bluuebunny/crossref_metadata_2025 huggingface dataset:
Application of New Primer-Enzyme Combinations to Terminal Restriction Fragment Length Polymorphism Profiling of Bacterial Populations in Human Feces
Authors: Nagashima Koji , Hisada Takayoshi , Sato Maremi , Mochizuki Jun
Published: February 2003

New primer-enzyme combinations for terminal restriction fragment length polymorphism (T-RFLP) targeting of the 16S rRNA gene were constructed by using the T-RFLP analysis program (designated TAP T-RFLP) located at the Ribosomal Database Project website, and their performance was examined empirically. By using the fluorescently labeled 516f primer (
Escherichia coli
positions 516 to 532) and 1510r primer (positions 1510 to 1492), the 16S rRNA gene was amplified from human fecal DNA. The resulting amplified product was digested with
Rsa
I plus
Bfa
I or with
Bsl
I. When the T-RFLP was carried out with fecal DNAs from eight individuals, eight predominant operational taxonomic units (OTUs) were detected with
Rsa
I and
Bfa
I digestion and 14 predominant OTUs were detected with
Bsl
I digestion. The distribution of the OTUs was consistent with the results of the computer simulations with TAP T-RFLP. The T-RFLP analyses of the fecal DNAs from individuals gave characteristic profiles, while the variability of the T-RFLP profiles between duplicate DNA preparations from the same samples were minimal. This new T-RFLP method made it easy to predict what kind of intestinal bacterial group corresponded to each OTU on the basis of the terminal restriction fragment length compared with the conventional T-RFLP and, moreover, made it possible to identify the bacterial species that an OTU represents by cloning and sequencing.
Impaired Temperature Stress Response of a
Streptococcus thermophilus deoD
Mutant
Authors: Varcamonti Mario , Graziano Maria R. , Pezzopane Romilde , Naclerio Gino , Arsenijevic Slavica , De Felice Maurilio
Published: February 2003

An insertional
deoD
mutant of
Streptococcus thermophilus
strain SFi39 had a reduced growth rate at 20°C and an enhanced survival capacity to heat shock compared to the wild type, indicating that the
deoD
product is involved in temperature shock adaptation. We report evidence that ppGpp is implicated in this dual response.
Genetic Differences between Two Strains of
Xylella fastidiosa
Revealed by Suppression Subtractive Hybridization
Authors: Harakava Ricardo , Gabriel Dean W.
Published: February 2003

Suppression subtractive hybridization was used to rapidly identify 18 gene differences between a citrus variegated chlorosis (CVC) strain and a Pierce's disease of grape (PD) strain of
Xylella fastidiosa
. The results were validated as being highly representative of actual differences by comparison of the completely sequenced genome of a CVC strain with that of a PD strain.
Isolation and Molecular Characterization of pMG160, a Mobilizable Cryptic Plasmid from
Rhodobacter blasticus
Authors: Inui Masayuki , Nakata Kaori , Roh Jung Hyeob , Vertès Alain A. , Yukawa Hideaki
Published: February 2003

A 3.4-kb cryptic plasmid was obtained from a new isolate of
Rhodobacter blasticus
. This plasmid, designated pMG160, was mobilizable by the conjugative strain
Escherichia coli
S17.1 into
Rhodobacter sphaeroides, Rhodobacter capsulatus
, and
Rhodopseudomonas palustris
. It replicated in the latter strains but not in
Rhodospirillum rubrum, Rhodocyclus gelatinosus
, or
Bradyrhizobium
species. Plasmid pMG160 was stably maintained in
R. sphaeroides
for more than 100 generations in the absence of selection but showed segregational instability in
R. palustris
. Instability in
R. palustris
correlated with a decrease in plasmid copy number compared to the copy number in
R. sphaeroides
. The complete nucleotide sequence of plasmid pMG160 contained three open reading frames (ORFs). The deduced amino acid sequences encoded by ORF1 and ORF2 showed high degrees of homology to the MobS and MobL proteins that are involved in plasmid mobilization of certain plasmids. Based on homology with the Rep protein of several other plasmids, ORF3 encodes a putative
rep
gene initiator of plasmid replication. The functions of these sequences were demonstrated by deletion mapping, frameshift analysis, and analysis of point mutations. Two 6.1-kb pMG160-based
E. coli-R. sphaeroides
shuttle cloning vectors were constructed and designated pMG170 and pMG171. These two novel shuttle vectors were segregationally stable in
R. sphaeroides
growing under nonselective conditions.
Cooperation between
Lactococcus lactis
and Nonstarter Lactobacilli in the Formation of Cheese Aroma from Amino Acids
Authors: Kieronczyk Agnieszka , Skeie Siv , Langsrud Thor , Yvon Mireille
Published: February 2003

In Gouda and Cheddar type cheeses the amino acid conversion to aroma compounds, which is a major process for aroma formation, is essentially due to lactic acid bacteria (LAB). In order to evaluate the respective role of starter and nonstarter LAB and their interactions in cheese flavor formation, we compared the catabolism of phenylalanine, leucine, and methionine by single strains and strain mixtures of
Lactococcus lactis
subsp.
cremoris
NCDO763 and three mesophilic lactobacilli. Amino acid catabolism was studied in vitro at pH 5.5, by using radiolabeled amino acids as tracers. In the presence of α-ketoglutarate, which is essential for amino acid transamination, the lactobacillus strains degraded less amino acids than
L. lactis
subsp.
cremoris
NCDO763
,
and produced mainly nonaromatic metabolites.
L. lactis
subsp.
cremoris
NCDO763 produced mainly the carboxylic acids, which are important compounds for cheese aroma. However, in the reaction mixture containing glutamate, only two lactobacillus strains degraded amino acids significantly. This was due to their glutamate dehydrogenase (GDH) activity, which produced α-ketoglutarate from glutamate. The combination of each of the GDH-positive lactobacilli with
L. lactis
subsp.
cremoris
NCDO763 had a beneficial effect on the aroma formation. Lactobacilli initiated the conversion of amino acids by transforming them mainly to keto and hydroxy acids, which subsequently were converted to carboxylic acids by the
Lactococcus
strain. Therefore, we think that such cooperation between starter
L. lactis
and GDH-positive lactobacilli can stimulate flavor development in cheese.
Hydrogen-Dependent Oxygen Reduction by Homoacetogenic Bacteria Isolated from Termite Guts
Authors: Boga Hamadi I. , Brune Andreas
Published: February 2003

