bluuebunny/crossref_metadata_2025 huggingface dataset:
A Chemically Modified Tetracycline (CMT-3) Is a New Antifungal Agent
Authors: Liu Yu , Ryan Maria E. , Lee Hsi-Ming , Simon Sanford , Tortora George , Lauzon Carol , Leung Michael K. , Golub Lorne M.
Published: July 2002

Several chemically modified tetracycline analogs (CMTs), which were chemically modified to eliminate their antibacterial efficacy, were unexpectedly found to have antifungal properties. Of 10 CMTs screened in vitro, all exhibited antifungal activities, although their efficacies varied. Among these compounds, CMT-315, -3, and -308 were found to be the most potent as antifungal agents. The MICs of CMT-3 against 47 strains of fungi in vitro were determined by using amphotericin B (AMB) and doxycycline as positive and negative controls, respectively. The MICs of CMT-3 were generally found to be between 0.25 and 8.00 μg/ml, a range that approximates the blood levels of this drug when administrated orally to humans. Of all the yeast species tested to date,
Candida albicans
showed the greatest sensitivity to CMT-3. The filamentous species most susceptible to CMT-3 were found to be
Epidermophyton floccosum
,
Microsporum gypseum
,
Pseudallescheria boydii
, a
Penicillium
sp.,
Scedosporium apiospermum
, a
Tricothecium
sp., and
Trichophyton rubrum
. Growth inhibition of
C. albicans
by CMT-3, determined by a turbidity assay, indicated a 50% inhibitory concentration of 1 μg/ml. Thirty-nine strains, including 20 yeasts and 19 molds, were used to measure viability (the ability to grow after treatment with a drug) inhibition by CMT-3 and AMB. CMT-3 exhibited fungicidal activity against most of these fungi, especially the filamentous fungi. Eighty-four percent (16 of 19) of the filamentous fungi tested showed more than 90% inhibition of viability by CMT-3. In contrast, AMB showed fungicidal activity against all yeasts tested. However, most of the filamentous fungi (16 of 19) showed less than 50% inhibition of viability by AMB, indicating that AMB is fungistatic against most of these filamentous fungi. To begin to identify the sites in fungal cells affected by CMT-3,
C. albicans
and a
Penicillium
sp. were incubated with the compound at 35°C, and then the fluorescence of CMT-3 was observed by confocal laser scanning electron microscopy. CMT-3 appeared to have widespread intracellular distribution throughout
C. albicans
and the
Penicillium
sp. The mechanisms of the antifungal activity of CMT-3 are now being explored.
In Vitro Activities of Free and Lipid Formulations of Amphotericin B and Nystatin against Clinical Isolates ofCoccidioides immitisat Various Saprobic Stages
Authors: González Gloria M. , Tijerina Rolando , Sutton Deanna A. , Graybill John R. , Rinaldi Michael G.
Published: July 2002

We investigated the susceptibilities of hyphal, mixed hyphal, ungerminated arthroconidial, and germinated arthroconidial populations ofCoccidioides immitisto lipid formulations of amphotericin B and nystatin and their conventional preparations, utilizing the National Committee for Clinical Laboratory Standards M38-P broth macrodilution method. The differences in effects of the three different growth stages of the saprobic phase ofC. immitison the MIC/minimum lethal concentration (MLC) ratio were not statistically significant for any of the antifungal agents tested. These results suggest that either inocula could be used for in vitro susceptibility studies withC. immitis.
Single-Dose Pharmacokinetics of Amprenavir Coadministered with Grapefruit Juice
Authors: Demarles D. , Gillotin C. , Bonaventure-Paci S. , Vincent I. , Fosse S. , Taburet A. M.
Published: July 2002

In this crossover study in 12 healthy volunteers, coadministration of amprenavir (1,200 mg; single dose) with grapefruit juice slightly reduced the maximum concentration of drug in serum (
C
max
) compared to administration with water (7.11 versus 9.10 μg/ml), slightly increased the time to
C
max
(1.13 versus 0.75 h), and did not affect the area under the concentration-time curve from 0 to 12 h (AUC
0-12
), the AUC
0-∞
, or the concentration at 12 h. Therefore, grapefruit juice does not clinically significantly affect amprenavir pharmacokinetics.
In Vitro Activity of Linezolid against
Clostridium difficile
Authors: Peláez T. , Alonso R. , Pérez C. , Alcalá L. , Cuevas O. , Bouza E.
Published: July 2002

We examined the in vitro activity of linezolid against
Clostridium difficile,
including isolates with reduced susceptibility to metronidazole or vancomycin. The MIC at which 50% of the isolates were inhibited (MIC
50
) and MIC
90
were 0.5 and 2 μg/ml, respectively (range, 0.03 to 4 μg/ml). MICs were always ≤ 4 μg/ml, and thus, all isolates were considered susceptible.
Histidine-Rich Protein II: a Novel Approach to Malaria Drug Sensitivity Testing
Authors: Noedl Harald , Wernsdorfer Walther H. , Miller Robert Scott , Wongsrichanalai Chansuda
Published: July 2002

