bluuebunny/crossref_metadata_2025 huggingface dataset:
The internal structure of vP in Ju|'hoansi and ‡Hoan
Authors: Collins Chris
Published: November 2003

In this article, I analyze ki (‡Hoan) and ko (Ju|'hoansi) as heading vP internal functional projections dominating VP and dominated by vP. I show how this analysis provides a natural account for the various possible and impossible word orders in constructions involving locative and instrumental phrases. Lastly, I discuss the general implications of my analysis for the theory of Case checking in Chomsky (2000, 2001a, b).
What is the Chance of Yuan Revaluation?
Authors: Rossi Vanessa , Knapp Simon
Published: November 2003

From a Chinese perspective, chances of any change in the Yuan exchange rate look pretty slim. If you were an official in Beijing, would you stick your neck out even recommending this idea? If the status quo looks fairly acceptable, and it is ‘stability oriented’ (a very Chinese preference), why go rocking the boat? You certainly do not make any changes just because Japan or the US tell you to. And financial markets are not that persuasive – after all they wanted a devaluation only a short while ago. In this article, Vanessa Rossi and Simon Knapp assess the likelihood of a Yuan revaluation and its implications for the economic outlook.
The Changing Nexus: Tertiary Education Institutions, the Marketplace and the State
Authors: Steier Francis A.
Published: November 2003

This article examines the evolving relationship between the marketplace, the state and tertiary education institutions. The context of these relations has evolved strikingly in recent years, which have seen three major developments: growing system differentiation, changing governance patterns and diminished direct involvement of governments in the funding and provision of tertiary education. This article first describes the key dimensions of the rise of market forces in tertiary education throughout the world and the main implications of this phenomenon. It then articulates the rationale for continuing public intervention in the sector and, in conclusion, outlines the nature of an appropriate enabling framework for the further development of tertiary education.
Making Gender Comparisons More Meaningful: A Call for More Attention to Social Context
Authors: Yoder Janice D. , Kahn Arnold S.
Published: November 2003

We challenge researchers to consider sex and gender as a marker for possible social contextual differences. Disappointed by both philosophical and empirical attempts to find coherence in research making gender comparisons, we selectively review studies showing both context-specific similarities between women and men where overall comparisons found differences as well as context-specific differences where general patterns of similarity existed. These examples cut across embedded levels of social context, ranging from those immediately proximal to the individual (interpersonal) to organizational and broad societal structures. They suggest that seemingly identical contexts can have sweepingly different impacts on women and men and that effective social interventions be gender-sensitive.
On the Content and Structure of the Gender Role Self-Concept: Including Gender-Stereotypical Behaviors in Addition to Traits
Authors: Athenstaedt Ursula
Published: November 2003

The present study introduces a conceptualization of gender role self-concept that implies not only the commonly measured socially desirable expressive and instrumental traits (F+ and M+) but also feminine and masculine behaviors (FBehav and MBehav), and socially undesirable gender traits (F- and M-). Three different models were tested using structural equation modeling. For both men and women, F+ and FBehav load together on one dimension whereas M+ and MBehav load on a second dimension. F- and M- are conceptualized as independent dimensions that are mainly related to the cross-gendered latent factor. With the exception of these similarities, the self-concepts of men and women differed in several important aspects that point to a different meaning of gender roles for the two sexes.
Enhanced Fitness Conferred by Naturally Occurring Variation in the Circadian Clock
Authors: Michael Todd P. , Salomé Patrice A. , Yu Hannah J. , Spencer Taylor R. , Sharp Emily L. , McPeek Mark A. , Alonso José M. , Ecker Joseph R. , McClung C. Robertson
Published: November 2003

Natural variation in clock parameters is necessary for the circadian clock to contribute to organismal fitness over a broad geographic range. Considerable variation is evident in the period, phase, and amplitude of 150
Arabidopsis
accessions, and the period length is correlated with the day length at the latitude of origin, implying the adaptive significance of correctly regulated circadian timing. Quantitative trait loci analysis of recombinant inbred lines indicates that multiple loci interact to determine period, phase, and amplitude. The loss-of-function analysis of each member of the ARABIDOPSIS PSEUDO-RESPONSE REGULATOR family suggests that they are candidates for clock quantitative trait loci.
Roles for Mating and Environment in
C. elegans
Sex Determination
Authors: Prahlad Veena , Pilgrim Dave , Goodwin Elizabeth B.
Published: November 2003

In
Caenorhabditis elegans
the two sexes, hermaphrodites and males, are thought to be irreversibly determined at fertilization by the ratio of X chromosomes to sets of autosomes: XX embryos develop as hermaphrodites and XO embryos as males. We show instead that both sex and genotype of
C. elegans
can be altered postembryonically and that this flexibility requires sexual reproduction. When grown in specific bacterial metabolites, some XX larvae generated by mating males and hermaphrodites develop as males and lose one X chromosome. However, XX larvae produced by hermaphrodite self-fertilization show no such changes. We propose that sexual reproduction increases developmental flexibility of progeny, allowing for better adaptation to changing environments.
Cold Shock Response and Major Cold Shock Proteins of
Vibrio cholerae
Authors: Datta Partha Pratim , Bhadra Rupak K.
Published: November 2003