Although homoacetogenic bacteria are generally considered to be obligate anaerobes, they colonize the intestinal tracts of termites and other environments that are not entirely anoxic in space or time. In this study, we investigated how homoacetogenic bacteria isolated from the hindguts of various termites respond to the presence of molecular oxygen. All strains investigated formed growth bands in oxygen gradient agar tubes under a headspace of H
2
-CO
2
. The position of the bands coincided with the oxic-anoxic interface and depended on the O
2
partial pressure in the headspace; the position of the bands relative to the meniscus remained stable for more than 1 month. Experiments with dense cell suspensions, performed with Clark-type O
2
and H
2
electrodes, revealed a large capacity for H
2
-dependent oxygen reduction in
Sporomusa termitida
and
Sporomusa
sp. strain TmAO3 (149 and 826 nmol min
−1
mg of protein
−1
, respectively). Both strains also reduced O
2
with endogenous reductants, albeit at lower rates. Only in
Acetonema longum
did the basal rates exceed the H
2
-dependent rates considerably (181 versus 28 nmol min
−1
mg of protein)
−1
). Addition of organic substrates did not stimulate O
2
consumption in any of the strains. Nevertheless, reductive acetogenesis by cell suspensions of strain TmAO3 was inhibited even at the lowest O
2
fluxes, and growth in nonreduced medium occurred only after the bacteria had rendered the medium anoxic. Similar results were obtained with
Acetobacterium woodii
, suggesting that the results are not unique to the strains isolated from termites. We concluded that because of their tolerance to temporary exposure to O
2
at low partial pressures (up to 1.5 kPa in the case of strain TmAO3) and because of their large capacity for O
2
reduction, homoacetogens can reestablish conditions favorable for growth by actively removing oxygen from their environment.
Tin-Carbon Cleavage of Organotin Compounds by Pyoverdine from
Pseudomonas chlororaphis
Authors: Inoue Hiroyuki , Takimura Osamu , Kawaguchi Ken , Nitoda Teruhiko , Fuse Hiroyuki , Murakami Katsuji , Yamaoka Yukiho
Published: February 2003

The triphenyltin (TPT)-degrading bacterium
Pseudomonas chlororaphis
CNR15 produces extracellular yellow substances to degrade TPT. Three substances (F-I, F-IIa, and F-IIb) were purified, and their structural and catalytic properties were characterized. The primary structure of F-I was established using two-dimensional nuclear magnetic resonance techniques; the structure was identical to that of suc-pyoverdine from
P. chlororaphis
ATCC 9446, which is a peptide siderophore produced by fluorescent pseudomonads. Spectral and isoelectric-focusing analyses revealed that F-IIa and F-IIb were also pyoverdines, differing only in the acyl substituent attached to the chromophore part of F-I. Furthermore, we found that the fluorescent pseudomonads producing pyoverdines structurally different from F-I showed TPT degradation activity in the solid extracts of their culture supernatants. F-I and F-IIa degraded TPT to monophenyltin via diphenyltin (DPT) and degraded DPT and dibutyltin to monophenyltin and monobutyltin, respectively. The total amount of organotin metabolites produced by TPT degradation was nearly equivalent to that of the F-I added to the reaction mixture, whereas DPT degradation was not influenced by monophenyltin production. The TPT degradation activity of F-I was remarkably inhibited by the addition of metal ions chelated with pyoverdine. On the other hand, the activity of DPT was increased 13- and 8-fold by the addition of Cu
2+
and Sn
4+
, respectively. These results suggest that metal-chelating ligands common to pyoverdines may play important roles in the Sn-C cleavage of organotin compounds in both the metal-free and metal-complexed states.
Evaluation of Broiler Litter with Reference to the Microbial Composition as Assessed by Using 16S rRNA and Functional Gene Markers
Authors: Lu Jingrang , Sanchez Susan , Hofacre Charles , Maurer John J. , Harmon Barry G. , Lee Margie D.
Published: February 2003

