bluuebunny/crossref_metadata_2025 huggingface dataset:
Searching for tubulin‐like genes in free‐living bacteria and symbionts of ciliates. Characterization of the genomic environment of tubulin genes from Prosthecobater
Authors: PILHOFER MARTIN , ROSATI GIOVANNA , LUDWIG WOLFGANG , SCHLEIFER KARL‐HEINZ , PETRONI GIULIO
Published: April 2005

Tubulins are typical eucaryotic genes, which have recently been found in the free‐living bacteria Prosthecobacter (Verrucomicrobiales). Nevertheless, microtubule‐like structures were never observed in Prostecobacter, whereas they were reported in epixenosomes which are symbiotic Verrucomicrobiales of the ciliate Euplotidium. In the present work, an extensive search for tubulin like genes was performed with several combinations of PCR‐primers, designed according to different criteria. Although some combinations of these primers are able to amplify simultaneously eukaryotic α‐ and β‐tubulin as well as Prosthecobacter tubulin genes, no bacterial‐like tubulin genes were identified in other Verrucomicrobiales species. Because of host derived contaminations, even a subtractive PCR approach, using microtiter‐plate‐subtractive‐hybridization, was used with the epixenosomes. The characterization of the genomic environment of the Prosthecobacter tubulin genes was performed by gene walking. The sequences obtained from P. debontii revealed a so‐far undiscovered gene duplication. The bacterial tubulin operon looks conserved among Prosthecobacter species and includes an α‐tubulin, a β‐tubulin and a kinesin like protein. Upstream of two operons a repetetive sequence could be found which could be a hint for horizontal gene transfer. The analysis of the genomic environments of the tubulin genes provided open reading frames, which have homology to Verrucomicrobium spinosum genes (ongoing genome project data), whereas up to now the blast search did not reveal tubulin‐like genes in this organism. Present results suggest, that the Prosthecobacter tubulin genes are possibly of recent origin, maybe acquired by horizontal gene transfer, or lost by other so far studied Verrucomicrobiales. The origin and nature of microtubular genetic determinants from epixenosomes remains open.
The application of molecular techniques to the surveillance and control of leishmaniasis in Italy: the facts and doubts
Authors: GRAMICCIA MARINA , MUCCIO TRENTINA DI , SCALONE ALDO , ROSSI ERIKA , BONGIORNO GIOIA , MAROLI MICHELE , POGLAYEN GIOVANNI , GRADONI LUIGI
Published: April 2005

Leishmaniases are parasitic diseases caused by various species of the protozoan Leishmania. In Italy human visceral and cutaneous leishmaniases are caused by Leishmania infantum, which has the dog as domestic reservoir, and different Phlebotominae species as the vector. Several molecular techniques are currently used with success for a rapid, very sensitive and non‐invasive detection of Leishmania organisms in specimens of infected organs. The high sensitivity, however, may raise doubts on the significance of target DNA detection, which may represent either an established infection—with or without epidemiological importance—or just a trace of parasite–host contacts. Three examples of the application of nested PCR technique using rDNA sequence primers on different biological samples, are reported here: (a) five different bioptic samples were examined from 153 dogs of an endemic focus of canine leishmaniasis (CanL) (Salina Island, Sicily), giving a Leishmania prevalence of 82.8%, in contrast with 31.4% seroprevalence; (b) in a new CanL focus of Rome province; 60/127 Phlebotomus perniciosus (45.7,0%) were found positive by nested PCR, in contrast with 1.4% microscopically positive; and (c) leishmanial DNA was detected in a natural case of leishmaniasis in a novel mammal host, a domestic ferret (Mustela putorius furo). These results raise the following doubts, respectively: (a) Does the high “infection” prevalence detected in dogs correspond to the “infectiveness‐to‐the vector” prevalence? (b) Can the positive sandflies transmit infective promastigotes? and (c) Can an infected domestic ferret play a role as parasite reservoir?
A METHOD FOR ESTIMATING GLOBAL CORPORATE CAPITAL COSTS: THE CASE OF BESTFOODS
Authors: Pettit Justin , Ferguson Mack , Gluck Robert
Published: April 2005

The financial management practices of many multinational corporations are at odds with both financial theory and the strategic case for global expansion. Despite the weight of academic literature, many financial executives still cling to ad hoc rules of thumb that discourage value‐enhancing global growth. In particular, they tend to require large risk premiums for making foreign investments while ignoring the diversification benefits of such investments for their shareholders.

This article presents a practical method for estimating the cost of capital for use by multinationals both in evaluating foreign investment opportunities and in measuring the ongoing performance of overseas business units. The method represents a kind of “hybrid” version of the global CAPM—one that attempts to reconcile some of corporate executives' concerns about the distinctive risks of foreign investment with the finance theorist's portfolio perspective and reliance on capital market information. More specifically, the framework uses information from capital markets to determine the appropriate risk premiums for currency and sovereign risks associated with each country in an MNC's portfolio. But, at the same time, these risk premiums are partly offset by taking account of any diversification benefits that foreign investment provides for the firm's shareholders.