The production of histidine-rich protein II (HRP2), a histidine- and alanine-rich protein produced by
Plasmodium falciparum
, is closely associated with the development and proliferation of the parasite and therefore is perfectly suited to reflect growth inhibition as a measure of drug susceptibility. It was the aim of the present study to develop a malaria drug sensitivity assay based on the measurement of HRP2 in a simple enzyme-linked immunosorbent assay (ELISA). The new test proved to be as reliable as traditional in vitro assays, while it was considerably easier to establish and perform. Parasites are incubated at an initial level of parasitemia of 0.01 to 0.1% on microculture plates predosed with ascending concentrations of antimalarial drugs. After incubation for 48 to 72 h, the samples are freeze-thawed and transferred to ELISA plates. The complete ELISA takes about 2.5 h to perform, may be carried out with commercially available test kits, and requires relatively little technical equipment. In correlation analysis, the results closely paralleled those obtained by the isotopic assay (
R
= 0.892;
P
< 0.0001) and World Health Organization schizont maturation tests (
R
= 0.959;
P
< 0.0001). The novel HRP2 drug susceptibility assay proved to be very sensitive, simple to establish, and highly reproducible. It can be used for a wide range of applications, from epidemiological studies to the screening of new drugs, and may have the potential to replace traditional in vitro techniques. Standard operating procedures, updated information, and analytical software are available from
http://malaria.farch.net
.
Comparison of the Effects of Deferiprone versus Deferoxamine on Growth and Virulence of
Yersinia enterocolitica
Authors: Lesic Biliana , Foulon Jeannine , Carniel Elisabeth
Published: July 2002

Deferoxamine, a drug used to treat patients with iron overload, has the capacity to promote systemic
Y. enterocolitica
infections in humans. The aim of this study was to determine whether deferiprone, the only orally active alternative treatment, has the same potential. When
Y. enterocolitica
IP864 was grown in an iron-poor chemically defined medium, addition of deferoxamine promoted its growth, while various concentrations of deferiprone did not display this activity. Similarly, on iron-poor agar plates, various
Y. enterocolitica
strains were able to grow around paper disks impregnated with deferoxamine in a dose-dependent manner, while no growth was observed around the deferiprone disks. In a mouse experimental model of infection, the 50% lethal dose (LD
50
) of strain IP864 was decreased by more than 5 log units in mice pretreated with deferoxamine, while a deferiprone pretreatment did not affect it. Therefore, in contrast to deferoxamine, deferiprone does not enhance growth of pathogenic
Y. enterocolitica
in vitro and does not have the potential to promote
Y. enterocolitica
septicemia in a mouse model of infection. Deferiprone may thus represent a useful alternative iron-chelation therapy during invasive
Y. enterocolitica
infections.
Antitrypanosomal Activities of Proteasome Inhibitors
Authors: Nkemgu-Njinkeng Joseph , Rosenkranz Vera , Wink Michael , Steverding Dietmar
Published: July 2002

Seven peptidyl proteasome inhibitors were tested for in vitro activity against
Trypanosoma brucei
bloodstream forms. Two compounds showed activity in the low nanomolar range. In general, trypanosomes were more susceptible to the compounds than were human HL-60 cells. The data support the potential of proteasome inhibitors for rational antitrypanosomal drug development.
Quantifying Translocation ofListeria monocytogenesin Rats by Using Urinary Nitric Oxide-Derived Metabolites
Authors: Sprong R. Corinne , Hulstein Marco F. E. , van der Meer Roelof
Published: July 2002

The urinary nitric oxide metabolites NO2−and NO3−(summed as NOx) are a noninvasive, quantitative biomarker of translocation of salmonella from the intestinal lumen to systemic organs.Listeria monocytogenesis a food-borne gram-positive pathogen that can also cross the intestinal epithelium. In this study, we tested the efficacy of urinary NOxas a marker of listeria translocation. Rats (eight per group) were orally infected with increasing doses ofL. monocytogenes; control rats received heat-killed listeria. The kinetics of urinary NOxand population levels of listeria in feces were determined for 7 days. Another group of rats was killed 1 day after infection to verify translocation by culturing viable listeria from systemic organs. Oral administration of increasing doses ofL. monocytogenesresulted in a time- and dose-dependent increase in urinary NOxexcretion. Translocation was a prerequisite for inducing a NOxresponse, since heat-killedL. monocytogenesdid not elevate NOxexcretion in urine. Fecal counts of listeria also showed dose and time dependency. Moreover, the number of viableL. monocytogenescells in mesenteric lymph nodes also increased in a dose-dependent manner and correlated with urinary NOx. In conclusion, urinary NOxis a quantitative, noninvasive biomarker of listeria translocation.
Loss of O157 O Antigenicity of Verotoxin-Producing
Escherichia coli
O157:H7 Surviving under Starvation Conditions
Authors: Hara-Kudo Yukiko , Miyahara Michiko , Kumagai Susumu
Published: July 2002

Verotoxin (VT)-producing
Escherichia coli
O157:H7 was culturable on agar media after being left in water for 21 months. However, there were a number of colonies which had lost O157 O antigenicity. These colonies produced VTs, which are pathogenic to humans. These observations suggest that the immunologic methods based on O157 O antigenicity are unable to detect and isolate VT-producing
E. coli
in foods and other environments if the organism has been under starvation conditions for a long period.
Genetically Modified
Vibrio harveyi
Strains as Potential Bioindicators of Mutagenic Pollution of Marine Environments
Authors: Czyz˙ Agata , Jasiecki Jacek , Bogdan Adam , Szpilewska Hanna , We˛grzyn Grzegorz
Published: July 2002

For biodetection of mutagenic pollution of marine environments, an organism naturally occurring in these habitats should be used. We found that marine bacterium
Vibrio harveyi
may be an appropriate bioindicator of mutagenic pollution. For positive selection of mutants, we developed a simple method for isolation of
V. harveyi
mutants resistant to neomycin. We constructed genetically modified
V. harveyi
strains that produce significantly more neomycin-resistant mutants upon treatment with low concentrations of mutagens than the wild-type counterpart. The sensitivity of the mutagenicity test with the
V. harveyi
strains is at least comparable to (if not higher than) that of the commonly used Ames test, which uses
Salmonella enterica
serovar Typhimurium strains. Therefore, we consider that the
V. harveyi
strains described in this report could be used as potential bioindicators of mutagenic pollution of marine environments.
Cotransformation of
Trichoderma harzianum
with β-Glucuronidase and Green Fluorescent Protein Genes Provides a Useful Tool for Monitoring Fungal Growth and Activity in Natural Soils
Authors: Bae Yeoung-Seuk , Knudsen Guy R.
Published: July 2002