When exponentially growing
Vibrio cholerae
cells were shifted from 37°C to various lower temperatures, it was found that the organism could adapt and grow at temperatures down to 15°C, below which the growth was completely arrested. There was no difference between the patterns of the cold shock responses in toxinogenic and nontoxinogenic strains of
V. cholerae
. Gel electrophoretic analyses of proteins of cold-exposed cells revealed significant induction of two major cold shock proteins (Csps), whose molecular masses were 7.7 kDa (CspA
VC
) and 7.5 kDa (CspV), and six other Csps, most of which were much larger. We cloned, sequenced, and analyzed the
cspV
gene encoding the CspV protein of
V. cholerae
O139 strain SG24. Although CspA
VC
and CspV have similar kinetics of synthesis and down-regulation, the corresponding genes,
cspA
and
cspV
, which are located in the small chromosome, are not located in the same operon. A comparative analysis of the kinetics of synthesis revealed that the CspV protein was synthesized de novo only during cold shock. Although both CspA
VC
and CspV were stable for several hours in the cold, the CspV protein was degraded rapidly when the culture was shifted back to 37°C, suggesting that this protein is probably necessary for adaptation at lower temperatures. Northern blot analysis confirmed that the
cspV
gene is cold shock inducible and is regulated tightly at the level of transcription. Interestingly, the
cspV
gene has a cold shock-inducible promoter which is only 12 nucleotides from the translational start site, and therefore, it appears that no unusually long 5′ untranslated region is present in its mRNA transcript. Thus, this promoter is an exception compared to other promoters of cold shock-inducible genes of different organisms, including
Escherichia coli
. Our results suggest that
V. cholerae
may use an alternative pathway for regulation of gene expression during cold shock.
Involvement of Two Cytosolic Enzymes and a Novel Intermediate, 5′-Oxoaverantin, in the Pathway from 5′-Hydroxyaverantin to Averufin in Aflatoxin Biosynthesis
Authors: Sakuno Emi , Yabe Kimiko , Nakajima Hiromitsu
Published: November 2003

During aflatoxin biosynthesis, 5′-hydroxyaverantin (HAVN) is converted to averufin (AVR). Although we had previously suggested that this occurs in one enzymatic step, we demonstrate here that this conversion is composed of two enzymatic steps by showing that the two enzyme activities in the cytosol fraction of
Aspergillus parasiticus
were clearly separated by Mono Q column chromatography. An enzyme, HAVN dehydrogenase, catalyzes the first reaction from HAVN to a novel intermediate, another new enzyme catalyzes the next reaction from the intermediate to AVR, and the intermediate is a novel substance, 5′-oxoaverantin (OAVN), which was determined by physicochemical methods. We also purified both of the enzymes, HAVN dehydrogenase and OAVN cyclase, from the cytosol fraction of
A. parasiticus
by using ammonium sulfate fractionation and successive chromatographic steps. The HAVN dehydrogenase is a homodimer composed of 28-kDa subunits, and it requires NAD, but not NADP, as a cofactor for its activity. Matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis of tryptic peptides of the purified HAVN dehydrogenase revealed that this enzyme coincides with a protein deduced from the
adhA
gene in the aflatoxin gene cluster of
A. parasiticus
. Also, the OAVN cyclase enzyme is a homodimer composed of 79-kDa subunits which does not require any cofactor for its activity. Further characterizations of both enzymes were performed.
Modification of the Monomer Composition of Polyhydroxyalkanoate Synthesized in
Saccharomyces cerevisiae
Expressing Variants of the β-Oxidation-Associated Multifunctional Enzyme
Authors: Marchesini Silvia , Erard Nadine , Glumoff Tuomo , Hiltunen J. Kalervo , Poirier Yves
Published: November 2003

Expression by
Saccharomyces cerevisiae
of a polyhydroxyalkanoate (PHA) synthase modified at the carboxy end by the addition of a peroxisome targeting signal derived from the last 34 amino acids of the
Brassica napus
isocitrate lyase (ICL) and containing the terminal tripeptide Ser-Arg-Met resulted in the synthesis of PHA. The ability of the terminal peptide Ser-Arg-Met and of the 34-amino-acid peptide from the
B. napus
ICL to target foreign proteins to the peroxisome of
S. cerevisiae
was demonstrated with green fluorescent protein fusions. PHA synthesis was found to be dependent on the presence of both the enzymes generating the β-oxidation intermediate 3-hydroxyacyl-coenzyme A (3-hydroxyacyl-[CoA]) and the peroxin-encoding
PEX5
gene, demonstrating the requirement for a functional peroxisome and a β-oxidation cycle for PHA synthesis. Using a variant of the
S. cerevisiae
β-oxidation multifunctional enzyme with a mutation inactivating the B domain of the R-3-hydroxyacyl-CoA dehydrogenase, it was possible to modify the PHA monomer composition through an increase in the proportion of the short-chain monomers of five and six carbons.
Viability of and Plasmid Retention in Frozen Recombinant
Escherichia coli
over Time: a Ten-Year Prospective Study
Authors: Koenig Gina L.
Published: November 2003