The method is illustrated using the case of Bestfoods, a Fortune 200 company with extensive overseas operations that recently adopted the method. For the purpose of evaluating new projects, Bestfoods produces quarterly updates of its cost‐of‐capital estimates for each country in which it has (or expects to have) major operations. For evaluating the ongoing performance of each country business unit, the relevant cost of capital is calculated annually (at the beginning of each fiscal year).
WHEN SHAREHOLDERS CHOOSE NOT TO MAXIMIZE VALUE: THE UNION BANK OF SWITZERLAND'S 1994 PROXY FIGHT
Authors: Loderer Claudio , Zgraggen Plus
Published: April 2005

This paper examines the fight over a share reunification plan that pitted Swiss financier Martin Ebner against Union Bank of Switzerland (UBS), once the largest Swiss bank and a global leader in asset management. In the U.S. corporate governance debate, large shareholders are often held up as a possible solution to corporate governance problems. But this examination of the UBS proxy fight shows why some large shareholders can themselves be a source of governance problems.

The share reunification plan was designed to combine the firm's two classes of stock: registered (voting) shares and bearer (non‐voting) shares. As its motive for the plan, the UBS board cited a desire both to increase liquidity and to prevent a control change. The majority of the holders of the company's registered stock—many of whom had other financial ties to the bank—ended up voting for a proposal that caused them to lose 11% of the value of their shares during the three trading days following its announcement, and eventually almost twice as much. Moreover, even the holders of the bearer stock hurt themselves by approving the reunification plan. Although the plan clearly transferred value from the registered to the bearer shares, the redistribution benefits from the plan for the bearer shareholders were not sufficient to offset their losses from the reduced probability of a control change.

Although it did not succeed in accomplishing its immediate goal, the UBS proxy fight is today recognized as a watershed event in Swiss corporate governance. Until recently, Switzerland's economy has been dominated by cartels, with closely overlapping boards of directors. Hostile takeovers were essentially unheard of, and criticism of management by shareholders was highly unusual. Ebner's activities have had the effect of stimulating public debate about shareholders' rights and shareholder activism. In so doing, they have made it more acceptable for shareholders to criticize management and established shareholder value as a “politically correct” goal for Swiss corporations.
CONVERTIBLE SECURITIES: A TOLLBOX OF FLEXIBLE FINANCIAL INSTRUMENTS FOR CORPORATE ISSUERS
Authors: Ganshaw Trevor , Dillon Derek
Published: April 2005

During the 1990s, convertible and equity‐linked securities emerged as a major source of financing for U.S. corporate issuers. Issuance volume grew steadily throughout the decade and the secondary market value of U.S. convertible securities now exceeds $200 billion. In this overview of the market, the authors discuss the following: (1) the growth of issuance volume in the U.S. equity‐linked market; (2) the basic characteristics of convertible securities; (3) convertible debt alternatives; and (4) convertible preferred alternatives.

As a result of the proliferation of new convertible structures, corporate issuers are now able to adjust coupon/dividend, conversion premium, and call protection in order to meet their tax, accounting, rating agency, and cost‐of‐capital objectives. Historically, the convertible new issue market has had a broad variety of issuers, spanning all industry sectors as well as both investment grade and high yield credits. But in the last two years, the most aggressive issuers have been technology‐oriented companies, including telecommunications, Internet, hardware, software, and biotechnology concerns. Such technology‐related issuers, which are often rated below investment grade and unable to secure straight debt capital, are generally in heavy‐spending phases and view convertible bonds as a source of inexpensive financing. At the same time, investment‐grade, “old‐economy” issuers have continued to use convertible securities selectively, in most cases as cheap “quasi‐equity” in the context of mergers and acquisitions, or as a tax‐deferred strategy for selling cross‐holdings of stock.
WHAT DO WE KNOW ABOUT STOCK REPURCHASES?
Authors: Grullon Gustavo , Ikenberry David L.
Published: April 2005

Stock repurchases by U.S. companies experienced a remarkable surge in the 1980s and ‘90s. Indeed, in 1998, the total value of all stock repurchased by U.S. companies exceeded for the first time the total amount paid out as cash dividends. And the U.S. repurchase movement has gone global in the past few years, spreading not only to Canada and the U.K., but also to countries like Japan and Germany, where such transactions were prohibited until recently.

Why are companies buying back their stock in such amounts? After dismissing the popular argument that stock repurchases boost earnings per share, the authors argue that repurchases serve to add value in two main ways: (1) they provide managers with a tax‐efficient means of returning excess capital to shareholders and (2) they allow managers to “signal” to investors their view that the firm is undervalued. Returning excess capital is value‐adding for two reasons: First, it helps prevent companies from pursuing growth and size at the expense of profitability and value. Second, by returning capital to investors, repurchases (like dividends) play the critically important economic function of allowing investors to channel their investment from mature or declining sectors of the economy to more promising ones.

But if stock repurchases and dividends serve the same basic economic function, why are repurchases growing more rapidly? Part of the explanation is that, because repurchases are taxed as capital gains and dividends as ordinary income, repurchases are a more tax‐efficient way of distributing excess capital. But perhaps even more important than their tax treatment is the flexibility that (at least) open market repurchases provide corporate managers‐flexibility to make small adjustments in capital structure, to exploit (or correct) perceived undervaluation of the firm's shares, and possibly even to increase the liquidity of the stock, which could be particularly valuable in bear markets.

For U.S. regulators, the growth in open market stock repurchases raises some interesting issues. Perhaps most important, companies are not required to (and rarely do) furnish their investors with details about a given program's structure, execution method, number of shares repurchased, or even its duration. Policy regulators (and corporate executives as well) should consider some of the benefits provided by other systems, notably Canada's, which provide greater transparency and more guidelines for the repurchase process.
CAPITAL FORMATION AND THE INTERNET
Authors: Aggarwal Reena , Dahiya Sandeep
Published: April 2005

The Internet is expected to play a significant role in the capital‐raising process. Internet investment banks like Wit Capital and WR Hambrecht are supposed to make the IPO process more equitable by giving retail investors access to deals and pricing deals more accurately, thereby leaving less “money on the table” and lowering the cost of going public.