Trichoderma harzianum
was cotransformed with genes encoding green fluorescent protein (GFP), β-glucuronidase (GUS), and hygromycin B (
hygB
) resistance, using polyethylene glycol-mediated transformation. One cotransformant (ThzID1-M3) was mitotically stable for 6 months despite successive subculturing without selection pressure. ThzID1-M3 morphology was similar to that of the wild type; however, the mycelial growth rate on agar was reduced. ThzID1-M3 was formed into calcium alginate pellets and placed onto buried glass slides in a nonsterile soil, and its ability to grow, sporulate, and colonize sclerotia of
Sclerotinia sclerotiorum
was compared with that of the wild-type strain. Wild-type and transformant strains both colonized sclerotia at levels above those of indigenous
Trichoderma
spp. in untreated controls. There were no significant differences in colonization levels between wild-type and cotransformant strains; however, the presence of the GFP and GUS marker genes permitted differentiation of introduced
Trichoderma
from indigenous strains. GFP activity was a useful tool for nondestructive monitoring of the hyphal growth of the transformant in a natural soil. The green color of cotransformant hyphae was clearly visible with a UV epifluorescence microscope, while indigenous fungi in the same samples were barely visible. Green-fluorescing conidiophores and conidia were observed within the first 3 days of incubation in soil, and this was followed by the formation of terminal and intercalary chlamydospores and subsequent disintegration of older hyphal segments. Addition of 5-bromo-4-chloro-3-indolyl-β-
d
-glucuronic acid (X-Gluc) substrate to recovered glass slides confirmed the activity of GUS as well as GFP in soil. Our results suggest that cotransformation with GFP and GUS can provide a valuable tool for the detection and monitoring of specific strains of
T. harzianum
released into the soil.
Antagonistic Activity ofLactobacillus acidophilusLB against IntracellularSalmonella entericaSerovar Typhimurium Infecting Human Enterocyte-Like Caco-2/TC-7 Cells
Authors: Coconnier Marie-Hélène , Liévin Vanessa , Lorrot Mathie , Servin Alain L.
Published: July 2002

To gain further insight into the mechanism by which lactobacilli develop antimicrobial activity, we have examined howLactobacillus acidophilusLB inhibits the promoted cellular injuries and intracellular lifestyle ofSalmonella entericaserovar Typhimurium SL1344 infecting the cultured, fully differentiated human intestinal cell line Caco-2/TC-7. We showed that the spent culture supernatant of strain LB (LB-SCS) decreases the number of apical serovar Typhimurium-induced F-actin rearrangements in infected cells. LB-SCS treatment efficiently decreased transcellular passage ofS. entericaserovar Typhimurium. Moreover, LB-SCS treatment inhibited intracellular growth of serovar Typhimurium, since treated intracellular bacteria displayed a small, rounded morphology resembling that of resting bacteria. We also showed that LB-SCS treatment inhibits adhesion-dependent serovar Typhimurium-induced interleukin-8 production.
Physiologic Determinants of Radiation Resistance in
Deinococcus radiodurans
Authors: Venkateswaran Amudhan , McFarlan Sara C. , Ghosal Debabrota , Minton Kenneth W. , Vasilenko Alexander , Makarova Kira , Wackett Lawrence P. , Daly Michael J.
Published: July 2002

Immense volumes of radioactive wastes, which were generated during nuclear weapons production, were disposed of directly in the ground during the Cold War, a period when national security priorities often surmounted concerns over the environment. The bacterium
Deinococcus radiodurans
is the most radiation-resistant organism known and is currently being engineered for remediation of the toxic metal and organic components of these environmental wastes. Understanding the biotic potential of
D. radiodurans
and its global physiological integrity in nutritionally restricted radioactive environments is important in development of this organism for in situ bioremediation. We have previously shown that
D. radiodurans
can grow on rich medium in the presence of continuous radiation (6,000 rads/h) without lethality. In this study we developed a chemically defined minimal medium that can be used to analyze growth of this organism in the presence and in the absence of continuous radiation; whereas cell growth was not affected in the absence of radiation, cells did not grow and were killed in the presence of continuous radiation. Under nutrient-limiting conditions, DNA repair was found to be limited by the metabolic capabilities of
D. radiodurans
and not by any nutritionally induced defect in genetic repair. The results of our growth studies and analysis of the complete
D. radiodurans
genomic sequence support the hypothesis that there are several defects in
D. radiodurans
global metabolic regulation that limit carbon, nitrogen, and DNA metabolism. We identified key nutritional constituents that restore growth of
D. radiodurans
in nutritionally limiting radioactive environments.
Three-Year Study To Assess Human Enteric Viruses in Shellfish
Authors: Le Guyader F. , Haugarreau L. , Miossec L. , Dubois E. , Pommepuy M.
Published: July 2002

The main pathogenic enteric viruses able to persist in the environment, such as hepatitis A virus (HAV), Norwalk-like virus (NLV), enterovirus (EV), rotavirus (RV), and astrovirus (AV), were detected by reverse transcription-PCR and hybridization in shellfish during a 3-year study. Oyster samples (
n
= 108), occasionally containing bacteria, were less frequently contaminated, showing positivity for AV (17%), NLV (23%), EV (19%), and RV (27%), whereas mussel samples, collected in areas routinely impacted by human sewage, were more highly contaminated: AV (50%), HAV (13%), NLV (35%), EV (45%), and RV (52%). Sequences obtained from HAV and NLV amplicons showed a great variety of strains, especially for NLV (strains close to Mexico, Snow Mountain Agent, or Norwalk virus). Viral contamination was mainly observed during winter months, although there were some seasonal differences among the viruses. This first study of virus detection over a fairly long period of time suggests that routine analysis of shellfish by a molecular technique is feasible.
Genotypic Characterization of
Bradyrhizobium
Strains Nodulating Small Senegalese Legumes by 16S-23S rRNA Intergenic Gene Spacers and Amplified Fragment Length Polymorphism Fingerprint Analyses
Authors: Doignon-Bourcier Florence , Willems Anne , Coopman Renata , Laguerre Gisele , Gillis Monique , de Lajudie Philippe
Published: July 2002