Very little is known about the microbial composition of animal bedding wastes, including poultry litter, and what is known has been deduced from standard culture methods, by which some fastidious organisms that exist in the environment may not be detected. We evaluated the bacterial composition of poultry litter by using a combination of culture and molecular detection. Total aerobic bacteria in poultry litter were detected by culture at 10
9
CFU/g of material. Enteric bacteria such as
Enterococcus
spp. and coliforms composed 0.1 and 0.01%, respectively, of the total aerobic cultivatable bacteria in poultry litter; no
Salmonella
strains were detected by culture. In order to characterize the most abundant bacterial groups, we sequenced 16S ribosomal DNA (rDNA) genes amplified by PCR with microbial community DNA isolated from poultry litter as the template. From the 16S rDNA library, 31 genera were identified. Twelve families or groups were identified with lactobacilli and
Salinococcus
spp. forming the most abundant groups. In fact, 82% of the total sequences were identified as gram-positive bacteria with 62% of total belonging to low G+C gram-positive groups. In addition to detection of 16S rDNA sequences associated with the expected fecal bacteria present in manure, we detected many bacterial sequences for organisms, such as
Globicatella sulfidofaciens
,
Corynebacterium ammoniagenes
,
Corynebacterium urealyticum
,
Clostridium aminovalericum
,
Arthrobacter
sp., and
Denitrobacter permanens
, that may be involved in the degradation of wood and cycling of nitrogen and sulfur. Several sequences were identified in the library for bacteria associated with disease in humans and poultry such as clostridia, staphylococci, and
Bordetella
spp. However, specific PCR targeting other human and veterinary pathogens did not detect the presence of
Salmonella
, pathogenic
Escherichia coli
,
Campylobacter
spp.,
Yersinia
spp.,
Listeria
spp., or toxigenic staphylococci. PCR and DNA hybridization revealed the presence of class 1 integrons with gene cassettes that specify resistance to aminoglycosides and chloramphenicol. Only from understanding the microbial community of animal wastes such as poultry litter can we manage animal disease and limit the impact of animal waste on the environment and human and animal health.
Production of a Doubly Chiral Compound, (4
R
,6
R
)-4-Hydroxy-2,2,6-Trimethylcyclohexanone, by Two-Step Enzymatic Asymmetric Reduction
Authors: Wada Masaru , Yoshizumi Ayumi , Noda Yumiko , Kataoka Michihiko , Shimizu Sakayu , Takagi Hiroshi , Nakamori Shigeru
Published: February 2003

A practical enzymatic synthesis of a doubly chiral key compound, (4
R
,6
R
)-4-hydroxy-2,2,6-trimethylcyclohexanone, starting from the readily available 2,6,6-trimethyl-2-cyclohexen-1,4-dione is described. Chirality is first introduced at the C-6 position by a stereoselective enzymatic hydrogenation of the double bond using old yellow enzyme 2 of
Saccharomyces cerevisiae
, expressed in
Escherichia coli
, as a biocatalyst. Thereafter, the carbonyl group at the C-4 position is reduced selectively and stereospecifically by levodione reductase of
Corynebacterium aquaticum
M-13, expressed in
E. coli
, to the corresponding alcohol. Commercially available glucose dehydrogenase was also used for cofactor regeneration in both steps. Using this two-step enzymatic asymmetric reduction system, 9.5 mg of (4
R
,6
R
)-4-hydroxy-2,2,6-trimethylcyclohexanone/ml was produced almost stoichiometrically, with 94% enantiomeric excess in the presence of glucose, NAD
+
, and glucose dehydrogenase. To our knowledge, this is the first report of the application of
S. cerevisiae
old yellow enzyme for the production of a useful compound.
Identification of a Specific Antigenic Region of the P82 Protein of
Babesia equi
and Its Potential Use in Serodiagnosis
Authors: Hirata Haruyuki , Xuan Xuenan , Yokoyama Naoaki , Nishikawa Yoshifumi , Fujisaki Kozo , Suzuki Naoyoshi , Igarashi Ikuo
Published: February 2003

The efficacy of the Be82 gene product fused with glutathione
S
-transferase (GST/Be82) in an enzyme-linked immunosorbent assay (ELISA) for the diagnosis of
Babesia equi
infection was reported previously (H. Hirata et al., J. Clin. Microbiol. 40:1470-1474, 2002). However, the ELISA with the GST/Be82 antigen cross-reacted with
Babesia caballi
-infected horse sera, despite the high rate of detection of
B. equi
. These results suggested that GST/Be82 has an antigen in common with
B. caballi
or antigenicity similar to that of
B. caballi
. In the present study, we constructed a series of five clones with deletions in the Be82 gene product, each of which was fused with GST, and used them in ELISAs in order to overcome the cross-reactivity seen with
B. caballi
. One of the deletion clones, a clone with a deletion of the Be82 gene from positions 236 to 381 (Be82/236-381), specifically and sensitively detected
B. equi
-infected horse sera without cross-reactivity with
B. caballi
-infected horse sera. Assays with clones from which other gene products were deleted showed decreased sensitivities or remained nonspecific for the detection of
B. equi
-infected horse sera. These results suggest that the Be82/236-381 gene product is a novel antigen for the diagnosis of
B. equi
infection in horses.
Highly Polymorphic Microsatellite for Identification of
Candida albicans
Strains
Authors: Sampaio Paula , Gusmão Leonor , Alves Cíntia , Pina-Vaz Cidália , Amorim António , Pais Célia
Published: February 2003

The polymorphism of a new microsatellite locus (CAI) was investigated in a total of 114
Candida albicans
strains, including 73 independent clinical isolates, multiple isolates from the same patient, isolates from several episodes of recurrent vulvovaginal infections, and two reference strains. PCR genotyping was performed automatically, using a fluorescence-labeled primer, and in the 73 independent isolates, 26 alleles and 44 different genotypes were identified, resulting in a discriminatory power of 0.97. CAI was revealed to be species specific and showed a low mutation rate, since no amplification product was obtained when testing other pathogenic
Candida
species and no genotype differences were observed when testing over 300 generations. When applying this microsatellite to the identification of strains isolated from recurrent vulvovaginal infections in eight patients, it was found that 13 out of 15 episodes were due to the same strain. When multiple isolates, obtained from the same patient and plated simultaneously, were typed for CAI, the same genotype was found in each case, confirming that the infecting population was clonal. Moreover, the same genotype appeared in isolates from the rectum and the vagina, revealing that the former could be a reservoir of potentially pathogenic strains. This new microsatellite proves to be a valuable tool to differentiate
C. albicans
strains. Furthermore, when compared to other molecular genotyping techniques, CAI proved to be very simple, highly efficient, and reproducible, being suitable for low-quantity and very-degraded samples and for application in large-scale epidemiological studies.
Multisite Reproducibility of Results Obtained by Two Broth Dilution Methods for Susceptibility Testing of
Mycobacterium avium
Complex
Authors: Woods Gail L. , Williams-Bouyer Natalie , Wallace, Richard J. , Brown-Elliott Barbara A. , Witebsky Frank G. , Conville Patricia S. , Plaunt Marianne , Hall Geraldine , Aralar Priscilla , Inderlied Clark
Published: February 2003

