bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Elevated Atmospheric CO
2
on Soil Microbial Biomass, Activity, and Diversity in a Chaparral Ecosystem
Authors: Lipson David A. , Wilson Richard F. , Oechel Walter C.
Published: December 2005

This study reports the effects of long-term elevated atmospheric CO
2
on root production and microbial activity, biomass, and diversity in a chaparral ecosystem in southern California. The free air CO
2
enrichment (FACE) ring was located in a stand dominated by the woody shrub
Adenostoma fasciculatum
. Between 1995 and 2003, the FACE ring maintained an average daytime atmospheric CO
2
concentration of 550 ppm. During the last two years of operation, observations were made on soil cores collected from the FACE ring and adjacent areas of chaparral with ambient CO
2
levels. Root biomass roughly doubled in the FACE plot. Microbial biomass and activity were related to soil organic matter (OM) content, and so analysis of covariance was used to detect CO
2
effects while controlling for variation across the landscape. Extracellular enzymatic activity (cellulase and amylase) and microbial biomass C (chloroform fumigation-extraction) increased more rapidly with OM in the FACE plot than in controls, but glucose substrate-induced respiration (SIR) rates did not. The metabolic quotient (field respiration over potential respiration) was significantly higher in FACE samples, possibly indicating that microbial respiration was less C limited under high CO
2
. The treatments also differed in the ratio of SIR to microbial biomass C, indicating a metabolic difference between the microbial communities. Bacterial diversity, described by 16S rRNA clone libraries, was unaffected by the CO
2
treatment, but fungal biomass was stimulated. Furthermore, fungal biomass was correlated with cellulase and amylase activities, indicating that fungi were responsible for the stimulation of enzymatic activity in the FACE treatment.
Amplified Expression of Fructose 1,6-Bisphosphatase in
Corynebacterium glutamicum
Increases In Vivo Flux through the Pentose Phosphate Pathway and Lysine Production on Different Carbon Sources
Authors: Becker Judith , Klopprogge Corinna , Zelder Oskar , Heinzle Elmar , Wittmann Christoph
Published: December 2005

The overexpression of fructose 1,6-bisphosphatase (FBPase) in
Corynebacterium glutamicum
leads to significant improvement of lysine production on different sugars. Amplified expression of FBPase via the promoter of the gene encoding elongation factor TU (EFTU) increased the lysine yield in the feedback-deregulated lysine-producing strain
C. glutamicum
lysC
fbr
by 40% on glucose and 30% on fructose or sucrose. Additionally formation of the by-products glycerol and dihydroxyacetone was significantly reduced in the P
EFTU
fbp mutant. As revealed by
13
C metabolic flux analysis on glucose the overexpression of FBPase causes a redirection of carbon flux from glycolysis toward the pentose phosphate pathway (PPP) and thus leads to increased NADPH supply. Normalized to an uptake flux of glucose of 100%, the relative flux into the PPP was 56% for
C. glutamicum
lysC
fbr
P
EFTU
fbp and 46% for
C. glutamicum
lysC
fb

r

. The flux for NADPH supply was 180% in the P
EFTU
fbp strain and only 146% in the parent strain. Amplification of FBPase increases the production of lysine via an increased supply of NADPH. Comparative studies with another mutant containing the
sod
promoter upstream of the
fbp
gene indicate that the expression level of FBPase relates to the extent of the metabolic effects. The overexpression of FBPase seems useful for starch- and molasses-based industrial lysine production with
C. glutamicum
. The redirection of flux toward the PPP should also be interesting for the production of other NADPH-demanding compounds as well as for products directly stemming from the PPP.
Proteome Analysis of
Streptococcus thermophilus
Grown in Milk Reveals Pyruvate Formate-Lyase as the Major Upregulated Protein
Authors: Derzelle Sylviane , Bolotin Alexander , Mistou Michel-Yves , Rul Françoise
Published: December 2005

We investigated the adaptation to milk of
Streptococcus thermophilus
LMG18311 using a proteomic approach. Two-dimensional electrophoresis of cytosolic proteins were performed after growth in M17 medium or in milk. A major modification of the proteome concerned proteins involved in the supply of amino acids, like the peptidase PepX, and several enzymes involved in amino acid biosynthesis. In parallel, we observed the upregulation of the synthesis of seven enzymes directly involved in the synthesis of purines, as well as formyl-tetrahydrofolate (THF) synthetase and serine hydroxy-methyl transferase, two enzymes responsible for the synthesis of compounds (THF and glycine, respectively) feeding the purine biosynthetic pathway. The analysis also revealed a massive increase in the synthesis of pyruvate formate-lyase (PFL), the enzyme which converts pyruvate into acetyl coenzyme A and formate. PFL has been essentially studied for its role in mixed-acid product formation in lactic acid bacteria during anaerobic fermentation. However, formate is an important methyl group donor for anabolic pathway through the formation of folate derivates. We hypothesized that PFL was involved in purine biosynthesis during growth in milk. We showed that PFL expression was regulated at the transcriptional level and that
pfl
transcription occurred during the exponential growth phase in milk. The complementation of milk with formate or purine bases was shown to reduce
pfl
expression, to suppress PFL synthesis, and to stimulate growth of
S. thermophilus.
These results show a novel regulatory mechanism controlling the synthesis of PFL and suggest an unrecognized physiological role for PFL as a formate supplier for anabolic purposes.
Transcriptional Activation of Quinoline Degradation Operons of
Pseudomonas putida
86 by the AraC/XylS-Type Regulator OxoS and Cross-Regulation of the P
qorM
Promoter by XylS
Authors: Carl Birgit , Fetzner Susanne
Published: December 2005