The long-term viability and plasmid retention of recombinant
Escherichia coli
strains were investigated by real-time testing of master cell banks (MCBs) stored at the Roche Molecular Systems Culture Collection (RMSCC). MCBs at the RMSCC were cryogenically frozen and stored at −80°C for long-term preservation. At regular intervals during a period of 5 to more than 10 years, representative cryovials of each MCB were tested for viability and plasmid retention. Plasmid retention and viability for all 30 MCBs were stable over time. Twenty-seven MCBs maintained high levels of plasmid retention (at or near 100%), while three MCBs showed lower plasmid retention rates (ranging from 13.9 to 96.5%) that were consistent over time. New MCBs with high plasmid retention were created from two of the MCBs with lower plasmid retention by selective pressure with high levels of antibiotics. These new MCBs have shown stable viability and high plasmid retention over the first 5 months of storage. In conclusion, this study shows that properly selected, frozen and stored MCBs retain viability and maintain plasmid retention over time. Moreover, it is possible to recover cultures with high plasmid retention from MCBs with low plasmid retention by selecting clones grown in the presence of high levels of antibiotics.
High-Resolution Differentiation of Cyanobacteria by Using rRNA-Internal Transcribed Spacer Denaturing Gradient Gel Electrophoresis
Authors: Janse Ingmar , Meima Marion , Kardinaal W. Edwin A. , Zwart Gabriel
Published: November 2003

For many ecological studies of cyanobacteria, it is essential that closely related species or strains can be discriminated. Since this is often not possible by using morphological features, cyanobacteria are frequently studied by using DNA-based methods. A powerful method for analysis of the diversity and dynamics of microbial populations and for checking the purity and affiliation of cultivated strains is denaturing gradient gel electrophoresis (DGGE). We realized high-resolution discrimination of a variety of cyanobacteria by means of DGGE analysis of sections of the internal transcribed spacer between the 16S and 23S rRNA genes (rRNA-ITS). A forward primer specific for cyanobacteria, targeted at the 3′ end of the 16S rRNA gene, was designed. The combination of this primer and three different reverse primers targeted to the rRNA-ITS or to the 23S rRNA gene yielded PCR products of different sizes from cultures of all 16 cyanobacterial genera that were tested but not from other bacteria. DGGE profiles produced from the shortest section of rRNA-ITS consisted of one band for all but one cyanobacterial genera, and those generated from longer stretches of rRNA-ITS yielded DGGE profiles containing one to four bands. The suitability of DGGE for detecting intrageneric and intraspecific variation was tested by using strains of the genus
Microcystis
. Many strains could be discriminated by means of rRNA-ITS DGGE, and the resolution of this method was strikingly higher than that obtained with previously described methods. The applicability of the developed DGGE assays for analysis of cyanobacteria in field samples was demonstrated by using samples from freshwater lakes. The advantages and disadvantages associated with the use of each developed primer set are discussed.
Binding of Pediocin PA-1 with Anionic Lipid Induces Model Membrane Destabilization
Authors: Gaussier Hélène , Lefèvre Thierry , Subirade Muriel
Published: November 2003

To obtain molecular insights into the action mode of antimicrobial activity of pediocin PA-1, the interactions between this bacteriocin and dimyristoylphosphatidylcholine (DMPC) or dimyristoylphosphatidylglycerol (DMPG) model membranes have been investigated in D2O at pD 6 by Fourier transform infrared spectroscopy. The interactions were monitored with respect to alteration of the secondary structure of pediocin, as registered by the amide I′ band, and phospholipid conformation, as revealed by the methylene νs(CH2) and carbonyl ν(C═;O) stretching vibrations. The results show that no interaction between pediocin and DMPC occurs. By contrast, pediocin undergoes a structural reorganization in the presence of DMPG. Upon heating, pediocin self-aggregates, which is not observed for this pD in aqueous solution. The gel-to-crystalline phase transition of DMPG shifts to higher temperatures with a concomitant dehydration of the interfacial region. Our results indicate that pediocin is an extrinsic peptide and that its action mechanism may lie in a destabilization of the cell membrane.
Microbial Community Dynamics Associated with Rhizosphere Carbon Flow
Authors: Butler Jessica L. , Williams Mark A. , Bottomley Peter J. , Myrold David D.
Published: November 2003

Root-deposited photosynthate (rhizodeposition) is an important source of readily available carbon (C) for microbes in the vicinity of growing roots. Plant nutrient availability is controlled, to a large extent, by the cycling of this and other organic materials through the soil microbial community. Currently, our understanding of microbial community dynamics associated with rhizodeposition is limited. We used a
13
C pulse-chase labeling procedure to examine the incorporation of rhizodeposition into individual phospholipid fatty acids (PLFAs) in the bulk and rhizosphere soils of greenhouse-grown annual ryegrass (
Lolium multiflorum
Lam. var. Gulf). Labeling took place during a growth stage in transition between active root growth and rapid shoot growth on one set of plants (labeling period 1) and 9 days later during the rapid shoot growth stage on another set of plants (labeling period 2). Temporal differences in microbial community composition were more apparent than spatial differences, with a greater relative abundance of PLFAs from gram-positive organisms (i15:0 and a15:0) in the second labeling period. Although more abundant, gram-positive organisms appeared to be less actively utilizing rhizodeposited C in labeling period 2 than in labeling period 1. Gram-negative bacteria associated with the 16:1ω5 PLFA were more active in utilizing
13
C-labeled rhizodeposits in the second labeling period than in the first labeling period. In both labeling periods, however, the fungal PLFA 18:2ω6,9 was the most highly labeled. These results demonstrate the effectiveness of using
13
C labeling and PLFA analysis to examine the microbial dynamics associated with rhizosphere C cycling by focusing on the members actively involved.
Comparative Genomics of Insect-Symbiotic Bacteria: Influence of Host Environment on Microbial Genome Composition
Authors: Rio Rita V. M. , Lefevre Cedric , Heddi Abdelaziz , Aksoy Serap
Published: November 2003