A multicenter study was conducted to assess the interlaboratory reproducibility of susceptibility testing of
Mycobacterium avium
complex (MAC) by broth microdilution using two different media (cation-adjusted Mueller-Hinton broth with 5% oleic acid-albumin-dextrose-catalase and 7H9 broth with casein) and by macrodilution using the BACTEC 460TB and 12B media at pH 6.8 and 7.3 to 7.4. Ten well-characterized strains of MAC (four macrolide susceptible, six macrolide resistant) were tested against clarithromycin and azithromycin (the latter only by BACTEC 460TB, pH 6.8). At each site, strains were tested against clarithromycin three times on each of three separate days (nine testing events per isolate) by using a common lot of microdilution trays and BACTEC 12B medium, pH 6.8; strains were tested once on three separate days against clarithromycin in 12B medium at pH 7.3 to 7.4 and against azithromycin. Agreement among MICs (i.e., mode ± 1 twofold dilution) was 100% for all strains and both drugs when BACTEC 460TB was used, regardless of the pH of the medium, but varied when microdilution with either medium was used, particularly with susceptible strains. Agreement based on interpretive category, with NCCLS-recommended breakpoints, was 100% for all strains with the BACTEC 460TB method (both drugs and both pH values) and with microdilution using 7H9 broth. With microdilution and Mueller-Hinton broth, agreement by interpretive category was 100% for eight isolates and >90% for two; errors occurred only in laboratories where personnel had minimal experience with this technique. MAC susceptibility testing may be performed by broth macrodilution or microdilution at either pH, with NCCLS-recommended interpretive breakpoints. However, because visual interpretation of broth microdilution end points is subjective, it is more prone to reader error; therefore, this method requires greater expertise than the BACTEC 460TB. Both techniques require appropriate validation and continued documentation of proficiency.
Mixed-Genome Microarrays Reveal Multiple Serotype and Lineage-Specific Differences among Strains of
Listeria monocytogenes
Authors: Call Douglas R. , Borucki Monica K. , Besser Thomas E.
Published: February 2003

Epidemiological studies and analysis of putative virulence genes have shown that
Listeria monocytogenes
has diverged into several phylogenetic divisions. We hypothesize that similar divergence has occurred for many genes that influence niche-specific fitness and virulence and that identifying these differences may offer new opportunities for the detection, treatment, and control of this important pathogen. To explore this issue further, we developed a microarray composed of fragmented DNA taken from 10 strains of
L. monocytogenes
. We then hybridized genomic DNA from 50 different strains to replicate arrays and analyzed the resulting hybridization patterns. A simple Euclidean distance metric permitted the reconstruction of previously described genetic relationships between serotypes, and only four microarray probes were needed to discriminate between the most important serotypes (1/2a, 1/2b, 1/2c, and 4). We calculated an index of linkage equilibrium from the microarray data and confirmed that
L. monocytogenes
has a strongly clonal population structure (

I
A

= 3.85). Twenty-nine informative probes were retrieved from the library and sequenced. These included genes associated with repairing UV-damaged DNA, salt tolerance, biofilm formation, heavy metal transport, ferrous iron transport, and teichoic acid synthesis. Several membrane-bound lipoproteins and one internalin were identified, plus three phage sequences and six sequences with unknown function. Collectively, these data confirm that many genes have diverged between lineages of
L. monocytogenes
. Furthermore, these results demonstrate the value of mixed-genome microarrays as a tool for deriving biologically useful information and for identifying and screening genetic markers for clinically important microbes.
Bartonella
Strains from Ground Squirrels Are Identical to
Bartonella washoensis
Isolated from a Human Patient
Authors: Kosoy Michael , Murray Mike , Gilmore, Robert D. , Bai Ying , Gage Kenneth L.
Published: February 2003

The most likely animal source of a human case of cardiac disease in Washoe County, Nev., was identified by comparison of DNA sequences of three genes (citrate synthase
gltA
, 60-kDa heat shock protein gene
groEL
, and 16S rRNA gene) of
Bartonella washoensis
cultured from the human patient in question and of
Bartonella
isolates obtained from the following Nevada rodents:
Peromyscus maniculatus
(17 isolates),
Tamias minimus
(11 isolates),
Spermophilus lateralis
(3 isolates), and
Spermophilus beecheyi
(7 isolates). Sequence analyses of
gltA
amplicons obtained from
Bartonella
from the rodents demonstrated considerable heterogeneity and resulted in the identification of 16 genetic variants that were clustered within three groups in phylogenetic analysis. Each of the three groups was associated with a rodent genus,
Peromyscus
,
Tamias,
or
Spermophilus.
The
gltA
, 16S rRNA gene, and
groEL
sequences of a
Bartonella
isolate obtained from a California ground squirrel (
S. beecheyi
) were completely identical to homologous sequences of
B. washoensis
, strongly suggesting that these animals were the source of infection in the human case.
Identification and Characterization of Putative Secreted Antigens fromBabesia microti
Authors: Homer Mary J. , Lodes Michael J. , Reynolds Lisa D. , Zhang Yanni , Douglass John F. , McNeill Patricia D. , Houghton Raymond L. , Persing David H.
Published: February 2003