The quinoline-degradative gene cluster (
oxoO
, open reading frames 1 to 6 [ORF1 to -6],
qorMSL
, ORF7 to -9,
oxoR
) of
Pseudomonas putida
86 consists of several overlapping operons controlled in response to quinoline by the master promoter P
oxoO
and internal promoters Porf3, P
qorM
, and P
oxoR
. ORF7 to -9, presumed to be important for maturation of the molybdenum hydroxylase quinoline 2-oxidoreductase, are also weakly transcribed independently of quinoline. Expression of the
oxoS
gene, located upstream of
oxoO
, is not influenced by the carbon source. OxoS shows 26% amino acid sequence identity to XylS, the transcriptional regulator of the
meta
pathway promoter Pm of TOL plasmid pWW0, and is required for quinoline-dependent transcription from P
oxoO
, Porf3, P
qorM
, and P
oxoR
. 5′ deletion analysis of P
oxoO
and P
qorM
suggested that a 5′-TGCPuCT-N
3
-GGGATA-3′ motif, which resembles the distal 5′-TGCA-N
6
-GGNTA-3′ half-site of the tandem XylS binding site, is essential for
oxoS
-dependent transcriptional activation. P
qorM
, which shows similarity to the tandem XylS recognition site of Pm, was cross-activated by the
xylS
gene product in response to benzoate. The distal half-site of P
qorM
is necessary, but probably not sufficient, for transcriptional activation by XylS. Despite conservation in P
oxoO
of a distal 5′-TGCA-N
6
-GGNTA-3′ sequence, cross-activation of P
oxoO
by XylS and benzoate was not observed. The
oxoS
gene product in the presence of quinoline weakly stimulated transcription from the Pm promoter. Involvement of an XylS-type protein in the regulation of genes encoding synthesis of a molybdenum hydroxylase is without precedent and may reflect the evolutionary origin of this pathway in the metabolism of aromatic compounds.
Evaluation of Gel-Pad Oligonucleotide Microarray Technology by Using Artificial Neural Networks
Authors: Pozhitkov Alex , Chernov Boris , Yershov Gennadiy , Noble Peter A.
Published: December 2005

Past studies have suggested that thermal dissociation analysis of nucleic acids hybridized to DNA microarrays would improve discrimination among duplex types by scanning through a broad range of stringency conditions. To more fully constrain the utility of this approach using a previously described gel-pad microarray format, artificial neural networks (NNs) were trained to recognize noisy or low-quality data, as might derive from nonspecific fluorescence, poor hybridization, or compromised data collection. The NNs were trained to classify dissociation profiles (melts) into groups based on selected characteristics (e.g., initial signal intensity, area under the curve) using a data set of 21,044 profiles derived from 186 probes hybridized to a study set of RNA extracted from 32 microbes common to the human oral cavity. Three melt profile groups were identified: one group consisted mostly of ideal melt profiles; another group consisted mostly of poor melt profiles; and, the remainder were difficult to classify. Screening of melting profiles of perfect-match hybrids revealed inconsistencies in the form of melting profiles even for identical probes on the same microarray hybridized to same target rRNA. Approximately 18% of perfect-match duplex types were correctly classified as poor. Experimental variability and deviation from ideal melt behavior were shown to be attributable primarily to a method of local background subtraction that was very sensitive to displacement of the grid frames used for image capture (both determined by the image analysis system) and duplexes with low binding constants. Additional results showed that long RNA fragments limit the discriminating power among duplex types.
Microcolony Cultivation on a Soil Substrate Membrane System Selects for Previously Uncultured Soil Bacteria
Authors: Ferrari Belinda C. , Binnerup Svend J. , Gillings Michael
Published: December 2005

Traditional microbiological methods of cultivation recover less than 1% of the total bacterial species, and the culturable portion of bacteria is not representative of the total phylogenetic diversity. Classical cultivation strategies are now known to supply excessive nutrients to a system and therefore select for fast-growing bacteria that are capable of colony or biofilm formation. New approaches to the cultivation of bacteria which rely on growth in dilute nutrient media or simulated environments are beginning to address this problem of selection. Here we describe a novel microcultivation method for soil bacteria that mimics natural conditions. Our soil slurry membrane system combines a polycarbonate membrane as a growth support and soil extract as the substrate. The result is abundant growth of uncharacterized bacteria as microcolonies. By combining microcultivation with fluorescent in situ hybridization, previously “unculturable” organisms belonging to cultivated and noncultivated divisions, including candidate division TM7, can be identified by fluorescence microscopy. Successful growth of soil bacteria as microcolonies confirmed that the missing culturable majority may have a growth strategy that is not observed when traditional cultivation indicators are used.
Cooccurrence of
ScDSP
Gene Expression, Cell Death, and DNA Fragmentation in a Marine Diatom,
Skeletonema costatum
Authors: Chung Chih-Ching , Hwang Sheng-Ping L. , Chang Jeng
Published: December 2005

A novel death-specific gene,
ScDSP
, was obtained from a death stage subtraction cDNA library of the diatom
Skeletonema costatum
. The full length of
ScDSP
cDNA was 921 bp in length, containing a 699-bp open reading frame encoding 232 amino acids and two stretches of 66 and 156 bp in the 5′ and 3′ untranslated regions, respectively. Analysis of the peptide structure revealed that ScDSP contained a signal peptide domain, a transmembrane domain, and a pair of EF-hand motifs. When
S. costatum
grew exponentially at a rate of 1.3 day
−1
, the
ScDSP
mRNA level was at 2 μmol · mole 18S rRNA
−1
. In contrast, when the culture entered the death phase with a growth rate decreasing to 0.5 day
−1
,
ScDSP
mRNA increased dramatically to 668 μmol · mole 18S rRNA
−1
, and a high degree of DNA fragmentation was simultaneously observed. Under the influence of a light-dark cycle,
ScDSP
expression in both exponential and stationary phases clearly showed a diel rhythm, but the daily mean mRNA level was significantly higher in the stationary phase. Our results suggest that
ScDSP
may play a role in the molecular mechanism of self-destructive autolysis in phytoplankton under stress.
Transcriptional Response of
Saccharomyces cerevisiae
to Desiccation and Rehydration
Authors: Singh Jatinder , Kumar Deept , Ramakrishnan Naren , Singhal Vibha , Jervis Jody , Garst James F. , Slaughter Stephen M. , DeSantis Andrea M. , Potts Malcolm , Helm Richard F.
Published: December 2005