Commensal symbionts, thought to be intermediary amid obligate mutualists and facultative parasites, offer insight into forces driving the evolutionary transition into mutualism. Using macroarrays developed for a close relative,
Escherichia coli
, we utilized a heterologous array hybridization approach to infer the genomic compositions of a clade of bacteria that have recently established symbiotic associations:
Sodalis glossinidius
with the tsetse fly (Diptera,
Glossina
spp.) and
Sitophilus oryzae
primary endosymbiont (SOPE) with the rice weevil (Coleoptera,
Sitophilus oryzae
). Functional biologies within their hosts currently reflect different forms of symbiotic associations. Their hosts, members of distant insect taxa, occupy distinct ecological niches and have evolved to survive on restricted diets of blood for tsetse and cereal for the rice weevil. Comparison of genome contents between the two microbes indicates statistically significant differences in the retention of genes involved in carbon compound catabolism, energy metabolism, fatty acid metabolism, and transport. The greatest reductions have occurred in carbon catabolism, membrane proteins, and cell structure-related genes for
Sodalis
and in genes involved in cellular processes (i.e., adaptations towards cellular conditions) for SOPE. Modifications in metabolic pathways, in the form of functional losses complementing particularities in host physiology and ecology, may have occurred upon initial entry from a free-living to a symbiotic state. It is possible that these adaptations, streamlining genomes, act to make a free-living state no longer feasible for the harnessed microbe.
Variation in Resistance of
Mycobacterium paratuberculosis
to Acid Environments as a Function of Culture Medium
Authors: Sung Nackmoon , Collins Michael T.
Published: November 2003

Acid resistance of
Mycobacterium paratuberculosis
was examined as a function of growth conditions (i.e., in vitro growth medium and pH).
M. paratuberculosis
was cultured in either fatty acid-containing medium (7H9-OADC) or glycerol-containing medium (WR-GD or 7H9-GD) at two culture pHs (pHs 6.0 and 6.8). Organisms produced in these six medium and pH conditions were then tested for resistance to acetate buffer at pHs 3, 4, 5, and 6 at 20°C. A radiometric culture method (BACTEC) was used to quantify viable
M. paratuberculosis
cell data at various acid exposure times, and
D
values (decimal reduction times, or the times required to kill a 1-log
10
concentration of bacteria) were determined. Soluble proteins of
M. paratuberculosis
grown under all six conditions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to identify proteins that may be associated with acid resistance or susceptibility. The culture medium affected growth rate and morphology: thin floating sheets of cells were observed in 7H9-OADC versus confluent, thick, waxy, and wrinkled pellicles in WR-GD. Culture medium pH affected growth rate (which was highest at pH 6.0), but it had little or no effect on
D
values for
M. paratuberculosis
at any test pH. When grown in 7H9-OADC,
M. paratuberculosis
was more acid resistant at all test pHs (higher
D
values) than when grown in WR-GD. Glycerol appeared to be the culture medium component most responsible for lower levels of
M. paratuberculosis
acid resistance. When glycerol was substituted for OADC in the 7H9 medium,
D
values were significantly lower than those of 7H9-OADC-grown
M. paratuberculosis
and were approximately the same as those for
M. paratuberculosis
grown in WR-GD medium. Comparison of the SDS-PAGE protein profiles for
M. paratuberculosis
cultures grown in 7H9-OADC, WR-GD, or 7H9-GD medium revealed that increased expression of 34.2- and 14.0-kDa proteins was associated with higher levels of acid resistance of
M. paratuberculosis
grown in 7H9-OADC medium and that 56.6- and 41.3-kDa proteins were associated with lower levels of acid resistance. This is the first report showing that in vitro culture conditions significantly affect growth characteristics, acid resistance, and protein expression of
M. paratuberculosis
, and the results emphasize the importance of culture conditions for in vitro susceptibility studies.
The Isotope Array, a New Tool That Employs Substrate-Mediated Labeling of rRNA for Determination of Microbial Community Structure and Function
Authors: Adamczyk Justyna , Hesselsoe Martin , Iversen Niels , Horn Matthias , Lehner Angelika , Nielsen Per Halkjaer , Schloter Michael , Roslev Peter , Wagner Michael
Published: November 2003

A new microarray method, the isotope array approach, for identifying microorganisms which consume a
14
C-labeled substrate within complex microbial communities was developed. Experiments were performed with a small microarray consisting of oligonucleotide probes targeting the 16S rRNA of ammonia-oxidizing bacteria (AOB). Total RNA was extracted from a pure culture of
Nitrosomonas eutropha
grown in the presence of [
14
C]bicarbonate. After fluorescence labeling of the RNA and microarray hybridization, scanning of all probe spots for fluorescence and radioactivity revealed that specific signals were obtained and that the incorporation of
14
C into rRNA could be detected unambiguously. Subsequently, we were able to demonstrate the suitability of the isotope array approach for monitoring community composition and CO
2
fixation activity of AOB in two nitrifying activated-sludge samples which were incubated with [
14
C]bicarbonate for up to 26 h. AOB community structure in the activated-sludge samples, as predicted by the microarray hybridization pattern, was confirmed by quantitative fluorescence in situ hybridization (FISH) and comparative
amoA
sequence analyses. CO
2
fixation activities of the AOB populations within the complex activated-sludge communities were detectable on the microarray by
14
C incorporation and were confirmed independently by combining FISH and microautoradiography. AOB rRNA from activated sludge incubated with radioactive bicarbonate in the presence of allylthiourea as an inhibitor of AOB activity showed no incorporation of
14
C and thus was not detectable on the radioactivity scans of the microarray. These results suggest that the isotope array can be used in a PCR-independent manner to exploit the high parallelism and discriminatory power of microarrays for the direct identification of microorganisms which consume a specific substrate in the environment.
Sequencing and Characterization of pBM400 from
Bacillus megaterium
QM B1551
Authors: Scholle Michael D. , White Christen A. , Kunnimalaiyaan Muthusamy , Vary Patricia S.
Published: November 2003