The need for improved diagnostic reagents to identify human long-term carriers of the zoonotic parasiteBabesia microtiis evidenced by numerous reported cases of transfusion-acquired infections. This report describes the identification and initial characterization of 27 clones representing seven genes or gene families that were isolated through serological expression cloning by using a technique that we specifically designed to screen for shed antigens. In this screen, sera fromB. microti-infected SCID mice, putatively containing secreted or shed antigens from the parasites, were harvested and used to immunize syngeneic immunocompetent mice (BALB/c). After boosting, the sera from the BALB/c mice, containing antibodies against the immunodominant secreted antigens, were used to screen aB. microtigenomic expression library. Analyses of the putative peptides encoded by the novel DNA sequences revealed characteristics indicating that these peptides might be secreted. Initial serological data obtained with recombinant proteins and a patient serum panel demonstrated that several of the proteins could be useful in developing diagnostic tests for detection ofB. microtiantibodies and antigens in serum.
Rapid Detection of
Clostridium difficile
in Feces by Real-Time PCR
Authors: Bélanger Simon D. , Boissinot Maurice , Clairoux Natalie , Picard François. J. , Bergeron Michel G.
Published: February 2003

Clostridium difficile
is the major causative agent of nosocomial antibiotic-associated diarrhea, colitis, and pseudomembranous colitis. The pathogenicity of
C. difficile
is closely related to the production of toxins A and B. Toxigenic
C. difficile
detection by a tissue culture cytotoxin assay is often considered the “gold standard.” However, this assay is time consuming, as it implies an incubation period of at least 24 h. We have developed a rapid real-time fluorescence-based multiplex PCR assay targeting the
C. difficile
toxin genes
tcdA
and
tcdB
, with the Smart Cycler. Two molecular beacons bearing different fluorophores were used as internal probes specific for each amplicon type. The analytical sensitivity of the assay was around 10 genome copies for all nine
C. difficile
strains tested, representing the 6 most common toxinotypes. The specificity was demonstrated by the absence of amplification with DNA purified from bacterial species other than
C. difficile
(
n
= 14), including
Clostridium sordellii
for which the lethal toxin gene sequence is closely related to the toxin genes of
C. difficile
. Following a rapid (15 min) and simple fecal sample preparation protocol, both
tcdA
and
tcdB
were efficiently amplified from 28 of 29 cytotoxin-positive feces samples. There was no amplification observed with all 27 cytotoxin-negative feces samples tested. This is the first real-time PCR assay for the detection of
C. difficile
. It is rapid, sensitive, and specific and allows detection of
C. difficile
directly from feces samples.
Molecular Diagnosis of Infective Endocarditis by PCR Amplification and Direct Sequencing of DNA from Valve Tissue
Authors: Gauduchon Valérie , Chalabreysse Lara , Etienne Jerome , Célard Marie , Benito Yvonne , Lepidi Hubert , Thivolet-Béjui Françoise , Vandenesch François
Published: February 2003

We used broad-range eubacterial PCR amplification followed by direct sequencing to identify microbial pathogens in heart valve material from 29 patients with histologically confirmed infective endocarditis and 23 patients free of infective endocarditis. Microorganisms cultured by conventional techniques matched those identified by PCR in 21 cases. PCR alone identified the causative agent in three cases (
Streptococcus bovis
,
Staphylococcus cohnii
, and
Coxiella burnetii
), allowing better patient management. PCR corrected the initial bacteriological diagnosis in three cases (
Streptococcus bovis
,
Streptococcus mutans
, and
Bartonella henselae
). Among the 29 cases of histologically confirmed infective endocarditis, PCR findings were positive in 27 cases and were consistent with the bacterial morphology seen at Gram staining (26 cases) or with the results obtained by immunohistologic analysis with an anti-
C. burnetii
monoclonal antibody (one case). In two other cases of histologically confirmed infective endocarditis, PCR remained negative in a blood culture-negative case for which no bacteria were seen at histological analysis and in one case with visualization of cocci and blood cultures positive for
Enterococcus faecalis
. Ten clinical diagnoses of possible infective endocarditis were ruled out by histopathological analysis of the valves and subsequently by PCR. PCR was negative in 13 of the 14 patients in whom infective endocarditis was rejected on clinical grounds; the other patient was found to have
Coxiella burnetii
infective endocarditis on the basis of PCR and histopathological analysis and was subsequently included in the group of 29 definite cases. In total, PCR contributed to the diagnosis and management of infective endocarditis in 6 of 29 (20%) cases.
Methods for Identification ofStaphylococcus aureusIsolates in Cases of Bovine Mastitis
Authors: Boerlin Patrick , Kuhnert Peter , Hüssy Daniela , Schaellibaum Melchior
Published: February 2003

A total of 272 staphylococcal isolates from cases of bovine mastitis (159Staphylococcus aureus) belonging to 12 different species were identified with ID32 STAPH galleries, and 51 of them were confirmed by 16S rRNA gene (rrs) sequencing. The same isolates were examined for their hemolytic activity on sheep blood agar, DNase activity, and coagulase activity and with two rapid identification kits (Slidex Staph Plus kit and RAPIDEC Staph from Bio-Merieux). The results of this study confirm those obtained by other groups with hemolysis, DNase, and coagulase. Only 50% ofS. aureusisolates from mastitis cases show coagulase activity after 4 h of incubation, and a 24-h incubation is necessary for the full sensitivity of this test. In contrast to results from other studies with human isolates, the Slidex Staph Plus kit was not sensitive enough for the identification ofS. aureusfrom bovine mastitis samples. The aurease test of the RAPIDEC Staph kit showed 100% sensitivity and 100% specificity. Used in conjunction with hemolysis patterns, the RAPIDEC Staph kit is therefore very well adapted to rapid, efficient, and cost-effective identification ofS. aureusin cultures from bovine mastitis samples. Sequencing ofrrsgenes also proved very efficient in identifying theStaphylococcusspecies encountered in these samples and confirming phenotypical identification results with unsatisfactory scores. With continuously improving technologies and decreasing costs, genetic identification methods likerrsgene sequencing will soon find a place in routine veterinary diagnostics.
Multicenter Quality Control for the Detection of Hepatitis C Virus RNA in Seminal Plasma Specimens
Authors: Bourlet Thomas , Levy Rachel , Laporte Silvy , Blachier Stéphane , Bocket Laurence , Cassuto Guy , Chollet Lionel , Leruez-Ville Marianne , Maertens Anne , Mousnier Fabienne , Pasquier Christophe , Payan Christopher , Pellegrin Bertrand , Schvoerer Evelyne , Zavadzki Patricia , Chouteau Jacques , Duverlie Gilles , Izopet Jacques , Lunel-Fabiani Françoise , Pawlotsky Jean-Michel , Profizi Nerina , Rouzioux Christine , Stoll-Keller Françoise , Thibault Vincent , Wattré Pierre , Pozzetto Bruno
Published: February 2003