A transcriptional analysis of the response of
Saccharomyces cerevisiae
strain BY4743 to controlled air-drying (desiccation) and subsequent rehydration under minimal glucose conditions was performed. Expression of genes involved in fatty acid oxidation and the glyoxylate cycle was observed to increase during drying and remained in this state during the rehydration phase. When the BY4743 expression profile for the dried sample was compared to that of a commercially prepared dry active yeast, strikingly similar expression changes were observed. The fact that these two samples, dried by different means, possessed very similar transcriptional profiles supports the hypothesis that the response to desiccation is a coordinated event independent of the particular conditions involved in water removal. Similarities between “stationary-phase-essential genes” and those upregulated during desiccation were also noted, suggesting commonalities in different routes to reduced metabolic states. Trends in extracellular and intracellular glucose and trehalose levels suggested that the cells were in a “holding pattern” during the rehydration phase, a concept that was reinforced by cell cycle analyses. Application of a “redescription mining” algorithm suggested that sulfur metabolism is important for cell survival during desiccation and rehydration.
SelectListeria monocytogenesSubtypes Commonly Found in Foods Carry Distinct Nonsense Mutations ininlA, Leading to Expression of Truncated and Secreted Internalin A, and Are Associated with a Reduced Invasion Phenotype for Human Intestinal Epithelial Cells
Authors: Nightingale K. K. , Windham K. , Martin K. E. , Yeung M. , Wiedmann M.
Published: December 2005

The surface protein internalin A (InlA) contributes to the invasion of human intestinal epithelial cells byListeria monocytogenes. Screening ofL. monocytogenesstrains isolated from human clinical cases (n= 46), foods (n= 118), and healthy animals (n= 58) in the United States revealed mutations ininlAleading to premature stop codons (PMSCs) inL. monocytogenesribotypes DUP-1052A and DUP-16635A (PMSC mutation type 1), DUP-1025A and DUP-1031A (PMSC mutation type 2), and DUP-1046B and DUP-1062A (PMSC mutation type 3). While all DUP-1046B, DUP-1062A, DUP-16635A, and DUP-1031A isolates (n= 76) containedinlAPMSCs, ribotypes DUP-1052A and DUP-1025A (n= 72) contained isolates with and withoutinlAPMSCs. Western immunoblotting showed that all threeinlAPMSCs result in the production of truncated and secreted InlA. Searches of the Pathogen Tracker database, which contains subtype and source information for more than 5,000L. monocytogenesisolates, revealed that the six ribotypes shown to contain isolates withinlAPMSCs were overall more commonly isolated from foods than from human listeriosis cases.L. monocytogenesstrains carryinginlAPMSCs also showed significantly (P= 0.0004) reduced invasion of Caco-2 cells compared to isolates with homologous 3′inlAsequences without PMSCs. Invasion assays with an isogenic PMSC mutant further supported the observation thatinlAPMSCs lead to reduced invasion of Caco-2 cells. Our data show that specificL. monocytogenessubtypes which are common among U.S. food isolates but rare among human listeriosis isolates carryinlAmutations that are associated with, and possibly at least partially responsible for, an attenuated invasion phenotype.
Symbiosis and Insect Diversification: an Ancient Symbiont of Sap-Feeding Insects from the Bacterial Phylum
Bacteroidetes
Authors: Moran Nancy A. , Tran Phat , Gerardo Nicole M.
Published: December 2005

Several insect groups have obligate, vertically transmitted bacterial symbionts that provision hosts with nutrients that are limiting in the diet. Some of these bacteria have been shown to descend from ancient infections. Here we show that the large group of related insects including cicadas, leafhoppers, treehoppers, spittlebugs, and planthoppers host a distinct clade of bacterial symbionts. This newly described symbiont lineage belongs to the phylum
Bacteroidetes
. Analyses of 16S rRNA genes indicate that the symbiont phylogeny is completely congruent with the phylogeny of insect hosts as currently known. These results support the ancient acquisition of a symbiont by a shared ancestor of these insects, dating the original infection to at least 260 million years ago. As visualized in a species of spittlebug (Cercopoidea) and in a species of sharpshooter (Cicadellinae), the symbionts have extraordinarily large cells with an elongate shape, often more than 30 μm in length; in situ hybridizations verify that these correspond to the phylum
Bacteroidetes
. “
Candidatus
Sulcia muelleri” is proposed as the name of the new symbiont.
Highly Divergent Genes for Methanopterin-Linked C
1
Transfer Reactions in Lake Washington, Assessed via Metagenomic Analysis and mRNA Detection
Authors: Kalyuzhnaya Marina G. , Bowerman Sarah , Nercessian Olivier , Lidstrom Mary E. , Chistoserdova Ludmila
Published: December 2005