We examined the genotypic diversity of 64
Bradyrhizobium
strains isolated from nodules from 27 native leguminous plant species in Senegal (West Africa) belonging to the genera
Abrus
,
Alysicarpus
,
Bryaspis
,
Chamaecrista
,
Cassia
,
Crotalaria
,
Desmodium
,
Eriosema
,
Indigofera
,
Moghania
,
Rhynchosia
,
Sesbania
,
Tephrosia
, and
Zornia
, which play an ecological role and have agronomic potential in arid regions. The strains were characterized by intergenic spacer (between 16S and 23S rRNA genes) PCR and restriction fragment length polymorphism (IGS PCR-RFLP) and amplified fragment length polymorphism (AFLP) fingerprinting analyses. Fifty-three reference strains of the different
Bradyrhizobium
species and described groups were included for comparison. The strains were diverse and formed 27 groups by AFLP and 16 groups by IGS PCR-RFLP. The sizes of the IGS PCR products from the
Bradyrhizobium
strains that were studied varied from 780 to 1,038 bp and were correlated with the IGS PCR-RFLP results. The grouping of strains was consistent by the three methods AFLP, IGS PCR-RFLP, and previously reported 16S amplified ribosomal DNA restriction analysis. For investigating the whole genome, AFLP was the most discriminative technique, thus being of particular interest for future taxonomic studies in
Bradyrhizobium
, for which DNA is difficult to obtain in quantity and quality to perform extensive DNA:DNA hybridizations.
Competitive Exclusion of Heterologous
Campylobacter
spp. in Chicks
Authors: Chen Hui-Cheng , Stern Norman J.
Published: July 2002

Chicken and human isolates of
Campylobacter jejuni
were used to provide oral challenge of day-old broiler chicks. The isolation ratio of the competing challenge strains was monitored and varied, depending upon the isolates used. A PCR-restriction fragment length polymorphism assay of the flagellin gene (
flaA
) was used to discriminate between the chick-colonizing isolates. Our observations indicated that the selected
C. jejuni
colonizers dominated the niche provided by the chicken ceca. Chicken isolates from the
flaA
type 7 grouping generally had numerical superiority over the human isolates when they were administered in our 1-day-old chick model. Our results suggest that it is possible to use combinations of
C. jejuni
chicken isolates as a defined bacterial preparation for the competitive exclusion of human-pathogenic
C. jejuni
in poultry.
Identification ofDekkera bruxellensis(Brettanomyces) from Wine by Fluorescence In Situ Hybridization Using Peptide Nucleic Acid Probes
Authors: Stender Henrik , Kurtzman Cletus , Hyldig-Nielsen Jens J. , Sørensen Ditte , Broomer Adam , Oliveira Kenneth , Perry-O'Keefe Heather , Sage Andrew , Young Barbara , Coull James
Published: July 2002

A new fluorescence in situ hybridization method using peptide nucleic acid (PNA) probes for identification ofBrettanomycesis described. The test is based on fluorescein-labeled PNA probes targeting a species-specific sequence of the rRNA ofDekkera bruxellensis. The PNA probes were applied to smears of colonies, and results were interpreted by fluorescence microscopy. The results obtained from testing 127 different yeast strains, including 78Brettanomycesisolates from wine, show that the spoilage organismBrettanomycesbelongs to the speciesD. bruxellensisand that the new method is able to identifyBrettanomyces(D. bruxellensis) with 100% sensitivity and 100% specificity.
Purification, Characterization, and Application of a Novel Dye-Linked
l
-Proline Dehydrogenase from a Hyperthermophilic Archaeon,
Thermococcus profundus
Authors: Sakuraba Haruhiko , Takamatsu Yoshinori , Satomura Takenori , Kawakami Ryushi , Ohshima Toshihisa
Published: July 2002

The distribution of dye-linked
l
-amino acid dehydrogenases was investigated in several hyperthermophiles, and the activity of dye-linked
l
-proline dehydrogenase (dye-
l
-proDH,
l
-proline:acceptor oxidoreductase) was found in the crude extract of some
Thermococcales
strains. The enzyme was purified to homogeneity from a hyperthermophilic archaeon,
Thermococcus profundus
DSM 9503, which exhibited the highest specific activity in the crude extract. The molecular mass of the enzyme was about 160 kDa, and the enzyme consisted of heterotetrameric subunits (α
2
β
2
) with two different molecular masses of about 50 and 40 kDa. The N-terminal amino acid sequences of the α-subunit (50-kDa subunit) and the β-subunit (40-kDa subunit) were MRLTEHPILDFSERRGRKVTIHF and XRSEAKTVIIGGGIIGLSIAYNLAK, respectively. Dye-
l
-proDH was extraordinarily stable among the dye-linked dehydrogenases under various conditions: the enzyme retained its full activity upon incubation at 70°C for 10 min, and ca. 40% of the activity still remained after heating at 80°C for 120 min. The enzyme did not lose the activity upon incubation over a wide range of pHs from 4.0 to 10.0 at 50°C for 10 min. The enzyme exclusively catalyzed
l
-proline dehydrogenation using 2,6-dichloroindophenol (Cl2Ind) as an electron acceptor. The Michaelis constants for
l
-proline and Cl2Ind were determined to be 2.05 and 0.073 mM, respectively. The reaction product was identified as Δ
1
-pyrroline-5-carboxylate by thin-layer chromatography. The prosthetic group of the enzyme was identified as flavin adenine dinucleotide by high-pressure liquid chromatography. In addition, the simple and specific determination of
l
-proline at concentrations from 0.10 to 2.5 mM using the stable dye-
l
-proDH was achieved.
Development and Application of a Most-Probable-Number–PCR Assay To Quantify Flagellate Populations in Soil Samples
Authors: Fredslund Line , Ekelund Flemming , Jacobsen Carsten Suhr , Johnsen Kaare
Published: July 2002