This article argues that the Internet will not replace, but will likely “supplement,” the current system. The certification function provided by traditional investment banks and their relationships with institutional investors will continue to be important determinants of a successful offering. Thus, although Internet banks will get pieces of IPO transactions, the lead managers of such deals will continue to be older firms with well‐established reputations and ties with institutions.

Nevertheless, the Internet is expected to play a larger role in the case of public bond offerings. Because the issuance of bonds is a repetitive business and the pricing is much simpler, the authors predict that the Internet will significantly reduce the costs of issuing bonds and perhaps limit the role of traditional investment banks in this process.
CAPITAL MARKET EXCESSES AND COMPETITIVE STRENGTH: THE CASE OF THE HARD DISK DRIVE INDUSTRY 1984–2000
Authors: Bygrave William D. , Lange Julian E. , Roedel J. R. , Wu Gary
Published: April 2005

In a much‐cited 1985 study of the U.S. hard disk drive industry entitled “Capital Market Myopia,” William Sahlman and Howard Stevenson concluded that “excesses in the capital markets turned an opportunity into a disaster” for investors in the emerging industry. Sahlman and Stevenson fretted about what their findings implied not only for investors but also for the industrial competitiveness of the U.S.A.

With the benefit of hindsight, the research presented in this article shows how the hard disk drive industry has evolved in the past 15 years to the point where disk drives are a vital part of the information technology revolution that has transformed the way we live, work, and play. The authors' research examines the financial performance of both hard disk drive manufacturers and storage system manufacturers completely dependent on hard disk drives; looks at improvements in the technical performance of hard drives; and then compares the competitiveness of U.S. companies with foreign manufacturers.

After the venture capital/IPO bubble of the early 1980s gave way to the traditional discipline of U.S. financial markets, many weak companies were driven out of business and others were forced to consolidate. Only a handful of the startup firms survived. But these U.S companies, together with IBM, now dominate the global hard drive market. Moreover, there are striking parallels between the 1982–84 hard drive bubble and the recent experience of U.S. Internet firms. Although the first half of 2000 saw a large number of closings, forced sales, and IPO cancellations by American dot‐coms, a number of the likely “survivors”—notably, Yahoo, eBay, and Amazon.com—now seem poised to dominate their global markets.
DECENTRALIZATION, INCENTIVES, AND VALUE CREATION: THE CASE OF JLG INDUSTRIES
Authors: Treml Heidi E. , Lehn Kenneth
Published: April 2005

From 1993 through 1998, JLG Industries, a manufacturer of aerial work platforms, achieved dramatic improvements in operating efficiency and created substantial value for shareholders. One dollar invested in JLG stock at the end of 1992 would have been worth $15.82 at the end of 1998, whereas one dollar invested in a portfolio of industry peers would have grown to only $1.48 over the same period. Most of the value created by JLG can be traced to three factors: a highly successful product development program that generated substantial revenue; large cost savings resulting from improvements in operations; and a dramatic reduction in inventories. The authors attribute JLG's success to a strategic initiative that focused attention on key value drivers, decentralized decision‐making, and strengthened incentives for employees throughout the organization.

In the early 1980s JLG adopted several knowledge‐based management techniques, including TQM and just‐in time inventory, but at that time the company's decision‐making was more centralized and employee incentives did not correspond to strategic goals. After the departure of its founder in 1993, JLG decentralized decision‐making, strengthened employees' incentives, and achieved enormous success. Evidence from the JLG case supports the argument that the benefits of TQM and related management techniques are most likely to be achieved when companies have organizational structures and incentives that encourage the most effective use of those techniques. It also is consistent with recent data compiled by Industry Week that shows a direct relation between employee empowerment and corporate performance.
Experimental and Numerical Evaluation of Crack Arresting Capability Due to a Dimple
Authors: Nishimura Toshihiko
Published: April 2005

Crack arresting capability is experimentally examined using center cracked tension specimens and varying the placement of the dimple ahead of the crack tip. It is confirmed that the dimple gives a greater crack arrest effect than that of the stop hole method. Next, the fatigue life of smooth specimens is compared, both with and without dimples. It is indicated that the dimple has a benefit only to the repair case after the crack is initiated. Finally, stress intensity factors of a crack due to a dimple are numerically evaluated, and a guiding principle is clarified for placing dimples.
Dual Symbiosis in a
Bathymodiolus
sp. Mussel from a Methane Seep on the Gabon Continental Margin (Southeast Atlantic): 16S rRNA Phylogeny and Distribution of the Symbionts in Gills
Authors: Duperron Sébastien , Nadalig Thierry , Caprais Jean-Claude , Sibuet Myriam , Fiala-Médioni Aline , Amann Rudolf , Dubilier Nicole
Published: April 2005