The origins and the evolutionary history of tetrahydromethanopterin-linked C
1
transfer reactions that are part of two environmentally important biotransformations, methylotrophy and methanogenesis, are still not well understood. In previous studies, we have expanded the known phylogenetic diversity of these reactions by identifying genes highly diverging from the ones associated with cultivated
Proteobacteria
,
Planctomycetes
, or
Archaea
(M. G. Kalyuzhnaya, M. E. Lidstrom, and L. Chistoserdova, Microb. Ecol. 48:463-472, 2004; M. G. Kalyuzhnaya, O. Nercessian, M. E. Lidstrom, and L. Chistoserdova, Environ. Microbiol. 7:1269-1274, 2005). Here we used a metagenomic approach to demonstrate that these divergent genes are present with high abundance in the microbial community inhabiting Lake Washington sediment. We also gained preliminary insights into the genomic composition of the organisms possessing these genes by sequencing genomic fragments from three uncultured microbes possessing the genes of interest. Phylogenetic analyses suggested that, although distantly related to each other, these organisms deeply diverge from known
Bacteria
and
Archaea
, with more relation to the former, suggesting their affiliation with a new bacterial phylum. We also demonstrate, via specific mRNA detection, that these divergent genes are expressed in the environment, pointing toward their potential role in local carbon cycling.
Comparison of Genome Sequences of Single-Stranded RNA Viruses Infecting the Bivalve-Killing Dinoflagellate
Heterocapsa circularisquama
Authors: Nagasaki Keizo , Shirai Yoko , Takao Yoshitake , Mizumoto Hiroyuki , Nishida Kensho , Tomaru Yuji
Published: December 2005

Heterocapsa circularisquama
RNA virus (HcRNAV) has at least two ecotypes (types UA and CY) that have intraspecies host specificities which are complementary to each other. We determined the complete genomic RNA sequence of two typical HcRNAV strains, HcRNAV34 and HcRNAV109, one of each ecotype. The nucleotide sequences of the viruses were 97.0% similar, and each had two open reading frames (ORFs), ORF-1 coding for a putative polyprotein having protease and RNA-dependent RNA polymerase (RdRp) domains and ORF-2 encoding a single major capsid protein. Phylogenetic analysis of the RdRp amino acid sequence suggested that HcRNAV belongs to a new previously unrecognized virus group. Four regions in ORF-2 had amino acid substitutions when HcRNAV34 was compared to HcRNAV109. We used a reverse transcription-nested PCR system to amplify the corresponding regions and also examined RNAs purified from six other HcRNAV strains with known host ranges. We also looked at natural marine sediment samples. Phylogenetic dendrograms for the amplicons correlated with the intraspecies host specificities of the test virus strains. The cloned sequences found in sediment also exhibited considerable similarities to either the UA-type or CY-type sequence. The tertiary structure of the capsid proteins predicted using computer modeling indicated that many of the amino acid substitutions were located in regions on the outside of the viral capsid proteins. This strongly suggests that the intraspecies host specificity of HcRNAV is determined by nanostructures on the virus surface that may affect binding to suitable host cells. Our study shows that capsid alterations can change the phytoplankton-virus (host-parasite) interactions in marine systems.
Cooccurrence of Aerobic and Anaerobic Methane Oxidation in the Water Column of Lake Plußsee
Authors: Eller Gundula , Känel Layla , Krüger Martin
Published: December 2005

Dissolved methane was investigated in the water column of eutrophic Lake Plußsee and compared to temperature, oxygen, and sulfide profiles. Methane concentrations and δ-
13
C signatures indicated a zone of aerobic methane oxidation and additionally a zone of anaerobic methane oxidation in the anoxic water body. The latter coincided with a peak in hydrogen sulfide concentration. High cell numbers of aerobic and anaerobic methane-oxidizing microorganisms were detected by fluorescence in situ hybridization (FISH) or the more sensitive catalyst-amplified reporter deposition-FISH, respectively, in these layers.
Two Tsetse Fly Species,
Glossina palpalis gambiensis
and
Glossina morsitans morsitans
, Carry Genetically Distinct Populations of the Secondary Symbiont
Sodalis glossinidius
Authors: Geiger Anne , Cuny Gérard , Frutos Roger
Published: December 2005

Genetic diversity among
Sodalis glossinidius
populations was investigated using amplified fragment length polymorphism markers. Strains collected from
Glossina palpalis gambiensis
and
Glossina morsitans morsitans
flies group into separate clusters, being differentially structured. This differential structuring may reflect different host-related selection pressures and may be related to the different vector competences of
Glossina
spp.
An Aflatoxin Biosynthesis Cluster Gene Encodes a Novel Oxidase Required for Conversion of Versicolorin A to Sterigmatocystin
Authors: Ehrlich Kenneth C. , Montalbano Beverly , Boué Stephen M. , Bhatnagar Deepak
Published: December 2005

Disruption of the aflatoxin biosynthesis cluster gene
aflY
(
hypA
) gave
Aspergillus parasiticus
transformants that accumulated versicolorin A. This gene is predicted to encode the Baeyer-Villiger oxidase necessary for formation of the xanthone ring of the aflatoxin precursor demethylsterigmatocystin.
Phage Tail-Like (High-Molecular-Weight) Bacteriocins of
Budvicia aquatica
and
Pragia fontium
(
Enterobacteriaceae
)
Authors: Šmarda Jan , Benada Oldřich
Published: December 2005

Electron microscopic analysis of contractile phage tail-like bacteriocins of three
Pragia fontium
strains and one
Budvicia aquatica
strain was performed. Fonticin and aquaticin are remarkably heat sensitive but trypsin resistant. Simultaneous production of contractile and flexible phage tail-like bacteriocins in the
P. fontium
64613 strain is shown for the first time.
Genetic Characterization of the
Bifidobacterium breve
UCC 2003
hrcA
Locus
Authors: Ventura Marco , Canchaya Carlos , Bernini Valentina , Del Casale Antonio , Dellaglio Franco , Neviani Erasmo , Fitzgerald Gerald F. , van Sinderen Douwe
Published: December 2005