The discrepant results available in the literature about the presence of hepatitis C virus (HCV) RNA in seminal plasma of men chronically infected by this agent are related, at least in part, to the molecular techniques used and particularly to the wide range of protocols dedicated to RNA extraction. In order to evaluate these protocols and to standardize the method of detection of HCV RNA in this fluid, a panel of coded specimens was tested blindly in 12 French laboratories; it included 14 seminal plasma specimens and four water controls spiked with HCV RNA ranging from 10 to 20,000 IU/ml and two HCV-negative seminal plasma specimens. The extraction step was performed according to methods using either silica beads (NucliSens [Organon Teknika S.A., Fresnes, France]; RNA viral kit [Qiagen, Courtaboeuf, France]) or guanidinium thiocyanate (Amplicor HCV assay; Roche Diagnostics, Meylan, France), preceded or not by a centrifugation of the seminal plasma. For the amplification step, all the laboratories performed the same reverse transcription-PCR technique (Amplicor HCV Cobas assay). The percentage of correct results ranged from 53.3 to 100, the poorest results being obtained when no centrifugation step preceded the Amplicor extraction protocol. The rate of correct results was significantly higher in laboratories using a preliminary centrifugation of the specimen (
P
= 0.034 by chi-square test). By contrast, the overall number of correct results was not correlated to the initial volume of sample used for the test. These results allowed us to validate standardized techniques adapted to the performance of this test on a routine basis, especially in men infected with HCV and involved in programs of medically assisted reproduction.
Evaluation of Four Commercially Available Rapid Serologic Tests for Diagnosis of Leptospirosis
Authors: Bajani Mary D. , Ashford David A. , Bragg Sandra L. , Woods Christopher W. , Aye Tin , Spiegel Richard A. , Plikaytis Brian D. , Perkins Bradley A. , Phelan Maureen , Levett Paul N. , Weyant Robbin S.
Published: February 2003

Four rapid tests for the serologic diagnosis of leptospirosis were evaluated, and the performance of each was compared with that of the current standard, the microscopic agglutination test (MAT). The four rapid tests were a microplate immunoglobulin M (IgM)-enzyme-linked immunosorbent assay (ELISA), an indirect hemagglutination assay (IHA), an IgM dipstick assay (LDS), and an IgM dot-ELISA dipstick test (DST). A panel of 276 sera from 133 cases of leptospirosis from four different geographic locations was tested as well as 642 sera from normal individuals or individuals with other infectious or autoimmune diseases. Acute-phase sera from cases (
n
= 148) were collected ≤14 days (median = 6.0) after the onset of symptoms, and convalescent-phase sera (
n
= 128) were collected ≥15 days after onset (median = 29.1). By a traditional method (two-by-two contingency table), the sensitivities for detection of leptospirosis cases were 93.2% by LDS, 92.5% by DST, 86.5% by ELISA, and 79.0% by IHA. Specificity was 98.8% by DST, 97% by ELISA and MAT, 95.8% by IHA, and 89.6% by LDS. With a latent class analysis (LCA) model that included all the rapid tests and the clinical case definition, sensitivity was 95.5% by DST, 94.5% by LDS, 89.9% by ELISA, and 81.1% by IHA. The sensitivity and specificity estimated by the traditional methods were quite close to the LCA estimates. However, LCA allowed estimation of the sensitivity of the MAT (98.2%), which traditional methods do not allow. For acute-phase sera, sensitivity was 52.7% by LDS, 50.0% by DST, 48.7% by MAT and ELISA, and 38.5% by IHA. The sensitivity for convalescent-phase sera was 93.8% by MAT, 84.4% by DST, 83.6% by LDS, 75.0% by ELISA, and 67.2% by IHA. A good overall correlation with the MAT was obtained for each of the assays, with the highest concordance being with the DST (kappa value, 0.85; 95% confidence interval [CI], 0.8 to 0.90). The best correlation was between ELISA and DST (kappa value, 0.86; 95% CI, 0.81 to 0.91). False-positive LDS results were frequent (≥20%) in sera from individuals with Epstein-Barr virus, human immunodeficiency virus, and periodontal disease and from healthy volunteers. The ease of use and significantly high sensitivity and specificity of DST and ELISA make these good choices for diagnostic testing.
Optimum Detection Times for Bacteria and Yeast Species with the BACTEC 9120 Aerobic Blood Culture System: Evaluation for a 5-Year Period in a Turkish University Hospital
Authors: Durmaz Gül , Us Tercan , Aydinli Aydin , Kiremitci Abdurrahman , Kiraz Nuri , Akgün Yurdanur
Published: February 2003