Bacillus
megaterium
QM B1551 plasmid pBM400, one of seven indigenous
plasmids, has been labeled with a selectable marker, isolated,
completely sequenced, and partially characterized. A sequence of 53,903
bp was generated, revealing a total of 50 predicted open reading frames
(ORFs); 33 were carried on one strand and 17 were carried on the other.
These ORFs comprised 57% of the pBM400 sequence. Besides the
replicon region and a complete rRNA operon that have previously been
described, several interesting genes were found, including genes for
predicted proteins for cell division (FtsZ and FtsK), DNA-RNA
interaction (FtsK, Int/Rec, and reverse transcriptase), germination
(CwlJ), styrene degradation (StyA), and heavy metal resistance (Cu-Cd
export and ATPase). Three of the ORF products had high similarities to
proteins from the
Bacillus anthracis
virulence plasmid pXO1.
An insertion element with similarity to the IS
256
family and
several hypothetical proteins similar to those from the chromosomes of
other
Bacillus
and
Lactococcus
species were present.
This study provides a basis for isolation and sequencing of other
high-molecular-weight plasmids from QM B1551 and for understanding the
role of megaplasmids in gram-positive bacteria. The genes carried by
pBM400 suggest a possible role of this plasmid in the survival of
B. megaterium
in hostile environments with heavy metals or
styrene and also suggest that there has been an exchange of genes
within the gram-positive bacteria, including
pathogens.
Growth of
Vibrio cholerae
O1 in Red Tide Waters off California
Authors: Mouriño-Pérez Rosa R. , Worden Alexandra Z. , Azam Farooq
Published: November 2003

Vibrio cholerae
serotype O1 is autochthonous to estuarine and coastal waters. However, its population dynamics in such environments are not well understood. We tested the proliferation of
V. cholerae
N16961 during a
Lingulodinium polyedrum
bloom, as well as other seawater conditions. Microcosms containing 100-kDa-filtered seawater were inoculated with
V. cholerae
or the 0.6-μm-pore-size filterable fraction of seawater assemblages. These cultures were diluted 10-fold with fresh 100-kDa-filtered seawater every 48 h for four cycles. Growth rates ranged from 0.3 to 14.3 day
−1
(4.2 day
−1
± 3.9) for
V. cholerae
and 0.1 to 9.7 day
−1
(2.2 ± 2.8 day
−1
) for bacterial assemblage. Our results suggest that dissolved organic matter during intense phytoplankton blooms has the potential to support explosive growth of
V. cholerae
in seawater. Under the conditions tested, free-living
V. cholerae
was able to reach concentrations per milliliter that were up to 3 orders of magnitude higher than the known minimum infectious dose (10
4
cell ml
−1
) and remained viable under many conditions. If applicable to the complex conditions in marine ecosystems, our results suggest an important role of the growth of free-living
V. cholerae
in disease propagation and prevention during phytoplankton blooms.
Development of a Synthetic Minimal Medium for
Listeria monocytogenes
Authors: Tsai Hsiang-Ning , Hodgson David A.
Published: November 2003

A defined solid and liquid minimal medium, HTM, which contained methionine and cysteine as the sole amino acids, was developed for
Listeria monocytogenes
. Complex broth-grown
L. monocytogenes
had to adapt to HTM by inducing amino acid biosyntheis. HTM is the simplest minimal medium available for growth of
L. monocytogenes
.
Mycobacterium xenopi
and Drinking Water Biofilms
Authors: Dailloux M. , Albert M. , Laurain C. , Andolfatto S. , Lozniewski A. , Hartemann P. , Mathieu L.
Published: November 2003

The ability of
Mycobacterium xenopi
to colonize an experimental drinking water distribution system (a Propella reactor) was investigated.
M. xenopi
was present in the biofilm within an hour following its introduction. After 9 weeks, it was always present in the outlet water (1 to 10 CFU 100 ml
−1
) and inside the biofilm (10
2
to 10
3
CFU cm
−2
). Biofilms may be considered reservoirs for the survival of
M. xenopi
.
Association of Genomic O Island 122 of
Escherichia coli
EDL 933 with Verocytotoxin-Producing
Escherichia coli
Seropathotypes That Are Linked to Epidemic and/or Serious Disease
Authors: Karmali Mohamed A. , Mascarenhas Mariola , Shen Songhai , Ziebell Kim , Johnson Shelley , Reid-Smith Richard , Isaac-Renton Judith , Clark Clifford , Rahn Kris , Kaper James B.
Published: November 2003