The bacterial heat shock response is characterized by the elevated expression of a number of chaperone complexes and transcriptional regulators, including the DnaJ and the HrcA proteins. Genome analysis of
Bifidobacterium breve
UCC 2003 revealed a second copy of a
dnaJ
gene, named
dnaJ
2
, which is flanked by the
hrcA
gene in a genetic constellation that appears to be unique to the actinobacteria. Phylogenetic analysis using 53 bacterial
dnaJ
sequences, including both
dnaJ
1
and
dnaJ
2
sequences, suggests that these genes have followed a different evolutionary development. Furthermore, the
B. breve
UCC 2003
dnaJ
2
gene seems to be regulated in a manner that is different from that of the previously characterized
dnaJ
1
gene. The
dnaJ
2
gene, which was shown to be part of a 2.3-kb bicistronic operon with
hrcA
, was induced by osmotic shock but not significantly by heat stress. This induction pattern is unlike those of other characterized
dnaJ
genes and may be indicative of a unique stress adaptation strategy by this commensal microorganism.
A Novel Real-Time PCR for
Listeria monocytogenes
That Monitors Analytical Performance via an Internal Amplification Control
Authors: Rodríguez-Lázaro David , Pla Maria , Scortti Mariela , Monzó Héctor J. , Vázquez-Boland José A.
Published: December 2005

We describe a novel quantitative real-time (Q)-PCR assay for
Listeria monocytogenes
based on the coamplification of a target
hly
gene fragment and an internal amplification control (IAC). The IAC is a chimeric double-stranded DNA containing a fragment of the rapeseed
BnACCg8
gene flanked by the
hly
-specific target sequences. This IAC is detected using a second TaqMan probe labeled with a different fluorophore, enabling the simultaneous monitoring of the
hly
and IAC signals. The
hly
-IAC assay had a specificity and sensitivity of 100%, as assessed using 49
L. monocytogenes
isolates of different serotypes and 96 strains of nontarget bacteria, including 51
Listeria
isolates. The detection and quantification limits were 8 and 30 genome equivalents, and the coefficients for PCR linearity (
R
2
) and efficiency (
E
) were 0.997 and 0.80, respectively. We tested the performance of the
hly
-IAC Q-PCR assay using various broth media and food matrices. Fraser and half-Fraser media, raw pork, and raw or cold-smoked salmon were strongly PCR-inhibitory. This Q-PCR assay for
L. monocytogenes
, the first incorporating an IAC to be described for quantitative detection of a food-borne pathogen, is a simple and robust tool facilitating the identification of false negatives or underestimations of contamination loads due to PCR failure.
Clonal Nature of
Campylobacter fetus
as Defined by Multilocus Sequence Typing
Authors: van Bergen Marcel A. P. , Dingle Kate E. , Maiden Martin C. J. , Newell Diane G. , van der Graaf-Van Bloois Linda , van Putten Jos P. M. , Wagenaar Jaap A.
Published: December 2005

Campylobacter
fetus
can be divided into the subspecies
C. fetus
subsp.
fetus
and
C. fetus
subsp.
venerealis. C. fetus
subsp.
fetus
causes
sporadic infections in humans and abortion in cattle and sheep and has
been isolated from a variety of sites in different hosts.
C.
fetus
subsp.
venerealis
is host restricted, being
isolated mainly from the genital tracts of cattle, and is the causative
agent of bovine genital campylobacteriosis. Despite differences in
niche preference, microbiological subspecies differentiation has proven
difficult. Different typing methods divided
C. fetus
isolates
into different subgroups, depending on the methods used. The relative
value of these methods can be assessed by the evolutionary relationship
of isolates belonging to the genus; therefore, we developed a
multilocus sequence typing (MLST) scheme for
C. fetus
. This
scheme was applied to 140
C. fetus
isolates previously typed
by amplified fragment length polymorphism (AFLP) analysis. A total of
14 different sequence types (STs) were identified, and these exhibited
low levels of inter-ST genetic diversity, with only 22 variable sites
in 3,312 nucleotides. These MLST data indicate that
C. fetus
is genetically homogeneous compared to the homogeneity of other
Campylobacter
species. The two
C. fetus
subspecies
were extremely closely related genetically, but ST-4 was associated
only with
C. fetus
subsp.
venerealis
, which
represents a “bovine” clone. The
C. fetus
subsp.
fetus
isolates studied were more diverse in terms of
their STs, and the STs correlated with epidemiological relationships.
Congruence was observed among
C. fetus
subspecies,
sap
type, and ST; therefore, MLST confirms that mammalian
C. fetus
is genetically stable, probably as result of the
introduction of a single ancestral clone into a mammalian
niche.
Molecular Characterization of
Theileria
Species Associated with Mortality in Four Species of African Antelopes
Authors: Nijhof A. M. , Pillay V. , Steyl J. , Prozesky L. , Stoltsz W. H. , Lawrence J. A. , Penzhorn B. L. , Jongejan F.
Published: December 2005

Pathogen DNA was isolated from roan antelope (
Hippotragus equinus
), sable antelope (
Hippotragus niger
), greater kudu (
Tragelaphus strepsiceros
), and common gray duiker (
Sylvicapra grimmia
) in South Africa whose deaths were attributed to either theileriosis or cytauxzoonosis. We developed
Theileria
species-specific probes used in combination with reverse line blot hybridization assays and identified three different species of
Theileria
in four African antelope species. The close phylogenetic relationship between members of the genera
Theileria
and
Cytauxzoon
, similarities in the morphologies of developmental stages, and confusion in the literature regarding theileriosis or cytauxzoonosis are discussed.
Use of Denaturing High-Performance Liquid Chromatography for Rapid Detection and Identification of Seven
Candida
Species
Authors: Goldenberg Oliver , Herrmann Stefanie , Adam Thomas , Marjoram Gina , Hong George , Göbel Ulf B. , Graf Barbara
Published: December 2005