This paper reports on the first successful molecular detection and quantification of soil protozoa. Quantification of heterotrophic flagellates and naked amoebae in soil has traditionally relied on dilution culturing techniques, followed by most-probable-number (MPN) calculations. Such methods are biased by differences in the culturability of soil protozoa and are unable to quantify specific taxonomic groups, and the results are highly dependent on the choice of media and the skills of the microscopists. Successful detection of protozoa in soil by DNA techniques requires (i) the development and validation of DNA extraction and quantification protocols and (ii) the collection of sufficient sequence data to find specific protozoan 18S ribosomal DNA sequences. This paper describes the development of an MPN-PCR assay for detection of the common soil flagellateHeteromita globosa, using primers targeting a 700-bp sequence of the small-subunit rRNA gene. The method was tested by use of gnotobiotic laboratory microcosms with sterile tar-contaminated soil inoculated with the bacteriumPseudomonas putidaOUS82 UCB55 as prey. There was satisfactory overall agreement betweenH. globosapopulation estimates obtained by the PCR assay and a conventional MPN assay in the three soils tested.
Novel Approach for Improving the Productivity of Antibiotic-Producing Strains by Inducing Combined Resistant Mutations
Authors: Hu Haifeng , Ochi Kozo
Published: July 2002

We developed a novel approach for improving the production of antibiotic from
Streptomyces coelicolor
A3(2) by inducing combined drug-resistant mutations. Mutants with enhanced (1.6- to 3-fold-higher) actinorhodin production were detected at a high frequency (5 to 10%) among isolates resistant to streptomycin (Str
r
), gentamicin (Gen
r
), or rifampin (Rif
r
), which developed spontaneously on agar plates which contained one of the three drugs. Construction of double mutants (
str gen
and
str rif
) by introducing gentamicin or rifampin resistance into an
str
mutant resulted in further increased (1.7- to 2.5-fold-higher) actinorhodin productivity. Likewise, triple mutants (
str gen rif
) thus constructed were found to have an even greater ability for producing the antibiotic, eventually generating a mutant able to produce 48 times more actinorhodin than the wild-type strain. Analysis of
str
mutants revealed that a point mutation occurred within the
rpsL
gene, which encodes the ribosomal protein S12.
rif
mutants were found to have a point mutation in the
rpoB
gene, which encodes the β-subunit of RNA polymerase. Mutation points in
gen
mutants still remain unknown. These single, double, and triple mutants displayed in hierarchical order a remarkable increase in the production of ActII-ORF4, a pathway-specific regulatory protein, as determined by Western blotting analysis. This reflects the same hierarchical order observed for the increase in actinorhodin production. The superior ability of the triple mutants was demonstrated by physiological analyses under various cultural conditions. We conclude that by inducing combined drug-resistant mutations we can continuously increase the production of antibiotic in a stepwise manner. This new breeding approach could be especially effective for initially improving the production of antibiotics from wild-type strains.
Zeamatin Inhibits Trypsin and α-Amylase Activities
Authors: Schimoler-O'Rourke Rebecca , Richardson Michael , Selitrennikoff Claude P.
Published: July 2002

Zeamatin is a 22-kDa protein isolated from
Zea mays
that has antifungal activity against human and plant pathogens. Unlike other pathogenesis-related group 5 proteins, zeamatin inhibits insect α-amylase and mammalian trypsin activities. It is of clinical significance that zeamatin did not inhibit human α-amylase activity and inhibited mammalian trypsin activity only at high molar concentrations.
Influence of Effluent Irrigation on Community Composition and Function of Ammonia-Oxidizing Bacteria in Soil
Authors: Oved Tamar , Shaviv Avi , Goldrath Tal , Mandelbaum Raphi T. , Minz Dror
Published: July 2002

The effect of effluent irrigation on community composition and function of ammonia-oxidizing bacteria (AOB) in soil was evaluated, using techniques of molecular biology and analytical soil chemistry. Analyses were conducted on soil sampled from lysimeters and from a grapefruit orchard which had been irrigated with wastewater effluent or fertilizer-amended water (FAW). Specifically, comparisons of AOB community composition were conducted using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified fragments of the gene encoding the α-subunit of the ammonia monooxygenase gene (amoA) recovered from soil samples and subsequent sequencing of relevant bands. A significant and consistent shift in the population composition of AOB was detected in soil irrigated with effluent. This shift was absent in soils irrigated with FAW, despite the fact that the ammonium concentration in the FAW was similar. At the end of the irrigation period,Nitrosospira-like populations were dominant in soils irrigated with FAW, whileNitrosomonas-like populations were dominant in effluent-irrigated soils. Furthermore, DGGE analysis of theamoAgene proved to be a powerful tool in evaluating the soil AOB community population and population shifts therein.
Location Effects of a Reporter Gene on Expression Levels and on Native Protein Synthesis inLactococcus lactisandSaccharomyces cerevisiae
Authors: Thompson Arthur , Gasson Michael J.
Published: July 2002

