 2002

We calculated an elastase activity index (EAI) by dividing the diameter of the elastin lysis halo by the fungal growth diameter. After 10 days' incubation at 37°C, all strains but one obtained from invasive aspergillosis showed an EAI ≥ 1. Of the 18 strains obtained from colonized patients, only 4 (22.2%) had an EAI ≥ 1, whereas neither of the strains isolated from patients with fungus ball reached this value. Overall, 44 out of the 142 strains obtained from the environment had an EAI ≥ 1 (30.9%).
Evidence that the 5′-End Cap Structure Is Essential for Encapsidation of Hepatitis B Virus Pregenomic RNA
Authors: Jeong Jong-Keun , Yoon Gye-Soon , Ryu Wang-Shick
Published: July 2002

Hepatitis B virus (HBV) replicates by reverse transcription of an RNA intermediate, the pregenomic RNA. The first step of HBV genome replication is the encapsidation of the pregenomic RNA encoding the encapsidation signal, termed ɛ, into the core particles, which is preceded by recognition and binding of HBV DNA polymerase to ɛ. The pregenomic RNA contains two identical ɛ elements due to its terminal redundancy: one near the 5′ end and another near the 3′ end. Despite the fact that both ɛ elements have an identical sequence, only the 5′ ɛ, but not the 3′ ɛ, is functional for encapsidation. To understand the molecular nature of this position effect, we made a series of
lacZ
RNA expression plasmids which contain the ɛ element at various positions from the 5′ end of the transcripts. Following transfection, the
lacZ
RNAs in cytoplasmic core particles were measured by RNase protection assay for encapsidation. The results indicated that the
lacZ
RNAs with ɛ positioned up to 65 nucleotides from the 5′ end were encapsidated, whereas the
lacZ
RNAs with ɛ positioned further downstream were not. Interestingly, the cap-free
lacZ
RNA transcribed by T7 RNA polymerase was not encapsidated, implying that the 5′ cap structure is required for encapsidation of the pregenomic RNA. We hypothesized that HBV DNA polymerase must somehow recognize the cap structure and/or its associated factors, as well as the 5′ ɛ, for encapsidation to occur.
Restricting Expression Prolongs Expression of Foreign Genes Introduced into Animals by Retroviruses
Authors: Pinto Valerian B. , Prasad Shiv , Yewdell Jonathan , Bennink Jack , Hughes Stephen H.
Published: July 2002

If foreign genes are ubiquitously expressed in mice using a viral vector, expression is abrogated by CD8+cells in 2 to 4 weeks. However, if the expression of the genes is confined to skeletal muscle cells, the CD8+T-cell response is much weaker and expression is maintained for more than 6 weeks. These data show that restricting the expression of foreign genes to skeletal muscle cells and presumably to other cells that are inefficient at antigen presentation can prolong the expression of a foreign gene product.
HLA Class I-Restricted Cytotoxic T-Cell Epitopes of the Respiratory Syncytial Virus Fusion Protein
Authors: Brandenburg A. H. , de Waal L. , Timmerman H. H. , Hoogerhout P. , de Swart R. L. , Osterhaus A. D. M. E.
Published: July 2002

Virus-specific cytotoxic T lymphocytes (CTL) play a major role in the clearance of respiratory syncytial virus (RSV) infection. We have generated cytotoxic T-cell clones (TCC) from two infants who had just recovered from severe RSV infection. These TCC were functionally characterized and used to identify HLA class I (B57 and C12)-restricted CTL epitopes of RSV.
Epstein-Barr Virus That Lacks Glycoprotein gN Is Impaired in Assembly and Infection
Authors: Lake Cathleen M. , Hutt-Fletcher Lindsey M.
Published: July 2002

The Epstein-Barr virus (EBV) glycoproteins N and M (gN and gM) are encoded by the BLRF1 and BBRF3 genes. To examine the function of the EBV gN-gM complex, recombinant virus was constructed in which the BLRF1 gene was interrupted with a neomycin resistance cassette. Recombinant virus lacked not only gN but also detectable gM. A significant proportion of the recombinant virus capsids remained associated with condensed chromatin in the nucleus of virus-producing cells, and cytoplasmic vesicles containing enveloped virus were scarce. Virus egress was impaired, and sedimentation analysis revealed that the majority of the virus that was released lacked a complete envelope. The small amount of virus that could bind to cells was also impaired in infectivity at a step following fusion. These data are consistent with the hypothesis that the predicted 78-amino-acid cytoplasmic tail of gM, which is highly charged and rich in prolines, interacts with the virion tegument. It is proposed that this interaction is important both for association of capsids with cell membrane to assemble and release enveloped particles and for dissociation of the capsid from the membrane of the newly infected cell on its way to the cell nucleus. The phenotype of EBV lacking the gN-gM complex is more striking than that of most alphaherpesviruses lacking the same complex but resembles in many respects the phenotype of pseudorabies virus lacking glycoproteins gM, gE, and gI. Since EBV does not encode homologs for gE and gI, this suggests that functions that may have some redundancy in alphaherpesviruses have been concentrated in fewer proteins in EBV.
Identification of Conserved Residues Contributing to the Activities of Adenovirus DNA Polymerase
Authors: Liu Huanting , Naismith James H. , Hay Ronald T.
Published: July 2002