A novel denaturing high-performance liquid chromatography (DHPLC)-based technique allows rapid high-resolution analysis of PCR products. We used this technique for unequivocal molecular identification of seven
Candida
species. We show the application of this PCR/DHPLC approach for direct detection and identification of yeast species from blood cultures and for detection of
Candida
colonization in the gastrointestinal tract of allogeneic transplant patients.
Polyphasic Characterization Reveals that the Human Pathogen
Mycobacterium peregrinum
Type II Belongs to the Bovine Pathogen Species
Mycobacterium senegalense
Authors: Wallace Richard J. , Brown-Elliott Barbara A. , Brown June , Steigerwalt Arnold G. , Hall Leslie , Woods Gail , Cloud Joann , Mann Linda , Wilson Rebecca , Crist Christopher , Jost Kenneth C. , Byrer Dorothy E. , Tang Jane , Cooper Jason , Stamenova Elena , Campbell Brian , Wolfe Joyce , Turenne Christine
Published: December 2005

Mycobacterium peregrinum
consists of two taxa: types I and II. We evaluated 43 clinical type II strains from throughout the United States. They were responsible for soft-tissue and bone infections, catheter-related infections, and possible pneumonitis. By carbohydrate utilization, they were indistinguishable from type I strains, being
d
-mannitol and trehalose positive. However, they had a distinct susceptibility pattern that included intermediate ciprofloxacin MICs but low clarithromycin and doxycycline MICs of ≤1 μg/ml. These features were also shared by reference isolates of
Mycobacterium senegalense
from African bovine cases of “farcy.” By 16S rRNA gene sequencing, the type II isolates shared 100% sequence identity with
M. senegalense
. Partial sequencing of the type II
hsp65
gene (441 bp) revealed four sequevars showing ≥98.4% identity with each other and ≥98.6% identity with the sequence of five bovine strains of
M. senegalense
. There was ≤97.1% identity with
M. peregrinum
type I isolates and other
Mycobacterium fortuitum
group species. Sequencing of additional gene targets including the 16S-23S rDNA internal transcribed spacer region and the
rpoB
gene (partial sequence) revealed a similar phylogenetic grouping. DNA-DNA hybridization showed 76 to 99% relatedness between the bovine and human strains. These studies demonstrate that type II isolates are not isolates of
M. peregrinum
but represent human strains of
M. senegalense
. This study is the first to demonstrate this species as a human pathogen. Representative human
M. senegalense
strains include ATCC 35755 and newly submitted strains ATCC BAA-849, ATCC BAA-850, and ATCC BAA-851.
Diagnostic Accuracy of Two rK39 Antigen-Based Dipsticks and the Formol Gel Test for Rapid Diagnosis of Visceral Leishmaniasis in Northeastern Uganda
Authors: Chappuis François , Mueller Yolanda , Nguimfack Alexandre , Rwakimari John Bosco , Couffignal Sophie , Boelaert Marleen , Cavailler Philippe , Loutan Louis , Piola Patrice
Published: December 2005

The development of an accurate, practical, and affordable diagnostic test is essential to improve the management of visceral leishmaniasis (VL) in remote health centers. We evaluated the Formol Gel test (FGT) and two rK39 antigen-based dipsticks, the DUAL-IT L/M, and the Kalazar Detect for VL diagnosis in Amudat Hospital in Uganda. The DUAL-IT L/M was also evaluated for the diagnosis of malaria. All patients clinically suspect of VL were prospectively included in the study between October 2003 and March 2004. The gold standard used to define a VL case was a positive spleen aspirate or a direct agglutination test titer of >1:12,800 with an appropriate clinical response to antileishmanial therapy. A total of 131 VL and 112 non-VL patients were included in the analysis. The DUAL IT L/M was found to be more sensitive than the Kalazar Detect: 97% (95% confidence interval [95%CI] = 92 to 99%) versus 82% (95%CI = 74 to 87%). The Kalazar Detect and the DUAL IT L/M were highly specific (99% [95%CI = 95 to 100%] and 97% [95%CI = 92 to 99%], respectively). The FGT lacked both sensitivity (66% [95%CI = 57 to 73%]) and specificity (90% [95%CI = 83 to 94%]). The sensitivity of the DUAL IT L/M for malaria was only 57% (95%CI = 37 to 76%). The two rK39 dipsticks can be used for diagnostic confirmation of VL in this region. The DUAL-IT L/M without its malaria diagnostic component (DiaMed-IT LEISH) will be adopted as first-line test for VL in Uganda.
Mistaken Identity:
Neosartorya pseudofischeri
and Its Anamorph Masquerading as
Aspergillus fumigatus
Authors: Balajee S. Arunmozhi , Gribskov Jennifer , Brandt Mary , Ito James , Fothergill Annette , Marr Kieren A.
Published: December 2005

Invasive fungal infections caused by
Neosartorya pseudofischeri
S. W. Peterson [anamorph
Aspergillus thermomutatus
(Paden) S. W. Peterson] are extremely rare. Phenotypically, the anamorphic state of
N. pseudofischeri
resembles
Aspergillus fumigatus
, the predominant agent of invasive aspergillosis in immunocompromised hosts. We report the recovery of three clinical isolates of
N. pseudofischeri
, all initially misidentified by morphological characteristics as
A. fumigatus
. All three isolates were correctly identified by sequencing portions of the β-tubulin and the rodlet A genes. Only one of the three isolates produced the confirmatory fruiting bodies and was thus classified as
N. pseudofischeri
; the other isolates did not produce asci and were therefore identified as
A. thermomutatus
. All three isolates had higher MICs to voriconazole in vitro compared to
A. fumigatus
Af293. This report emphasizes that phenotypic identification of filamentous fungi may not identify morphologically similar, but genetically distinct, members of the genus
Aspergillus
section
Fumigati
. Accurate identification of these organisms may be clinically meaningful, given their potential differences in antifungal susceptibilities.
Isolation of Nontuberculous Mycobacteria in Zambia: Eight Case Reports
Authors: Buijtels Patricia C. A. M. , Petit Pieter L. C. , Verbrugh Henri A. , van Belkum Alex , van Soolingen Dick
Published: December 2005

