bluuebunny/crossref_metadata_2025_split huggingface dataset:
Hydrogen-Dependent Oxygen Reduction by Homoacetogenic Bacteria Isolated from Termite Guts
Authors: Boga Hamadi I. , Brune Andreas 
Published: February 2003

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACTThe 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

ABSTRACT

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

ABSTRACT

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.