Deep-sea mussels of the genus
Bathymodiolus
(Bivalvia: Mytilidae) harbor symbiotic bacteria in their gills and are among the dominant invertebrate species at cold seeps and hydrothermal vents. An undescribed
Bathymodiolus
species was collected at a depth of 3,150 m in a newly discovered cold seep area on the southeast Atlantic margin, close to the Zaire channel. Transmission electron microscopy, comparative 16S rRNA analysis, and fluorescence in situ hybridization indicated that this
Bathymodiolus
sp. lives in a dual symbiosis with sulfide- and methane-oxidizing bacteria. A distinct distribution pattern of the symbiotic bacteria in the gill epithelium was observed, with the thiotrophic symbiont dominating the apical region and the methanotrophic symbiont more abundant in the basal region of the bacteriocytes. No variations in this distribution pattern or in the relative abundances of the two symbionts were observed in mussels collected from three different mussel beds with methane concentrations ranging from 0.7 to 33.7 μM. The 16S rRNA sequence of the methanotrophic symbiont is most closely related to those of known methanotrophic symbionts from other bathymodiolid mussels. Surprisingly, the thiotrophic
Bathymodiolus
sp. 16S rRNA sequence does not fall into the monophyletic group of sequences from thiotrophic symbionts of all other
Bathymodiolus
hosts. While these mussel species all come from vents, this study describes the first thiotrophic sequence from a seep mussel and shows that it is most closely related (99% sequence identity) to an environmental clone sequence obtained from a hydrothermal plume near Japan.
Characterization of Cry34/Cry35 Binary Insecticidal Proteins from Diverse
Bacillus thuringiensis
Strain Collections
Authors: Schnepf H. Ernest , Lee Stacey , Dojillo JoAnna , Burmeister Paula , Fencil Kristin , Morera Lisa , Nygaard Linda , Narva Kenneth E. , Wolt Jeff D.
Published: April 2005

Bacillus thuringiensis
crystal proteins of the Cry34 and Cry35 classes function as binary toxins showing activity on the western corn rootworm,
Diabrotica virgifera virgifera
LeConte. We surveyed 6,499
B. thuringiensis
isolates by hybridization for sequences related to
cry35A
genes, identifying 78 strains. Proteins of the appropriate molecular mass (ca. 44 kDa) for Cry35 were observed in 42 of the strains. Full-length, or nearly full-length, sequences of 34
cry34
genes and 16
cry35
genes were also obtained from cloning, PCR analysis, and DNA sequencing. These included representatives of all known Cry34A, Cry34B, Cry35A, and Cry35B classes, as well as a novel Cry34A/Cry35A-like pair. Bioassay analysis indicated that
cry35
-hybridizing strains not producing a ca. 14-kDa protein, indicative of Cry34, were not active on corn rootworms, and that the previously identified Cry34A/Cry35A pairs were more active than the Cry34B/Cry35B pairs. The
cry35
-hybridizing
B. thuringiensis
strains were found in locales and materials typical for other
B. thuringiensis
strains. Comparison of the sequences with the geographic origins of the strains showed that identical, or nearly identical, sequences were found in strains from both Australasia and the Americas. Sequence similarity searches revealed that Cry34 proteins are similar to predicted proteins in
Photorhabdus luminescens
and
Dictyostelium discoidium
, and that Cry35Ab1 contains a segment similar to beta-trefoil domains that may be a binding motif. The binary Cry34/Cry35
B. thuringiensis
crystal proteins thus appear closely related to each other, are environmentally ubiquitous, and share sequence similarities consistent with activity through membrane disruption in target organisms.
Roles of Four Chitinases (ChiA, ChiB, ChiC, and ChiD) in the Chitin Degradation System of Marine Bacterium
Alteromonas
sp. Strain O-7
Authors: Orikoshi Hideyuki , Nakayama Shigenari , Miyamoto Katsushiro , Hanato Chiaki , Yasuda Masahide , Inamori Yoshihiko , Tsujibo Hiroshi
Published: April 2005

Alteromonas
sp. strain O-7 secretes four chitinases (ChiA, ChiB, ChiC, and ChiD) in the presence of chitin. To elucidate why the strain produces multiple chitinases, we studied the expression levels of the four genes and proteins, their enzymatic properties, and their synergistic effects on chitin degradation. Among the four chitinases, ChiA was produced in the largest quantities, followed by ChiD, and the production of ChiB and ChiC changed at lower levels than those of ChiA and ChiD. The expression of the
chiA
,
chiB
,
chiC
, and
chiD
genes was investigated at the transcriptional level. The RNA transcript of
chiA
was most strongly induced in the presence of chitin, the expression of
chiD
followed, and the RNA transcripts of
chiB
and
chiC
changed at low levels. The hydrolyzing activities of the four chitinases against various substrates were examined. ChiA was the most active enzyme against powdered chitin, whereas ChiC was the most active against soluble chitin among the four chitinases. ChiD had activities closer to those of ChiA than to those of ChiB and ChiC. ChiB showed no distinctive feature against the chitinous substrates tested. When powdered chitin was treated with the proper combination of four chitinases, an approximately 2.0-fold increase in the hydrolytic activity was observed. These results, together with the results described above, indicate that ChiA plays a central role in chitin degradation for this strain.
Rapid and Sensitive Detection of Noroviruses by Using TaqMan-Based One-Step Reverse Transcription-PCR Assays and Application to Naturally Contaminated Shellfish Samples
Authors: Jothikumar Narayanan , Lowther James A. , Henshilwood Kathleen , Lees David N. , Hill Vincent R. , Vinjé Jan
Published: April 2005