The engineering of industrially important genetically modified organisms by the integration of heterologous genes into the chromosome is often the method of choice for several reasons concerned with long-term stability, homogeneous population distribution, and the enabling of selection without the addition of antibiotics. However, integration may disrupt endogenous gene expression, giving rise to increased levels of toxic metabolic byproducts or activating otherwise silent genes. The position of integration of a foreign gene in the chromosome can also influence its expression levels, and this effect will be of relevance in terms of optimizing protein production parameters. In this study, we determine how the random integration of a foreign reporter gene might affect expression levels and assess the use of proteome analysis to investigate possible effects on synthesis of endogenous proteins in two important food-relevant microorganisms,Saccharomyces cerevisiaeandLactococcus lactis. ElevenL. lactisintegrants carrying thegusAgene were analyzed, and expression levels were found to vary by a factor of threefold in contrast to expression levels oflacZin 18S. cerevisiaeintegrants, which showed a 14-fold variation. Of relevance to industry is whether any changes in expression levels might occur as a consequence of storage of the modified strains. Here it is also shown that the above differences in expression levels were not significantly affected by storage of frozen cultures over a period of several months. Analysis of the protein composition of the yeast and lactococcal integrant strains by separation on one-dimensional (1D) and 2D gels showed no significant variations in position beyond those observed in control samples.
Isolation and Genotyping of
Helicobacter pylori
from Untreated Municipal Wastewater
Authors: Lu Yingzhi , Redlinger Thomas E. , Avitia Raquel , Galindo Adriana , Goodman Karen
Published: July 2002

For this study, we isolated
Helicobacter pylori
from wastewater by a series of steps beginning with immunomagnetic separation and cell culture. After Gram staining and three standard microbial tests, the 16S rRNA sequences of a total of 23 out of 37 putative
H. pylori
isolates were verified by PCR. Eleven
H. pylori
isolates were genotyped and fell into four
vacA
classes: those with the
vacA
allelic variants
s1a
and
m1
,
s1b
and
m1
,
s2
and
m2
, or
s2
and
m1
. Most
H. pylori
isolates were of the
vacA s1a
/
m1
type, which has been shown to be associated with advanced diseases based on genotyping of
H. pylori
from gastric cancer patients. These results demonstrated that
H. pylori
survives in water and may be a potential source of
H. pylori
transmission, especially where water is not adequately treated.
Incidence ofMycobacterium paratuberculosisin Bulk Raw and Commercially Pasteurized Cows' Milk from Approved Dairy Processing Establishments in the United Kingdom
Authors: Grant Irene R. , Ball Hywel J. , Rowe Michael T.
Published: July 2002

Over a 17-month period (March 1999 to July 2000), a total of 814 cows' milk samples, 244 bulk raw and 567 commercially pasteurized (228 whole, 179 semiskim, and 160 skim), from 241 approved dairy processing establishments throughout the United Kingdom were tested for the presence ofMycobacterium paratuberculosisby immunomagnetic PCR (to detect all cells living and dead) and culture (to detect viable cells). Overall,M. paratuberculosisDNA was detected by immunomagnetic PCR in 19 (7.8%; 95% confidence interval, 4.3 to 10.8%) and 67 (11.8%; 95% confidence interval, 9.0 to 14.2%) of the raw and pasteurized milk samples, respectively. ConfirmedM. paratuberculosisisolates were cultured from 4 (1.6%; 95% confidence interval, 0.04 to 3.1%) and 10 (1.8%; 95% confidence interval, 0.7 to 2.8%) of the raw and pasteurized milk samples, respectively, following chemical decontamination with 0.75% (wt/vol) cetylpyridinium chloride for 5 h. The 10 culture-positive pasteurized milk samples were from just 8 (3.3%) of the 241 dairy processing establishments that participated in the survey. Seven of the culture-positive pasteurized milk samples had been heat treated at 72 to 74°C for 15 s; the remainder had been treated at 72 to 75°C for the extended holding time of 25 s. When typed by restriction fragment length polymorphism and pulsed-field gel electrophoresis methods, some of the milk isolates were shown to be types distinct from those of laboratory strains in regular use within the testing laboratory. From information gathered at the time of milk sample collection, all indications were that pasteurization had been carried out effectively at all of the culture-positive dairies. That is, pasteurization time and temperature conditions complied with the legal minimum high-temperature, short-time process; all pasteurized milk samples tested phosphatase negative; and postprocess contamination was considered unlikely to have occurred. It was concluded that viableM. paratuberculosisis occasionally present at low levels in commercially pasteurized cows' milk in the United Kingdom.
Interleukin 7 Can Enhance Antigen-Specific Cytotoxic-T-Lymphocyte and/or Th2-Type Immune Responses In Vivo
Authors: Sin Jeong-Im , Kim Jong , Patchuk Catherine , Weiner David B.
Published: July 2002

Interleukin 7 (IL-7) protein has been reported to be important in the development of cytotoxic-T-lymphocyte (CTL) responses. However, other studies also support a partial Th2 phenotype for this cytokine. In an effort to clarify this unusual conflict, we compared IL-7 along with IL-12 (Th1 control) and IL-10 (Th2 control) for its ability to induce antigen (Ag)-specific CTL and Th1- versus Th2-type immune responses using a well established DNA vaccine model. In particular, IL-7 codelivery showed a significant increase in immunoglobulin G1 (IgG1) levels compared to IgG2a levels. IL-7 coinjection also decreased production of Th1-type cytokine IL-2, gamma interferon, and the chemokine RANTES but increased production of the Th2-type cytokine IL-10 and the similarly biased chemokine MCP-1. In herpes simplex virus (HSV) challenge studies, IL-7 coinjection decreased the survival rate after lethal HSV type 2 (HSV-2) challenge compared with gD plasmid vaccine alone in a manner similar to IL-10 coinjection, whereas IL-12 coinjection enhanced the protection, further supporting that IL-7 drives immune responses to the Th2 type, resulting in reduced protection against HSV-2 challenge. Moreover, coinjection with human immunodeficiency virus type 1envandgag/polgenes plus IL-12 or IL-7 cDNA enhanced Ag-specific CTLs, while coinjection with IL-10 cDNA failed to influence CTL induction. Thus, IL-7 could drive Ag-specific Th2-type cellular responses and/or CTL responses. These results support that CTLs could be induced by IL-7 in a Th2-type cytokine and chemokine environment in vivo. This property of IL-7 allows for an alternative pathway for CTL development which has important implications for host-pathogen responses.
Antibody Responses of Cattle Immunized with the Tf190 Adhesin ofTritrichomonas foetus
Authors: Voyich Jovanka M. , Ansotegui Raymond , Swenson Connie , Bailey John , Burgess Donald E.
Published: July 2002