The distribution of EDL 933 O island 122 (OI-122) was investigated in 70 strains of Verocytotoxin-producing
Escherichia coli
(VTEC) of multiple serotypes that were classified into five “seropathotypes” (A through E) based on the reported occurrence of serotypes in human disease, in outbreaks, and/or in the hemolytic-uremic syndrome (HUS). Seropathotype A comprised 10 serotype O157:H7 and 3 serotype O157:NM strains. Seropathotype B (associated with outbreaks and HUS but less commonly than serotype O157:H7) comprised three strains each of serotypes O26:H11, O103:H2, O111:NM, O121:H19, and O145:NM. Seropathotype C comprised four strains each of serotypes O91:H21 and O113:H21 and eight strains of other serotypes that have been associated with sporadic HUS but not typically with outbreaks. Seropathotype D comprised 14 strains of serotypes that have been associated with diarrhea but not with outbreaks or HUS, and seropathotype E comprised animal VTEC strains of serotypes not implicated in human disease. All strains were tested for four EDL 933 OI-122 virulence genes (Z4321, Z4326, Z4332, and Z4333) by PCR. Negative PCRs were confirmed by Southern hybridization. Overall, 28 (40%) strains contained OI-122 (positive for all four virulence genes), 27 (38.6%) contained an “incomplete” OI-122 (positive for one to three genes), and 15 (21.4%) strains did not contain OI-122. The seropathotype distribution of complete OI-122 was as follows: 100% for seropathotype A, 60% for B, 36% for C, 15% for D, and 0% for E. The differences in the frequency of OI-122 between seropathotypes A, B, and C (associated with HUS) and seropathotypes D and E (not associated with HUS) and between seropathotypes A and B (associated with epidemic disease) and seropathotypes C, D, and E (not associated with epidemic disease) were highly significant (
P
< 0.0001).
C
6
Test as an Indicator of Therapy Outcome for Patients with Localized or Disseminated Lyme Borreliosis
Authors: Philipp Mario T. , Marques Adriana R. , Fawcett Paul T. , Dally Leonard G. , Martin Dale S.
Published: November 2003

Management of Lyme disease would benefit from a test to assess therapy outcome. Such a test could be employed to ascertain if treatment of early Lyme disease was successful and would be helpful to clinicians assessing patients with lingering posttreatment symptoms. We reported recently that levels of the antibody to C
6
, a
Borrelia burgdorferi-
derived peptide that is used as an antigen in the C
6
-Lyme diagnostic test, declined after successful antibiotic treatment of Lyme borreliosis patients. We assessed retrospectively the change in anti-C
6
antibody titers in 131 patients with either early localized disease (erythema migrans) or disseminated disease. All of these patients were treated with antibiotics and were free of the clinical signs shown at presentation within 12 weeks after the initiation of treatment. Decreases in reciprocal geometric mean titers (rGMT) of the anti-C
6
antibody were quantified for the subpopulation of 45 patients whose baseline rGMT were ≥80 and whose second serum specimens were obtained at least 6 months after the baseline specimen. Eighty percent of this patient group (36 of 45) experienced a ≥4-fold decrease in their rGMT (
P
< 0.0003). These results suggest that a change in the anti-C
6
antibody titer may serve as an indicator of therapy outcome for patients with localized or disseminated Lyme borreliosis.
Study of Animal-Borne Infections in the Mucosas of Patients with Inflammatory Bowel Disease and Population-Based Controls
Authors: Bernstein Charles N. , Nayar Gopi , Hamel Andre , Blanchard James F.
Published: November 2003

Crohn's disease may be triggered by an infection, and it is plausible to consider that such an infection may be animal borne and ingested with our food. There has been considerable interest in the past in determining whether
Mycobacterium avium
subsp.
paratuberculosis
(
M. avium
) might be the etiologic agent in Crohn's disease since it causes a disease in cattle that is similar to Crohn's disease in humans. We aimed to determine if there was an association between Crohn's disease and infection with
M. avium
or other zoonotic agents and compared the findings with those for patients with ulcerative colitis, unaffected siblings of Crohn's disease patients, or population-based controls without inflammatory bowel disease. Patients under age 50 years with Crohn's disease or ulcerative colitis, unaffected siblings of patients, or healthy controls drawn from a population-based age- and gender-matched registry were enrolled in a study in which subjects submitted to a questionnaire survey and venipuncture. A nested cohort underwent colonoscopy plus biopsy. Samples were batched and submitted to PCR for the detection of
M. avium
and other zoonotic agents known to cause predominately intestinal disease in cattle, sheep, or swine. Only one patient with ulcerative colitis, no patients with Crohn's disease, and none of the sibling controls were positive for
M. avium
, whereas 6 of 19 healthy controls were positive for
M. avium
. Since the control subjects were significantly older than the case patients, we studied another 11 patients with inflammatory bowel disease who were older than age 50 years, and another single subject with ulcerative colitis was positive for
M. avium
. One other subject older than age 50 years with ulcerative colitis was positive for circovirus, a swine-borne agent of infection. In conclusion, by performing PCR with mucosal samples from patients with Crohn's disease and controls, no association between Crohn's disease and infection with
M. avium
or any of the other six zoonotic agents studied could be found.
Evaluation of the Polymorphisms Associated with Tandem Repeats for
Pseudomonas aeruginosa
Strain Typing
Authors: Onteniente Lucie , Brisse Sylvain , Tassios Panayotis T. , Vergnaud Gilles
Published: November 2003