Noroviruses (NoV), which are members of the family
Caliciviridae
, are the most important cause of outbreaks of acute gastroenteritis worldwide and are commonly found in shellfish grown in polluted waters. In the present study, we developed broadly reactive one-step TaqMan reverse transcription (RT)-PCR assays for the detection of genogroup I (GI) and GII NoV in fecal samples, as well as shellfish samples. The specificity and sensitivity of all steps of the assays were systematically evaluated, and in the final format, the monoplex assays were validated by using RNA extracted from a panel of 84 stool specimens, which included NoV strains representing 19 different genotypes (7 GI, 11 GII, and 1 GIV strains). The assays were further validated with 38 shellfish cDNA extracts previously tested by nested PCR. Comparison with a recently described real-time assay showed that our assay had significantly higher sensitivity and was at least as sensitive as the nested PCR. For stool specimens, a one-step duplex TaqMan RT-PCR assay performed as well as individual genogroup-specific monoplex assays. All other enteric viruses examined were negative, and no cross-reaction between genogroups was observed. These TaqMan RT-PCR assays provide rapid (less than 90 min), sensitive, and reliable detection of NoV and should prove to be useful for routine monitoring of both clinical and shellfish samples.
Diversity of Transcripts of Nitrite Reductase Genes (
nirK
and
nirS
) in Rhizospheres of Grain Legumes
Authors: Sharma Shilpi , Aneja Manish Kumar , Mayer Jochen , Munch Jean Charles , Schloter Michael
Published: April 2005

Transcription of the
nirK
and
nirS
genes coding for dissimilatory bacterial nitrite reductases was analyzed by reverse transcription PCR (RT-PCR) of mRNA isolated from rhizosphere samples of three economically important grain legumes at maturity:
Vicia faba
,
Lupinus albus
, and
Pisum sativum
. The
nirK
gene and transcripts could be detected in all the rhizosphere samples. In contrast,
nirS
could not be detected. Sampling variations were analyzed by comparing denaturing gradient gel electrophoresis profiles derived from
nirK
RT-PCR products. High similarity was observed between the replicates, and so one representative product per legume was cloned. Clones with the correct insert size were screened by restriction fragment length polymorphism by using the restriction enzyme MspI. The clones could be distributed into 12 different patterns. Patterns 1, 3, 4, 5, and 7 were common in clone libraries of the three rhizosphere types under study. Patterns 2, 9, 10, and 11 were absent from
Pisum
rhizospheres, while patterns 6, 8, and 12 were absent from the
Vicia
library. Pattern 1, which was the most dominant in the
Vicia
and
Lupinus
libraries, constituted about 25% of all clones. The
Lupinus
library had clones representing all 12 patterns, indicating it to be the most diverse among the three. Clones representative of each pattern were sequenced. All patterns grouped together forming a distinct cluster, which was divergent from previously described
nirK
sequences in the database. The study revealed a hitherto unknown diversity of denitrifiers in legume rhizospheres. A plant-dependent rhizosphere effect on the transcripts of a gene was evident.
Xanthomonas axonopodis
pv. phaseoli var. fuscans Is Aggregated in Stable Biofilm Population Sizes in the Phyllosphere of Field-Grown Beans
Authors: Jacques M.-A. , Josi K. , Darrasse A. , Samson R.
Published: April 2005

The occurrence of “
Xanthomonas axonopodis
pv. phaseoli var. fuscans” (proposed name) populations as biofilms on bean leaves was investigated during three field experiments on plots established with naturally contaminated bean seeds. Behavior of aggregated versus solitary populations was determined by quantification of culturable cells in different fractions of the epiphytic population separated by particle size.
X. axonopodis
pv. phaseoli var. fuscans population dynamic studies confirmed an asymptomatic and epiphytic colonization of the bean phyllosphere. For all years of experiment and cultivars tested, biofilms and solitary components of the populations were always detected. Biofilm population sizes remained stable throughout the growing season (around 10
5
CFU/g of fresh weight) while solitary population sizes were more abundant and varied with climate. According to enterobacterial repetitive intergenic consensus fingerprinting, aggregated bacterial isolates were not different from solitary isolates. In controlled conditions, application of a hydric stress resulted in a decrease of the solitary populations on the leaf surface while the biofilm fraction remained stable. Suppression of the hydric stress allowed solitary bacterial populations to increase again. Aggregation in biofilms on leaf surfaces provides protection to the bacterial cells against hydric stress.
Improved Assessment of Denitrifying, N
2
-Fixing, and Total-Community Bacteria by Terminal Restriction Fragment Length Polymorphism Analysis Using Multiple Restriction Enzymes
Authors: Rösch Christopher , Bothe Hermann
Published: April 2005

A database of terminal restriction fragments (tRFs) of the 16S rRNA gene was set up utilizing 13 restriction enzymes and 17,327 GenBank sequences. A computer program, termed TReFID, was developed to allow identification of any of these 17,327 sequences by means of polygons generated from the specific tRFs of each bacterium. The TReFID program complements and exceeds in its data content the Web-based phylogenetic assignment tool recently described by A. D. Kent, D. J. Smith, B. J. Benson, and E. W. Triplett (Appl. Environ. Microb.
69:
6768-6766, 2003). The method to identify bacteria is different, as is the region of the 16S rRNA gene employed in the present program. For the present communication the software of the tRF profiles has also been extended to allow screening for genes coding for N
2
fixation (
nifH
) and denitrification (
nosZ
) in any bacterium or environmental sample. A number of controls were performed to test the reliability of the TReFID program. Furthermore, the TReFID program has been shown to permit the analysis of the bacterial population structure of bacteria by means of their 16S rRNA,
nifH
, and
nosZ
gene content in an environmental habitat, as exemplified for a sample from a forest soil. The use of the TReFID program reveals that noncultured denitrifying and dinitrogen-fixing bacteria might play a more dominant role in soils than believed hitherto.
Nonbioluminescent Strains ofPhotobacterium phosphoreumProduce the Cell-to-Cell Communication SignalN-(3-Hydroxyoctanoyl)homoserine Lactone
Authors: Flodgaard L. R. , Dalgaard P. , Andersen J. B. , Nielsen K. F. , Givskov M. , Gram L.
Published: April 2005