We tracked and documented the time of positivity of blood cultures by using the BACTEC 9120 (Becton Dickinson Diagnostic Instrument Systems) blood culture system over a 5-year study period. A 7-day protocol of the incubation period was selected, and a total of 11,156 blood cultures were evaluated. The clinically significant microorganisms (32.95%) were isolated in 3,676 specimens. Gram-positive and -negative bacterial isolation rates were found to be 41.07 and 44.88%, respectively. Yeasts were found in 14.03% of all pathogens. Both the false-positivity and -negativity rates were very low (0.1 and 0.3%, respectively). The mean detection times for all of the pathogens were determined to be 19.45 h. Yeasts, nonfermentative gram-negative bacteria, and
Brucella melitensis
strains were isolated within 5 days. By taking these data into account, we decided to establish a 5-day-incubation protocol in our laboratory instead of the 7 days that are commonly used.
Identification of Dermatophyte Species by 28S Ribosomal DNA Sequencing with a Commercial Kit
Authors: Ninet Béatrice , Jan Isabelle , Bontems Olympia , Léchenne Barbara , Jousson Olivier , Panizzon Renato , Lew Daniel , Monod Michel
Published: February 2003

We have shown that dermatophyte species can be easily identified on the basis of a DNA sequence encoding a part of the large-subunit (LSU) rRNA (28S rRNA) by using the MicroSeq D2 LSU rRNA Fungal Sequencing Kit. Two taxa causing distinct dermatophytoses were clearly distinguished among isolates of the
Trichophyton mentagrophytes
species complex.
Genetic Analysis of Crimean-Congo Hemorrhagic Fever Virus in Russia
Authors: Yashina Lyudmila , Vyshemirskii Oleg , Seregin Sergei , Petrova Irina , Samokhvalov Evgeny , Lvov Dmitry , Gutorov Valery , Kuzina Irina , Tyunnikov Georgy , Tang Yi-Wei , Netesov Sergei , Petrov Vladimir
Published: February 2003

Genetic analysis of wild-type Crimean-Congo hemorrhagic fever (CCHF) virus strains recovered in the European part of Russia was performed. Reverse transcriptase PCR followed by direct sequencing was used to recover partial sequences of the CCHF virus medium (M) genome segment (M segment) from four pools of
Hyalomma marginatum
ticks and six human patients. Phylogenetic analysis of the M-segment sequences from Russian strains revealed a close relatedness of the strains (nucleotide sequence diversity, ≤5.0%). The strains differed significantly from CCHF viruses from other regions of the world (nucleotide sequence diversity, 10.3 to 20.4%), suggesting that CCHF virus strains recovered in the European part of Russia form a distinct group.
Comparative Analysis of Multidrug-Resistant, Non-Multidrug-Resistant, and Archaic Methicillin-Resistant
Staphylococcus aureus
Isolates from Central Sydney, Australia
Authors: Watson Jason , Givney Rod , Beard-Pegler Mary , Rose Barbara , Merlino John , Vickery Alison , Gottlieb Tom , Bradbury Ross , Harbour Colin
Published: February 2003

In this study, the phenotypic and genotypic characteristics of 50 methicillin-resistant
Staphylococcus aureus
(MRSA) isolates (43 contemporary and 7 archaic strains from the mid-1960s) from four Sydney hospitals in the central Sydney area were compared. Phenotypic analysis based on antibiotic profiles and phage typing patterns categorized the MRSA isolates into three major groups: multidrug resistant (mMRSA), non-multidrug resistant (nmMRSA), and archaic. The nmMRSA isolates could be further subdivided into nmMRSA group 1, which was phage typeable and similar to the archaic group; nmMRSA group 2, which was non-phage typeable and only resistant to ciprofloxacin; and nmMRSA group 3, which was also nontypeable and generally resistant to other antibiotics. The characterization of all five phenotypic groups was then extended by genetic analysis. Restriction fragment length polymorphism (RFLP) analysis showed the 50 isolates could be sorted into 20 group-specific pulsotypes.
mecI
gene deletions and mutations at various percentages among the five MRSA groups were detected by sequencing. Several
mec
promoter mutations were also found. The overall findings indicated that nmMRSA strains may have independently acquired
mec
DNA and are more likely to be newly emergent strains than nmMRSA variants.
Transcriptional Analysis of
p30
Major Outer Membrane Protein Genes of
Ehrlichia canis
in Naturally Infected Ticks and Sequence Analysis of
p30-10
of
E. canis
from Diverse Geographic Regions
Authors: Felek Suleyman , Greene Russell , Rikihisa Yasuko
Published: February 2003

Rhipicephalus sanguineus
ticks transmit
Ehrlichia canis
, the etiologic agent of canine ehrlichiosis. In experimentally infected ticks, only
p30-10
transcript was detected among 22
p30
paralogs encoding immunodominant major outer membrane P30 proteins of
E. canis
. The present study revealed transcription of
p30-10
by
E. canis
in naturally infected ticks and sequence conservation of
p30-10
genes for
E. canis
from diverse geographic regions.
Consent and confidentiality in genetics: whose information is it anyway?
Authors: Kent A
Published: February 2003

Against a background of increasing regulation regarding access to medical information and the presentation of patients' confidentiality, the case of genetic information raises interesting questions about whether the application of general rules is appropriate in all situations. Whilst all genetic information is not equally sensitive, some of it is highly predictive. It also allows deductions to be made about other family members. It may not be regarded as particularly sensitive when compared to other types of medical information and those to whom it applies may not be as anxious about preserving their confidentiality as compared with—for example, the prospect of seeing research into cause and cures for rare diseases put in hand. These distinctions also find resonance with the general public. Resolving conflicting tensions will require subtlety, not a blunt “one size fits all” model.
Research on the mentally incompetent
Authors: Cuénod M , Gasser J
Published: February 2003