The isolation of nontuberculous mycobacteria (NTM) raises the question of their clinical significance, especially in an African setting. We found a high percentage of NTM isolated from various specimens, including ones that are normally sterile, among 213 patients in Zambia. Because tuberculosis can affect all parts of the body, we decided to include patients who had signs and symptoms in any part of the body for more than 2 weeks. Most patients had tractus respiratorius (80%) and tractus digestivus (10%) symptoms. During three consecutive days, sputum was collected and two separate sputum specimens were cultured for mycobacteria. Depending on the clinical picture, pleural effusion, ascites, abscess material, or enlarged lymph nodes were also cultured for mycobacteria. A specimen from one sterile body site was collected from 25 patients (60% human immunodeficiency virus [HIV] positive). NTM were isolated from 8 of these 25 specimens.
Mycobacterium lentiflavum
was isolated from four patients, and
Mycobacterium goodii
was isolated from one patient. In order to exclude the possibility of laboratory cross-contamination, a novel amplified fragment length polymorphism DNA typing method for
M. lentiflavum
was developed. Genetic variation was detected, rendering the likelihood of laboratory contamination unlikely. Clinically relevant infection due to NTM occurs in both HIV-positive and HIV-negative patients in Zambia, and their clinical impact seems to be underestimated. This is the first report of
M. lentiflavum
and
M. goodii
infections in Africa.
Presence of
Rickettsia conorii
subsp.
israelensis
, the Causative Agent of Israeli Spotted Fever, in Sicily, Italy, Ascertained in a Retrospective Study
Authors: Giammanco Giovanni M. , Vitale Giustina , Mansueto Serafino , Capra Giuseppina , Caleca Maria Pia , Ammatuna Pietro
Published: December 2005

A retrospective analysis by molecular-sequence-based techniques was performed to correctly identify the etiological agent of 24 Mediterranean spotted fever cases occurring in Western Sicily, Italy, from 1987 to 2001. Restriction analysis of a 632-bp PCR-amplified portion of the
ompA
gene allowed presumptive identification of five clinical isolates as belonging to
Rickettsia conorii
subsp.
israelensis
, the etiological agent of Israeli spotted fever (ISF). The remaining 19 rickettsial isolates were
Rickettsia conorii
subsp.
conorii
, the only pathogenic rickettsia of the spotted fever group reported in Italy until the present. Sequence analysis of the
ompA
gene confirmed the identification of all the
R. conorii
subsp.
israelensis
isolates and demonstrated that rickettsiosis caused by
R. conorii
subsp.
israelensis
can be traced back to 1991 in Sicily. The recorded clinical data of the five ISF patients support the idea that these strains could correlate to more-severe forms of human disease. Three of five patients experienced severe disease, and one of them died.
Recognition of SCC
mec
Types According to Typing Pattern Determined by Multienzyme Multiplex PCR-Amplified Fragment Length Polymorphism Analysis of Methicillin-Resistant
Staphylococcus aureus
Authors: van der Zee Anneke , Heck Max , Sterks Marcel , Harpal Airien , Spalburg Emile , Kazobagora Leonard , Wannet Wim
Published: December 2005

Multienzyme multiplex PCR-amplified fragment length polymorphism (ME-AFLP) typing is a reliable and simple method for typing of bacterial species. In this study we analyzed two well-documented strain collections of
Staphylococcus aureus
and compared ME-AFLP typing results with results of various other typing methods. The discriminatory power of ME-AFLP was found comparable to pulsed-field gel electrophoresis, and typing results were highly concordant. ME-AFLP typing presents a suitable method for prescreening of large strain collections. Furthermore, the obtained typing patterns were found to cluster according to the staphylococcal cassette chromosome mec types of the strains.
Distribution of
Streptococcus pneumoniae
Serotypes Responsible for Penicillin Resistance and the Potential Role of New Conjugate Vaccines in New Caledonia
Authors: Michel N. , Watson M. , Baumann F. , Perolat P. , Garin B.
Published: December 2005

Invasive pneumococcal disease is a significant cause of morbidity and mortality worldwide. The aim of this study was to establish the serotypes responsible for pneumococcal disease and the serotypes responsible for penicillin resistance in Noumea, New Caledonia. Isolates of
Streptococcus pneumoniae
from all body sites referred to the Microbiology Department of the Pasteur Institute in New Caledonia between May 1999 and May 2001 had serotyping and susceptibility testing performed. Basic demographic data on patients were also collected. A total of 298 isolates were included in the study. The most common serotypes were types 1 (20%), 23F (10%), 12F (8%), 19F (8%), and 6B (5%). The serotype distribution differed significantly with age, site of collection, and ethnicity. Overall, 280 of 298 (94%) of the isolates had serogroups that are included in the 23-valent vaccine. However, only 14 of 20 (70%) of the isolates associated with invasive disease from children younger than 5 years of age and 26 of 113 (23%) of invasive isolates from patients more than 5 years of age were included in the new 7-valent conjugate vaccine. Overall, reduced susceptibility to penicillin was found in 43 of 298 (14.4%) of the isolates, with 13% falling into the intermediate resistance category and only 5 (2%) being high-level resistant. A higher prevalence of penicillin resistance occurred in younger age groups and in European patients and involved specifically certain serotypes. The 7-valent conjugate pneumococcal vaccine has potential benefit for the New Caledonian population under 5 years of age and should be considered for future vaccines schedules.
Use of Ramification Amplification Assay for Detection of
Escherichia coli
O157:H7 and Other
E. coli
Shiga Toxin-Producing Strains
Authors: Li Fan , Zhao Chunyan , Zhang Wandi , Cui Shenghui , Meng Jianghong , Wu Josephine , Zhang David Y.
Published: December 2005