Bioluminescence is a common phenotype in marine bacteria, such asVibrioandPhotobacteriumspecies, and can be quorum regulated byN-acylated homoserine lactones (AHLs). We extracted a molecule that induced a bacterial AHL monitor (Agrobacterium tumefaciensNT1 [pZLR4]) from packed cod fillets, which spoil due to growth ofPhotobacterium phosphoreum. Interestingly, AHLs were produced by 13 nonbioluminescent strains ofP. phosphoreumisolated from the product. Of 177 strains ofP. phosphoreum(including 18 isolates from this study), none of 74 bioluminescent strains elicited a reaction in the AHL monitor, whereas 48 of 103 nonbioluminescent strains did produce AHLs. AHLs were also detected inAeromonasspp., but not inShewanellastrains. Thin-layer chromatographic profiles of cod extracts andP. phosphoreumculture supernatants identified a molecule similar in relative mobility (Rfvalue) and shape toN-(3-hydroxyoctanoyl)homoserine lactone, and the presence of this molecule in culture supernatants from a nonbioluminescent strain ofP. phosphoreumwas confirmed by high-performance liquid chromatography-positive electrospray high-resolution mass spectrometry. Bioluminescence (in a non-AHL-producing strain ofP. phosphoreum) was strongly up-regulated during growth, whereas AHL production in a nonbioluminescent strain ofP. phosphoreumappeared constitutive. AHLs apparently did not influence bioluminescence, as the addition of neither synthetic AHLs nor supernatants delayed or reduced this phenotype in luminescent strains ofP. phosphoreum. The phenotypes of nonbioluminescentP. phosphoreumstrains regulated by AHLs remains to be elucidated.
Opposite Enantioselectivities of Two Phenotypically and Genotypically Similar Strains of
Pseudomonas frederiksbergensis
in Bacterial Whole-Cell Sulfoxidation
Authors: Adam Waldemar , Heckel Frank , Saha-Möller Chantu R. , Taupp Marcus , Meyer Jean-Marie , Schreier Peter
Published: April 2005

Soil samples were screened to select microorganisms with the capability to oxidize organic sulfides into the corresponding sulfoxides with differential enantioselectivities. Several bacterial strains that preferentially produced the
S-
configured sulfoxide enantiomer were isolated. Surprisingly, one bacterial strain, genotypically and phenotypically characterized as
Pseudomonas frederiksbergensis
, selectively gave the
R
enantiomer. The finding that two apparently identical organisms displayed opposite enantioselectivities is novel for non-genetically modified organisms.
Use of Specific rRNA Oligonucleotide Probes for Microscopic Detection of
Mycobacterium avium
Complex Organisms in Tissue
Authors: St. Amand Allison L. , Frank Daniel N. , De Groote Mary Ann , Pace Norman R.
Published: April 2005

Members of the
Mycobacterium avium
complex (MAC) are important environmental pathogens that are implicated in several chronic, idiopathic diseases. Diagnosis of MAC-based diseases is compromised by the need to cultivate these fastidious and slowly growing organisms in order to identify which mycobacterial species are present. Detection is particularly difficult when MAC is intracellular or embedded within mammalian tissues. We report on the development of culture-independent, in situ hybridization (ISH) assays for the detection of MAC in culture, sputum, and tissue. This assay includes a highly reliable technique for the permeabilization of mycobacterial cells within culture and tissues. We describe a set of rRNA-based oligonucleotide probes that specifically detect either
M. intracellulare
, the two
M. avium
subspecies associated with human disease, or all members of MAC. The results call into question the validity of ISH results derived by the use of other gene loci, such as IS
900
.
Identification of Clinically Relevant Viridans Streptococci by an Oligonucleotide Array
Authors: Chen Chao Chien , Teng Lee Jene , Kaiung Seng , Chang Tsung Chain
Published: April 2005

Viridans streptococci (VS) are common etiologic agents of subacute infective endocarditis and are capable of causing a variety of pyogenic infections. Many species of VS are difficult to differentiate by phenotypic traits. An oligonucleotide array based on 16S-23S rRNA gene intergenic spacer (ITS) sequences was developed to identify 11 clinically relevant VS. These 11 species were
Streptococcus anginosus
,
S. constellatus
,
S. gordonii
,
S. intermedius
,
S. mitis
,
S. mutans
,
S. oralis
,
S. parasanguinis
,
S. salivarius
,
S. sanguinis
, and
S. uberis
. The method consisted of PCR amplification of the ITS regions by using a pair of universal primers, followed by hybridization of the digoxigenin-labeled PCR products to a panel of species-specific oligonucleotides immobilized on a nylon membrane. After 120 strains of the 11 species of VG and 91 strains of other bacteria were tested, the sensitivity and specificity of the oligonucleotide array were found to be 100% (120 of 120 strains) and 95.6% (87 of 91 strains), respectively.
S. pneumoniae
cross-hybridized to the probes used for the identification of
S. mitis
, and simple biochemical tests such as optochin susceptibility or bile solubility should be used to differentiate
S. pneumoniae
from
S. mitis
. In conclusion, identification of species of VS by use of the present oligonucleotide array is accurate and could be used as an alternative reliable method for species identification of strains of VS.
European Multicenter Study of the LIAISON Automated Diagnostic System for Determination of
Toxoplasma gondii
-Specific Immunoglobulin G (IgG) and IgM and the IgG Avidity Index
Authors: Petersen Eskild , Borobio Maria Victoria , Guy Edward , Liesenfeld Oliver , Meroni Valeria , Naessens Anne , Spranzi Emma , Thulliez Philippe
Published: April 2005