We report on the development of a scheme for the typing of
Pseudomonas aeruginosa
, multiple-locus variable number of tandem repeat (VNTR) analysis (MLVA). We first evaluated the polymorphisms of 201 tandem repeat loci selected from more than 3,000 such sequences present in strain PAO1 with a test collection of 12 genotypically distinct clinical strains. Seven VNTR loci which can be easily scored with the technology used here were identified and used to genotype a collection of 89 clinical isolates that had previously been classified into 46 ribotypes, including 2 widespread ribotypes. Seventy-one different MLVA genotypes could be distinguished. With only two exceptions, strains with identical ribotypes were grouped together upon cluster analysis of the MLVA data. The 27 isolates with the most frequent ribotype were divided into 14 MLVA types, and the 18 isolates with the second most frequent ribotype were divided into 15 MLVA types. Analysis of a subset of 17 strains belonging to the major ribotype by pulsed-field gel electrophoresis with the enzyme
Spe
I distinguished seven types, identical to the number of MLVA types in this subset. Our data show that MLVA typing of
P. aeruginosa
based on the first set of loci has a high discriminatory power. Because MLVA is highly reproducible and easily portable among laboratories, it represents a very promising tool for the molecular surveillance of
P. aeruginosa
. A free, online strain identification service based on the genotyping data produced herein has been developed.
Comparison of the NucliSens Basic Kit (Nucleic Acid Sequence-Based Amplification) and the Argene Biosoft Enterovirus Consensus Reverse Transcription-PCR Assays for Rapid Detection of Enterovirus RNA in Clinical Specimens
Authors: Landry Marie L. , Garner Robin , Ferguson David
Published: November 2003

Samples were tested for enterovirus by nucleic acid sequence-based amplification (NASBA) (NucliSens Basic kit; BioMerieux), reverse transcription-PCR (RT-PCR) (Enterovirus Consensus RT-PCR kit; Argene Biosoft), and virus isolation. Eighty-two samples were tested, and 44 were positive, 34 by both NASBA and RT-PCR and 5 each by NASBA or RT-PCR only. Two nasopharyngeal samples positive only by RT-PCR were determined to be rhinovirus. Of 42 enterovirus-positive samples, NASBA detected 39 (92.9%) and RT-PCR detected 37 (88.1%). The NucliSens Basic kit and the Argene Biosoft RT-PCR had comparable sensitivities for detection of enterovirus RNA, and both molecular methods were more sensitive than culture, which detected only 60.5% of positive samples. NASBA could be completed in 6.5 h versus 9 h for the Argene Biosoft RT-PCR kit.
Pulsed-Field Gel Electrophoresis Typing of Oxacillin-ResistantStaphylococcus aureusIsolates from the United States: Establishing a National Database
Authors: McDougal Linda K. , Steward Christine D. , Killgore George E. , Chaitram Jasmine M. , McAllister Sigrid K. , Tenover Fred C.
Published: November 2003

Oxacillin-resistantStaphylococcus aureus(ORSA) is a virulent pathogen responsible for both health care-associated and community onset disease. We usedSmaI-digested genomic DNA separated by pulsed-field gel electrophoresis (PFGE) to characterize 957S. aureusisolates and establish a database of PFGE patterns. In addition to PFGE patterns of U.S. strains, the database contains patterns of representative epidemic-type strains from the United Kingdom, Canada, and Australia; previously described ORSA clonal-type isolates; 13 vancomycin-intermediateS. aureus(VISA) isolates, and two high-level vancomycin-resistant,vanA-positive strains (VRSA). Among the isolates from the United States, we identified eight lineages, designated as pulsed-field types (PFTs) USA100 through USA800, seven of which included both ORSA and oxacillin-susceptibleS. aureusisolates. With the exception of the PFT pairs USA100 and USA800, and USA300 and USA500, each of the PFTs had a unique multilocus sequence type andspatype motif. The USA100 PFT, previously designated as the New York/Tokyo clone, was the most common PFT in the database, representing 44% of the ORSA isolates. USA100 isolates were typically multiresistant and included all but one of the U.S. VISA strains and both VRSA isolates. Multiresistant ORSA isolates from the USA200, -500, and -600 PFTs have PFGE patterns similar to those of previously described epidemic strains from Europe and Australia. The USA300 and -400 PFTs contained community isolates resistant only to β-lactam drugs and erythromycin. Noticeably absent from the U.S. database were isolates with the previously described Brazilian and EMRSA15 PFGE patterns. These data suggest that there are a limited number of ORSA genotypes present in the United States.
Microarrays for Genotyping Human Group A Rotavirus by Multiplex Capture and Type-Specific Primer Extension
Authors: Lovmar Lovisa , Fock Caroline , Espinoza Felix , Bucardo Filemon , Syvänen Ann-Christine , Bondeson Kåre
Published: November 2003

Human group A rotavirus (HRV) is the major cause of severe gastroenteritis in infants worldwide. HRV shares the feature of a high degree of genetic diversity with many other RNA viruses, and therefore, genotyping of this organism is more complicated than genotyping of more stable DNA viruses. We describe a novel microarray-based method that allows high-throughput genotyping of RNA viruses with a high degree of polymorphism by multiplex capture and type-specific extension on microarrays. Denatured reverse transcription (RT)-PCR products derived from two outer capsid genes of clinical isolates of HRV were hybridized to immobilized capture oligonucleotides representing the most commonly occurring P and G genotypes on a microarray. Specific primer extension of the type-specific capture oligonucleotides was applied to incorporate the fluorescent nucleotide analogue cyanine 5-labeled dUTP as a detectable label. Laser scanning and fluorescence detection of the microarrays was followed by visual or computer-assisted interpretation of the fluorescence patterns generated on the microarrays. Initially, the method detected HRV in all 40 samples and correctly determined both the G and the P genotypes of 35 of the 40 strains analyzed. After modification by inclusion of additional capture oligonucleotides specific for the initially unassigned genotypes, all genotypes could be correctly defined. The results of genotyping with the microarray fully agreed with the results obtained by nucleotide sequence analysis and sequence-specific multiplex RT-PCR. Owing to its robustness, simplicity, and general utility, the microarray-based method may gain wide applicability for the genotyping of microorganisms, including highly variable RNA and DNA viruses.
Rapid Colorimetric Method for Testing Susceptibility of
Mycobacterium tuberculosis
to Isoniazid and Rifampin in Liquid Cultures
Authors: Syre H. , Phyu S. , Sandven P. , Bjorvatn B. , Grewal H. M. S.
Published: November 2003