The antibody response patterns of cattle after subcutaneous and intranasal immunizations with adhesin Tf190 ofTritrichomonas foetuswere investigated. Reactions of antibody from cattle parenterally immunized with Tf190 revealed antigen specificity and Tf190 sensitization in the majority of the animals, as determined by Western blotting. The results also demonstrated strong preimmune immunoglobulin G2 (IgG2) binding toT. foetusantigens not seen in IgG1 profiles. Subcutaneous injections of Tf190 resulted in significant (P< 0.05) increases in serum IgG1 and IgG2 titers over time, as determined by parasite specific enzyme-linked immunosorbent assay. Immune sera also significantly inhibited parasite adhesion to mammalian cell lines compared to the level of inhibition obtained with preimmune sera (P< 0.05). Intranasal immunization with Tf190 failed to produce measurable parasite-specific antibody in serum; however, this immunization route did result in significant (P< 0.05) increases in parasite-specific IgA titers in cervical mucus secretions from immunized animals that were more resistant to intravaginal challenge withT. foetusthan controls. These results suggest that systemic immunization with Tf190 results in serum antibody production and antiparasitic adhesin antibodies. Additionally, the results of challenge experiments with intranasally immunized animals suggests that Tf190 primes protective immune responses that lead to lower rates of infection among these animals.
ListeriaPathogenesis and Molecular Virulence Determinants
Authors: Vázquez-Boland José A. , Kuhn Michael , Berche Patrick , Chakraborty Trinad , Domı́nguez-Bernal Gustavo , Goebel Werner , González-Zorn Bruno , Wehland Jürgen , Kreft Jürgen
Published: July 2002

The gram-positive bacterium Listeria monocytogenes is the causative agent of listeriosis, a highly fatal opportunistic foodborne infection. Pregnant women, neonates, the elderly, and debilitated or immunocompromised patients in general are predominantly affected, although the disease can also develop in normal individuals. Clinical manifestations of invasive listeriosis are usually severe and include abortion, sepsis, and meningoencephalitis. Listeriosis can also manifest as a febrile gastroenteritis syndrome. In addition to humans, L. monocytogenes affects many vertebrate species, including birds. Listeria ivanovii, a second pathogenic species of the genus, is specific for ruminants. Our current view of the pathophysiology of listeriosis derives largely from studies with the mouse infection model. Pathogenic listeriae enter the host primarily through the intestine. The liver is thought to be their first target organ after intestinal translocation. In the liver, listeriae actively multiply until the infection is controlled by a cell-mediated immune response. This initial, subclinical step of listeriosis is thought to be common due to the frequent presence of pathogenic L. monocytogenes in food. In normal indivuals, the continual exposure to listerial antigens probably contributes to the maintenance of anti-Listeria memory T cells. However, in debilitated and immunocompromised patients, the unrestricted proliferation of listeriae in the liver may result in prolonged low-level bacteremia, leading to invasion of the preferred secondary target organs (the brain and the gravid uterus) and to overt clinical disease. L. monocytogenes and L. ivanovii are facultative intracellular parasites able to survive in macrophages and to invade a variety of normally nonphagocytic cells, such as epithelial cells, hepatocytes, and endothelial cells. In all these cell types, pathogenic listeriae go through an intracellular life cycle involving early escape from the phagocytic vacuole, rapid intracytoplasmic multiplication, bacterially induced actin-based motility, and direct spread to neighboring cells, in which they reinitiate the cycle. In this way, listeriae disseminate in host tissues sheltered from the humoral arm of the immune system. Over the last 15 years, a number of virulence factors involved in key steps of this intracellular life cycle have been identified. This review describes in detail the molecular determinants of Listeria virulence and their mechanism of action and summarizes the current knowledge on the pathophysiology of listeriosis and the cell biology and host cell responses to Listeria infection. This article provides an updated perspective of the development of our understanding of Listeria pathogenesis from the first molecular genetic analyses of virulence mechanisms reported in 1985 until the start of the genomic era of Listeria research.
Identification of a Siderophore Receptor Required for Ferric Ornibactin Uptake in
Burkholderia cepacia
Authors: Sokol P. A. , Darling P. , Lewenza S. , Corbett C. R. , Kooi C. D.
Published: July 2002

Ornibactins are linear hydroxamate siderophores produced by
Burkholderia cepacia
with peptide structures similar to that of pyoverdines produced by the fluorescent pseudomonads. The gene encoding the outer membrane receptor (
orbA
) was identified, sequenced, and demonstrated to have significant homology with hydroxamate receptors produced by other organisms. The
orbA
precursor was predicted to be a protein with a molecular mass of 81 kDa. An
orbA
mutant was constructed and demonstrated to be unable to take up
59
Fe-ornibactins or to grow in medium supplemented with ornibactins. Outer membrane protein profiles from the parent strain, K56-2, revealed an iron-regulated outer membrane protein of 78 kDa that was not detectable in the K56
orbA
::tp mutant. When this mutant harbored a plasmid containing the
orbA
gene, the 78-kDa protein was present in the outer membrane protein profiles and the mutant was able to utilize ornibactin to acquire iron. The
orbA
mutant was less virulent in a chronic respiratory infection model than the parent strain, indicating that ornibactin uptake and utilization are important in the pathogenesis of
B. cepacia
respiratory infections.
Antigenicity, Expression, and Molecular Characterization of Surface-Located Pullulanase of
Streptococcus pneumoniae
Authors: Bongaerts Roger J. M. , Heinz Hans-Peter , Hadding Ulrich , Zysk Gregor
Published: July 2002