The LIAISON system is a fully automated system based on chemiluminescence and antigen bound to magnetic microparticles. The system allows fast and precise measurement of
Toxoplasma
-specific immunoglobulin G (IgG) and IgM antibody levels and measurement of the IgG avidity index even at low levels of
Toxoplasma
-specific IgG antibodies in a single step without manual interference. Seven European centers participated in a multicenter evaluation of the LIAISON system. The sensitivity and specificity of the LIAISON system compared to the Sabin-Feldman dye test were 99.3 and 96.8%, respectively. In a comparison of the LIAISON
Toxoplasma
-specific IgM assay with an immunosorbent agglutination assay, the LIAISON assay had a sensitivity of 96.7% and a specificity of 95.4%. The LIAISON IgG assay showed agreements of 91, 100, and 100% with the AXSYM IgG (Abbott), VIDAS IgG (bioMérieux), and Platelia IgG (Bio-Rad) assays, respectively. The LIAISON IgM assay showed agreements of 95% with the AXSYM IgM and Platelia IgM assays, 96% with the ISAGA IgM assay (bioMérieux), and 97% with the VIDAS IgM assay. The coefficient of correlation between the LIAISON system and the VIDAS
Toxoplasma
-specific IgG avidity index was 0.81. By use of the
Toxoplasma
-specific IgG avidity index assay with specific IgM-positive samples, the diagnosis of infection with
Toxoplasma gondii
in early pregnancy has been improved significantly. The LIAISON avidity assay is a valuable assay for the exclusion of recently acquired infection with
T. gondii
(less than 4 months) in pregnant women, and it decreases significantly the necessity for follow-up testing.
Rapid Detection of Ofloxacin Resistance in
Mycobacterium tuberculosis
by Two Low-Cost Colorimetric Methods: Resazurin and Nitrate Reductase Assays
Authors: Martin Anandi , Palomino Juan Carlos , Portaels Françoise
Published: April 2005

We have evaluated the performance of two rapid, low-cost methods for the detection of ofloxacin (OFX) resistance with 95
Mycobacterium tuberculosis
isolates from countries with high multidrug-resistant tuberculosis endemicity. Results obtained by nitrate reductase and resazurin assays showed 100% agreement with those of the proportion method on 7H11 agar using 2 μg of OFX/ml. We confirmed the resistance of all isolates found to be resistant to OFX by the Mycobacterium Growth Indicator Tube system, and complete agreement among all methods was observed. Nitrate reductase and resazurin assays are rapid, simple, low-cost methods and might become inexpensive alternative procedures for rapid detection of OFX resistance in low-resource countries.
Identification and Distribution of
Acanthamoeba
Species Genotypes Associated with Nonkeratitis Infections
Authors: Booton Gregory C. , Visvesvara Govinda S. , Byers Thomas J. , Kelly Daryl J. , Fuerst Paul A.
Published: April 2005

Acanthamoeba
is a free-living protozoan genus found in a wide variety of natural habitats, including water, soil, and air. Pathogenic isolates of
Acanthamoeba
are medically relevant as the causative agent of sight- threatening
Acanthamoeba
keratitis (AK), serious infections of other organs, and fatal granulomatous amebic encephalitis. Previous work employing DNA sequences of nuclear and mitochondrial small-subunit rRNA genes (SSU rRNA genes) determined the genotypic diversity of
Acanthamoeba
and found that many named species of
Acanthamoeba
are associated with particular genotypes. These studies also concluded that nearly all AK infections result from a single molecular genotype: T4. Here, we asked whether
Acanthamoeba
clinical isolates from non-AK infections are also associated with particular genotypes. DNA sequence determination of nuclear SSU rRNA genes was employed for genotypic identification of 29 isolates of
Acanthamoeba
from non-AK infections. Sequence analysis demonstrates that T4 is the predominant genotype in non-AK infections, including those in brain, cerebrospinal fluid, nasal passages, skin, and lung. Rare genotypes (T1, T10, and T12) have been isolated from brain infections. We conclude that genotype T4 is the primary genotype in non-AK
Acanthamoeba
infections, as was the case in AK infections. However, the genotypes that were isolated from brains have not been observed in environmental isolates of
Acanthamoeba
, and their natural ecological niche is unknown.
Evaluation of PCR Testing of Ethanol-Fixed Nasal Swab Specimens as an Augmented Surveillance Strategy for Influenza Virus and Adenovirus Identification
Authors: Krafft A. E. , Russell K. L. , Hawksworth A. W. , McCall S. , Irvine M. , Daum L. T. , Connoly J. L. , Reid A. H. , Gaydos J. C. , Taubenberger J. K.
Published: April 2005