We have developed a rapid colorimetric method for testing the susceptibility of
M. tuberculosis
to isoniazid (INH) and rifampin (RIF) based on incorporation of nitrate in broth cultures containing growth supplements. The performance of this colorimetric nitrate reductase-based antibiotic susceptibility (CONRAS) test was compared with that of the radiometric BACTEC 460TB system in determining the susceptibilities of 74
M. tuberculosis
strains to INH and RIF. By using the BACTEC 460TB system as the “gold standard,” the sensitivity (i.e., the ability to detect true drug resistance) and specificity (i.e., the ability to detect true drug susceptibility) of the CONRAS test were 100 and 95% for INH and 94 and 100% for RIF, respectively. The repeatability of the CONRAS test was excellent (for INH, kappa = 1 and
P
< 0.001; for RIF, kappa = 0.88 and
P
< 0.001). For the majority of strains, results were obtained within 5 days. The CONRAS test is rapid, accurate, and inexpensive and is an adequate alternative, particularly for resource-poor countries.
agr
-Dependent Bacterial Interference Has No Impact on Long-Term Colonization of
Staphylococcus aureus
during Persistent Airway Infection of Cystic Fibrosis Patients
Authors: Kahl Barbara C. , Becker Karsten , Friedrich Alexander W. , Clasen Julia , Sinha Bhanu , von Eiff Christof , Peters Georg
Published: November 2003

The
agr
specificity group distribution of persistent
Staphylococcus aureus
clones recovered from the airways of cystic fibrosis (CF) patients did not differ from that of isolates recovered from various clinical infections and healthy nasal carriers. The success of CF clones in terms of cocolonization and/or infection with
S. aureus
, prevalence of clones, or persistence appeared to be independent of
agr
group specificity.
Characterization of Genetic Differences between
Mycobacterium avium
subsp.
paratuberculosis
Type I and Type II Isolates
Authors: Dohmann Karen , Strommenger Birgit , Stevenson Karen , de Juan Lucía , Stratmann Janin , Kapur Vivek , Bull Tim J. , Gerlach Gerald-Friedrich
Published: November 2003

A combination of representational difference analysis and comparative DNA sequencing revealed that four type I (sheep) isolates of
Mycobacterium avium
subsp.
paratuberculosis
were differentiated from nine type II (bovine) isolates by the presence of an 11-bp insertion in a novel
M. avium
subsp.
paratuberculosis
-specific region of genomic DNA. Further, our studies show that
M. avium
subsp.
paratuberculosis
type I isolates contain three type-specific loci that are missing in
M. avium
subsp.
paratuberculosis
type II but are present in
M. avium
subsp.
avium
. Taken together, the results are consistent with the hypothesis that
M. avium
subsp.
paratuberculosis
type I strains are an evolutionary intermediate between
M. avium
subsp.
avium
and
M. avium
subsp.
paratuberculosis
type II isolates or share a subset of
M. avium
subsp.
avium
type-specific loci through horizontal transfer.
Risk of Fungemia Due to
Rhodotorula
and Antifungal Susceptibility Testing of
Rhodotorula
Isolates
Authors: Zaas Aimee K. , Boyce Molly , Schell Wiley , Lodge Barbara Alexander , Miller Jackie L. , Perfect John R.
Published: November 2003

Rhodotorula
infections occur among patients with immunosuppression and/or central venous catheters. Using standardized methods (NCCLS M27-A), we determined the antifungal susceptibilities of 10
Rhodotorula
bloodstream infection isolates. Patient information was collected for clinical correlation. The MICs of amphotericin B and posaconazole were the lowest, and the MICs of triazoles and echinocandins were higher than those of other antifungal agents.
Interlaboratory Evaluation of Hematocytometer Method of Inoculum Preparation for Testing Antifungal Susceptibilities of Filamentous Fungi
Authors: Rodriguez-Tudela J. L. , Chryssanthou Erja , Petrikkou Evangelia , Mosquera Juan , Denning David W. , Cuenca-Estrella Manuel
Published: November 2003

A three-laboratory study was performed to evaluate conidium counting in a hematocytometer as a technique of inoculum preparation for susceptibility testing of
Aspergillus
spp. In addition, inocula were quantified by colony counting and optical density determination. The agreement and correlation coefficient between conidium and colony quantifications were 89.2% and 0.73 (
P
< 0.01). Correlations with optical density determination were not significant.
Deep Infection by
Trichophyton rubrum
in an Immunocompromised Patient
Authors: Nir-Paz Ran , Elinav Hila , Pierard Gerald E. , Walker David , Maly Alexander , Shapiro Mervyn , Barton Richard C. , Polacheck Itzhack
Published: November 2003

Dermatophytes are common pathogens of skin but rarely cause invasive disease. We present a case of deep infection by
Trichophyton rubrum
in an immunocompromised patient.
T. rubrum
was identified by morphological characteristics and confirmed by PCR. Invasiveness was apparent by histopathology and immunohistochemistry. The patient was treated successfully with itraconazole.