Adenovirus codes for a DNA polymerase that is a member of the DNA polymerase α family and uses a protein primer for initiation of DNA synthesis. It contains motifs characteristic of a proofreading 3′-5′-exonuclease domain located in the N-terminal region and several polymerase motifs located in the C-terminal region. To determine the role of adenovirus DNA polymerase in DNA replication, 22 site-directed mutations were introduced into the conserved DNA polymerase motifs in the C-terminal region of adenovirus DNA polymerase and the mutant forms were expressed in insect cells using a baculovirus expression system. Each mutant enzyme was tested for DNA binding activity, the ability to interact with pTP, DNA polymerase catalytic activity, and the ability to participate in the initiation of adenovirus DNA replication. The mutant phenotypes identify functional domains within the adenovirus DNA polymerase and allow discrimination between the roles of conserved residues in the various activities carried out by the protein. Using the functional data in this study and the previously published structure of the bacteriophage RB69 DNA polymerase (J. Wang et al., Cell 89:1087–1099, 1997), it is possible to envisage how the conserved domains in the adenovirus DNA polymerase function.
Containment of Simian Immunodeficiency Virus Infection: Cellular Immune Responses and Protection from Rechallenge following Transient Postinoculation Antiretroviral Treatment
Authors: Lifson Jeffrey D. , Rossio Jeffrey L. , Arnaout Ramy , Li Li , Parks Thomas L. , Schneider Douglas K. , Kiser Rebecca F. , Coalter Vicky J. , Walsh Geneva , Imming Robert J. , Fisher Bradley , Flynn Bernard M. , Bischofberger Norbert , Piatak Michael , Hirsch Vanessa M. , Nowak Martin A. , Wodarz Dominik
Published: July 2002

To better understand the viral and host factors involved in the establishment of persistent productive infection by primate lentiviruses, we varied the time of initiation and duration of postinoculation antiretroviral treatment with tenofovir {9-[2-(R)-(phosphonomethoxy)propyl]adenine}while performing intensive virologic and immunologic monitoring in rhesus macaques, inoculated intravenously with simian immunodeficiency virus SIVsmE660. Postinoculation treatment did not block the initial infection, but we identified treatment regimens that prevented the establishment of persistent productive infection, as judged by the absence of measurable plasma viremia following drug discontinuation. While immune responses were heterogeneous, animals in which treatment resulted in prevention of persistent productive infection showed a higher frequency and higher levels of SIV-specific lymphocyte proliferative responses during the treatment period compared to control animals, despite the absence of either detectable plasma viremia or seroconversion. Animals protected from the initial establishment of persistent productive infection were also relatively or completely protected from subsequent homologous rechallenge. Even postinoculation treatment regimens that did not prevent establishment of persistent infection resulted in downmodulation of the level of plasma viremia following treatment cessation, compared to the viremia seen in untreated control animals, animals treated with regimens known to be ineffective, or the cumulative experience with the natural history of plasma viremia following infection with SIVsmE660. The results suggest that the host may be able to effectively control SIV infection if the initial exposure occurs under favorable conditions of low viral burden and in the absence of ongoing high level cytopathic infection of responding cells. These findings may be particularly important in relation to prospects for control of primate lentiviruses in the settings of both prophylactic and therapeutic vaccination for prevention of AIDS.
Kaposi's Sarcoma-Associated Herpesvirus Open Reading Frame 57 Encodes a Posttranscriptional Regulator with Multiple Distinct Activities
Authors: Kirshner Jessica R. , Lukac David M. , Chang Jean , Ganem Don
Published: July 2002

Open reading frame (ORF) 57 of Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a homolog of known posttranscriptional regulators that are essential for replication in other herpesviruses. Here, we examined the expression of this gene and the function(s) of its product. KSHV ORF 57 is expressed very early in infection from a 1.6-kb spliced RNA bearing several in-frame initiation codons. Its product is a nuclear protein that, in transient assays, has little effect on the expression of luciferase reporter genes driven by a variety of KSHV and heterologous promoters. However, ORF 57 protein enhances the accumulation of several viral transcripts, in a manner suggesting posttranscriptional regulation. These transcripts include not only known cytoplasmic mRNAs (e.g., ORF 59) but also a nuclear RNA (nut-1) that lacks coding potential. Finally, ORF 57 protein can also modulate the effects of the ORF 50 gene product, a classical transactivator known to be required for lytic induction. The expression from some (e.g., nut-1) but not all (e.g., tk) ORF 50-responsive promoters can be synergistically enhanced by coexpression of ORF 50 and ORF 57. This effect is not due to upregulation of ORF 50 expression but rather to a posttranslational enhancement of the transcriptional activity of ORF 50. These data indicate that ORF 57 is a powerful pleiotropic effector that can act on several posttranscriptional levels to modulate the expression of viral genes in infected cells.