Viral culture isolation has been widely accepted as the “gold standard” for laboratory confirmation of viral infection; however, it requires ultralow temperature specimen storage. Storage of specimens in ethanol at room temperature could expand our ability to conduct active surveillance and retrospective screenings of viruses with rapid and inexpensive real-time PCR tests, including isolates from remote regions where freezing specimens for culture is not feasible. Molecular methods allow for rapid identification of viral pathogens without the need to maintain viability. We hypothesized that ethanol, while inactivating viruses, can preserve DNA and RNA for PCR-based methods. To evaluate the use of ethanol-stored specimens for augmenting surveillance for detection of influenza viruses A and B and adenoviruses (AdV), paired nasal swab specimens were collected from 384 recruits with febrile respiratory illness at Fort Jackson, S.C., in a 2-year study. One swab was stored at ambient temperature in 100% ethanol for up to 6 months, and the other swab was stored at −70°C in viral medium. For viral detection, frozen specimens were cultured for a variety of respiratory viruses, and ethanol-fixed specimens were tested with TaqMan (TM) probe and LightCycler SYBR green (SG) melting curve assays with at least two different PCR targets for each virus. The sensitivities of the TM and SG assays on specimens stored in ethanol for 1 month were 75% and 58% for influenza A, 89% and 67% for influenza B, and 93 to 98% and 57% for AdV, respectively. Lower specificities of the real-time assays corresponded to the increased detection of PCR-positive but culture-negative specimens. Influenza virus RNA was detected as well or better after 6 months of storage in ethanol.
Detection and Genotyping of
Mycobacterium
Species from Clinical Isolates and Specimens by Oligonucleotide Array
Authors: Park Heekyung , Jang Hyunjung , Song Eunsil , Chang Chulhun L. , Lee Minki , Jeong Seokhoon , Park Junhyung , Kang Byeongchul , Kim Cheolmin
Published: April 2005

Identification of pathogenic
Mycobacterium
species is important for a successful diagnosis of mycobacteriosis. The purpose of this study was to develop an oligonucleotide array which could detect and differentiate mycobacteria to the species level by using the internal transcribed spacer (ITS) sequence. Using a genus-specific probe and 20 species-specific probes including two
M. avium-intracellulare
complex (MAC)-specific probes, we have developed an ITS-based oligonucleotide array for the rapid and reliable detection and discrimination of
M. tuberculosis
, MAC,
M. fortuitum
,
M. chelonae
,
M. abscessus
,
M. kansasii
,
M. gordonae
,
M. scrofulaceum
,
M. szulgai
,
M. vaccae
,
M. xenopi
,
M. terrae
,
M. flavescens
,
M. smegmatis
,
M. malmoense
,
M. simiae
,
M. marinum
,
M. ulcerans
,
M. gastri
, and
M. leprae
. All mycobacteria were hybridized with a genus-specific probe (PAN-03) for detection of the genus
Mycobacterium
. Mycobacterial species were expected to show a unique hybridization pattern with species-specific probes, except for
M. marinum
and
M. ulcerans
, which were not differentiated by ITS-based probe. Among the species-specific probes, two kinds of species-specific probes were designed for MAC in which there were many subspecies. The performance of the oligonucleotide array assay was demonstrated by using 46 reference strains, 149 clinical isolates, and 155 clinical specimens. The complete procedure (DNA extraction, PCR, DNA hybridization, and scanning) was carried out in 4.5 h. Our results indicated that the oligonucleotide array is useful for the identification and discrimination of mycobacteria from clinical isolates and specimens in an ordinary clinical laboratory.
Evaluation and Updating of the Osiris Expert System for Identification of
Escherichia coli
β-Lactam Resistance Phenotypes
Authors: Bert Frédéric , Juvin Manette , Ould-Hocine Zahia , Clarebout Gervais , Keller Emmanuelle , Lambert Nicole , Arlet Guillaume
Published: April 2005

Osiris is a video zone size reader for disk diffusion tests that includes a built-in extended expert system (EES). We evaluated the efficacy of the Osiris EES for the identification of β-lactam susceptibility phenotypes of
Escherichia coli
isolates. Fifteen β-lactam agents and three β-lactam-β-lactamase inhibitor combinations were tested by the disk diffusion method against 50
E. coli
strains with well-characterized resistance mechanisms. The strains were screened for the production of extended-spectrum β-lactamase (ESBL) by the double-disk synergy test using a disk of amoxicillin-clavulanic acid with disks of the extended-spectrum cephalosporins and aztreonam. Overall, the EES accurately identified the phenotype for 78% of the strains, indicated an inexact phenotype for 17% of the strains, and could not find a matching phenotype for the remaining 5% of the strains. The percentage of correct identification for each resistance mechanism was 100% for inhibitor-resistant TEM and for TEM plus cephalosporinase, 88.9% for TEM and for ESBL, 70.8% for cephalosporinase overproduction, and 25% for oxacillinase. The main cause of discrepancy was the misidentification of oxacillinase as inhibitor-resistant TEM. The conventional double-disk synergy test failed to detect ESBL production in two strains (one producing VEB-1 and one producing CTX-M-14), but synergy between cefepime and amoxicillin-clavulanic acid was visible after the distance between the disks was reduced to 20 mm. After the interpretative guidelines of the EES were updated according to our results, the percentage of correct phenotype identification increased from 78 to 96%.