A putative pullulanase-encoding gene from
Streptococcus pneumoniae
was identified by screening a genomic expression library with human convalescent-phase serum. The 3,864-bp gene encoded a 143-kDa protein. Surface location and pullulanase activity of the protein, designated SpuA, was demonstrated. SpuA was present in all investigated pneumococcal isolates of different serotypes. The
spuA
5′ end was highly conserved among clinical isolates except for a 75-bp region. The properties of SpuA reported here indicate that this novel immunogenic surface protein might have potential as a vaccine target.
Epitope Mapping of Immunogenic and Adhesive Structures in Repetitive Domains of
Mycoplasma bovis
Variable Surface Lipoproteins
Authors: Sachse K. , Helbig J. H. , Lysnyansky I. , Grajetzki C. , Müller W. , Jacobs E. , Yogev D.
Published: July 2002

The family of variable surface lipoproteins (Vsps) of the bovine pathogen
Mycoplasma bovis
includes some of the most immunogenic antigens of this microorganism. Vsps were shown to undergo high-frequency phase and size variations and to possess extensive reiterated coding sequences extending from the N-terminal end to the C-terminal end of the Vsp molecule. In the present study, mapping experiments were conducted to detect regions with immunogenicity and/or adhesion sites in repetitive domains of four Vsp antigens of
M. bovis
, VspA, VspB, VspE, and VspF. In enzyme-linked immunosorbent assay experiments, sera obtained from naturally infected cattle showed antibodies to different repeating peptide units of the Vsps, particularly to units R
A
1, R
A
2, R
A
4.1, R
B
2.1, R
E
1, and R
F
1, all of which were found to contain immunodominant epitopes of three to seven amino acids. Competitive adherence trials revealed that a number of oligopeptides derived from various repeating units of VspA, VspB, VspE, and VspF partially inhibited cytoadhesion of
M. bovis
PG45 to embryonic bovine lung cells. Consequently, putative adherence sites were identified in the same repeating units (R
A
1, R
A
2, R
A
4.1, R
B
2.1, R
E
1, and R
F
1) and in R
F
2. The positions and lengths of the antigenic determinants were mostly identical to those of adhesion-mediating sites in all short repeating units, whereas in the considerably longer R
F
1 unit (84 amino acid residues), there was only one case of identity among four immunogenic epitopes and six adherence sites. The identification of epitopes and adhesive structures in repetitive domains of Vsp molecules is consistent with the highly immunogenic nature observed for several members of the Vsp family and suggests a possible function for these Vsp molecules as complex adherence-mediating regions in pathogenesis.
Relative Roles of Pneumolysin and Hydrogen Peroxide fromStreptococcus pneumoniaein Inhibition of Ependymal Ciliary Beat Frequency
Authors: Hirst Robert A. , Sikand Kulvinder S. , Rutman Andrew , Mitchell Timothy J. , Andrew Peter W. , O'Callaghan Christopher
Published: July 2002

Ciliated ependymal cells line the ventricular system of the brain and the cerebral aqueducts. This study characterizes the relative roles of pneumolysin and hydrogen peroxide (H2O2) in pneumococcal meningitis, using the in vitro ependymal ciliary beat frequency (CBF) as an indicator of toxicity. We have developed an ex vivo model to examine the ependymal surface of the brain slices cut from the fourth ventricle. The ependymal cells had cilia beating at a frequency of between 38 and 44Hz. D39 (wild-type) and PLN-A (pneumolysin-negative) pneumococci at 108CFU/ml both caused ciliary slowing. Catalase protected against PLN-A-induced ciliary slowing but afforded little protection from D39. Lysed PLN-A did not reduce CBF, whereas lysed D39 caused rapid ciliary stasis. There was no effect of catalase, penicillin, or catalase plus penicillin on the CBF. H2O2at a concentration as low as 100 μM caused ciliary stasis, and this effect was abolished by coincubation with catalase. An additive inhibition of CBF was demonstrated using a combination of both toxins. A significant inhibition of CBF at between 30 and 120 min was demonstrated with both toxins compared with either H2O2(10 μM) or pneumolysin (1 HU/ml) alone. D39 released equivalent levels of H2O2to those released by PLN-A, and these concentrations were sufficient to cause ciliary stasis. The brain slices did not produce H2O2, and in the presence of 108CFU of D39 or PLN-A per ml there was no detectable bacterially induced increase of H2O2release from the brain slice. Coincubation with catalase converted the H2O2produced by the pneumococci to H2O. Penicillin-induced lysis of bacteria dramatically reduced H2O2production. The hemolytic activity released from D39 was sufficient to cause rapid ciliary stasis, and there was no detectable release of hemolytic activity from the pneumolysin-negative PLN-A. These data demonstrate that D39 bacteria released pneumolysin, which caused rapid ciliary stasis. D39 also released H2O2, which contributed to the toxicity, but this was masked by the more severe effects of pneumolysin. H2O2released from intact PLN-A was sufficient to cause rapid ciliary stasis, and catalase protected against H2O2-induced cell toxicity, indicating a role for H2O2in the response. There is also a slight additive effect of pneumolysin and H2O2on ependymal toxicity; however, the precise mechanism of action and the role of these toxins in pathogenesis remain unclear.