The specific problems of consent for the mentally incompetent are reviewed. Scientific research is essential to test the validity of present treatments and to develop new ones. The respective roles of the physician and the researcher have to be clearly defined. The vulnerability of psychiatric patients has to be taken into consideration in such a way that some research can be conducted. It is emphasised that the ethical restrictions for research, although highly justified and necessary, are in part responsible for the relatively slow progress in the application of modern neurosciences to psychiatric diseases.
Misled and confused? Telling the public about MMR vaccine safety: Table 1
Authors: Clements C J , Ratzan S
Published: February 2003

The extraordinary events surrounding the measles, mumps, and rubella (MMR) vaccine in the United Kingdom have not only placed in jeopardy the use of this triple vaccine but have also spread concern to other parts of the world. Examination of the public’s worry about MMR vaccine reveals they have been exposed to a range of conflicting views resulting in the feeling of having been misled about the safety of the vaccine. There are various groups and individuals who have legitimate roles in informing the public about such subjects. But is each one behaving in an ethically responsible way? And if confidence falters, vaccine coverage dips, and an outbreak of measles, mumps, or rubella ensues, who, if anyone, will stand and say “I misled them, I confused them, this is my responsibility”? We examine the ethical issues of each group with a voice in the debate about vaccine safety.
Organ retention and return: problems of consent
Authors: Brazier M
Published: February 2003

This paper explores difficulties around consent in the context of organ retention and return. It addresses the proposals of the Independent Review Group in Scotland on theRetention of Organs at Post Mortemto speak ofauthorisationrather than consent. Practical problems about whose consent determines disputes in relation to organ retention are explored. If a young child dies and his mother refuses consent but his father agrees what should ensue? Should the expressed wishes of a deceased adult override the objections of surviving relatives? The paper suggests much broader understanding of the issues embedded in organ retention is needed to provide solutions which truly meet families' and society's needs.
Confidentiality and the duties of care: Table 1
Authors: O’Brien J , Chantler C
Published: February 2003

Doctors have an ethical and legal duty to respect patient confidentiality. We consider the basis for this duty, looking particularly at the meaning and value of autonomy in health care. Enabling patients to decide how information about them is disclosed is an important element in autonomy and helps patients engage as active partners in their care.

Good quality data is, however, essential for research, education, public health monitoring, and for many other activities essential to provision of health care. We discuss whether it is necessary to choose between individual rights and the wider public interest and conclude that this should only rarely be necessary. The paper makes some recommendations on practical steps which could help ensure that good quality information is available for work which benefits society and the public health, while still enabling patients’ autonomy to be respected.
Psychiatric research: what ethical concerns do LRECs encounter? A postal survey: Table 1
Authors: Osborn D P J , Fulford K W M
Published: February 2003

Background and methods:Psychiatric research can occasionally present particular ethical dilemmas, but it is not clear what kind of problems local research ethics committees (LRECs) actually experience in this field. We aimed to assess the type of problems that committees encounter with psychiatric research, using a postal survey of 211 LRECs.

Results:One hundred and seven (51%) of those written to replied within the time limit. Twenty eight (26%) experienced few problems with psychiatric applications. Twenty six (24%) emphasised the value of a psychiatric expert on the committee. The most common issues raised were informed consent (n=64, 60%) and confidentiality (n=17, 16%). The use of placebos (and washout periods) (n=18, 17%), the validity of psychiatric questionnaires (n=16, 15%) and overuse of psychiatric “jargon” (n=14, 13%) in psychiatric applications also raised concern.

Conclusions:Our results suggest that LRECs have specific concerns regarding methodology, consent, and confidentiality in psychiatric research, and that they find psychiatric input invaluable.
A simple field method for manipulating ultraviolet reflectance of flowers
Authors: Johnson Steven D , Andersson Staffan
Published: February 2003

It has been difficult to manipulate the ultraviolet (UV) reflectance of flowers independently of other wavelengths to study the response of insect pollinators to this trait. One effective solution is to paint flower corollas with human sunscreen that absorbs UV wavelengths. Honeybees (Apis mellifera scutellata) foraging on the strongly UV-reflecting flowers of Hypoxis hemerocallidea Fisch. & C.A. Mey. rejected flowers that had UV reflectance eliminated by a sunscreen coating, but continued to visit control flowers painted with sunscreen solution that did not contain the UV absorbing compound. The sunscreen technique could be useful for determining the response of a wide range of pollinators to the UV component of spectral reflectance in flowers and could be used to test the functional significance of UV-contrasting "nectar guide" patterns.Key words: Apis mellifera scutellata, choice experiment, flower colour, Hypoxis hemerocallidea, pollination, foraging behaviour.
A new phenolic metabolite, involutone, isolated from the mushroom Paxillus involutus
Authors: Antkowiak Róza , Antkowiak Wieslaw Z , Banczyk Izabela , Mikolajczyk Lucyna
Published: February 2003

A new optically active metabolite, involutone, was found in methanol, ethanol, or n-butanol extracts of freshly collected Paxillus involutus. The structure of this compound was proved to be 5-(3,4-dihydroxyphenyl)-2-(4-hydroxyphenyl)-2-hydroxy-4-cyclopenten-1,3-dione on the basis of the spectral and chemical properties of involutone and its tetraacetyl derivative. In addition, a number of compounds of known structures such as linoleic and crotonic acids, mannitol, ergosterol, involutin, as well as methyl, ethyl, or n-butyl-β-D-glucopyranosides, and methyl, ethyl, or n-butyl linoleates, depending in both cases on the alcohol used in the extraction, were also isolated in a pure state from the mushroom.Key words: Paxillus involutus, Brown Roll-rim, metabolite isolation, structure study, quaternary hydroxycyclopentanedione, NMR of mushroom pigments, glucosides and linoleates, enzymic glucosylation and esterification.