Escherichia coli
O157:H7 and other Shiga toxin-producing
E. coli
(STEC) strains are important human pathogens that are mainly transmitted through the food chain. These pathogens have a low infectious dose and may cause life-threatening illnesses. However, detection of this microorganism in contaminated food or a patient's stool specimens presents a diagnostic challenge because of the low copy number in the sample. Often, a more sensitive nucleic acid amplification method, such as PCR, is required for rapid detection of this microorganism. Ramification amplification (RAM) is a recently introduced isothermal DNA amplification technique that utilizes a circular probe for target detection and achieves exponential amplification through the mechanism of primer extension, strand displacement, and ramification. In this study, we synthesized a circular probe specific for the Shiga toxin 2 gene (
stx
2
). Our results showed that as few as 10 copies of
stx
2
could be detected, indicating that the RAM assay was as sensitive as conventional PCR. We further tested 33 isolates of
E coli
O157:H7, STEC,
Shigella dysenteriae
, and nonpathogenic
E. coli
by RAM assay. Results showed that all 27 STEC isolates containing the
stx
2
gene were identified by RAM assay, while
S. dysenteriae
and nonpathogenic
E. coli
isolates were undetected. The RAM results were 100% in concordance with those of PCR. Because of its simplicity and isothermal amplification, the RAM assay could be a useful method for detecting STEC in food and human specimens.
Association of Virulence Genotype with Phylogenetic Background in Comparison to Different Seropathotypes of Shiga Toxin-Producing
Escherichia coli
Isolates
Authors: Girardeau Jean Pierre , Dalmasso Alessandra , Bertin Yolande , Ducrot Christian , Bord Séverine , Livrelli Valérie , Vernozy-Rozand Christine , Martin Christine
Published: December 2005

The distribution of virulent factors (VFs) in 287 Shiga toxin-producing
Escherichia coli
(STEC) strains that were classified according to Karmali et al. into five seropathotypes (M. A. Karmali, M. Mascarenhas, S. Shen, K. Ziebell, S. Johnson, R. Reid-Smith, J. Isaac-Renton, C. Clark, K. Rahn, and J. B. Kaper, J. Clin. Microbiol. 41:4930-4940, 2003) was
investigated. The associations of VFs with phylogenetic background were
assessed among the strains in comparison with the different
seropathotypes. The phylogenetic analysis showed that STEC strains
segregated mainly in phylogenetic group B1 (70%) and revealed the
substantial prevalence (19%) of STEC belonging to phylogenetic group A
(designated STEC-A). The presence of virulent clonal groups in
seropathotypes that are associated with disease and their absence from
seropathotypes that are not associated with disease support the concept
of seropathotype classification. Although certain VFs (
eae
,
stx
2-EDL933
,
stx
2-vha
, and
stx
2-vhb
) were concentrated in seropathotypes
associated with disease, others (
astA
, HPI,
stx
1c
, and
stx
2-NV206
)
were concentrated in seropathotypes that are not associated with
disease. Taken together with the observation that the STEC-A group was
exclusively composed of strains lacking
eae
recovered from
seropathotypes that are not associated with disease, the“
atypical” virulence pattern suggests that STEC-A
strains comprise a distinct category of STEC strains. A practical
benefit of our phylogenetic analysis of STEC strains is that
phylogenetic group A status appears to be highly predictive of“
nonvirulent”
seropathotypes.
Noroviruses as a Cause of Traveler's Diarrhea among Students from the United States Visiting Mexico
Authors: Ko GwangPyo , Garcia Coralith , Jiang Zhi-Dong , Okhuysen Pablo C. , Belkind-Gerson Jaime , Glass Roger I. , DuPont Herbert L.
Published: December 2005

Stool specimens from 124 international travelers with acute diarrhea were tested for the presence of enteropathogens. Noroviruses (NoVs) were the second most commonly identified enteric pathogen in diarrheal stool samples (21/124, 17%), exceeded only by enterotoxigenic
Escherichia coli
(50/106, 47%). This study indicates that NoV is an underappreciated cause of traveler's diarrhea.
Multiplex PCR Assay That Identifies the Major Lipooligosaccharide Serotype Expressed by
Moraxella catarrhalis
Clinical Isolates
Authors: Edwards Katie J. , Schwingel Johanna M. , Datta Anup K. , Campagnari Anthony A.
Published: December 2005

A heterologous cluster of glycosyltransferase genes was identified in the three
Moraxella catarrhalis
LOS serotype strains. Multiple PCR primers designed to this region amplified products that differentiate between the serotypes more rapidly and efficiently than previously described serological analyses. This assay will be valuable for clinical and research-based studies.
Evaluation of Ribotyping as a Tool for Molecular Typing of
Yersinia pseudotuberculosis
Strains of Worldwide Origin
Authors: Voskressenskaya Ekaterina , Leclercq Alexandre , Tseneva Galina , Carniel Elisabeth
Published: December 2005

Yersinia pseudotuberculosis
is a gram-negative bacterium that infects a wide range of animals, including humans, and is transmitted by the fecal-oral route. This species is found globally and is responsible for human outbreaks, mainly in cold countries. The aim of this study was to evaluate the potential of ribotyping for the molecular typing of worldwide isolates. For this purpose, 80 strains of
Y. pseudotuberculosis
belonging to the six classical serotypes and nine subserotypes and isolated from various countries and different hosts were analyzed. Combination of the EcoRI and EcoRV ribopatterns allowed the delineation of 27 ribotypes. In most instances, ribotypes were associated with specific subserotypes and allowed their subdivision. No association between the ribotype and the geographical origin of the strains was observed, arguing for a global spread of this organism. Similarly, no marked association between the ribotype and the type of host was noted, confirming the circulation of this pathogen in the environment, different animal species, and human hosts.
Y. pseudotuberculosis
exhibited ribopatterns very close to those of
Y. pestis
, although not completely identical. Altogether, the present study demonstrates that ribotyping may be a useful tool for molecular typing of global isolates of
Y. pseudotuberculosis
but that it has some limitations due to the small number of hybridizing bands that generate the diversity of the profiles.