bluuebunny/crossref_metadata_2025 huggingface dataset:
An integrated view of foresight: integrated foresight management model
Authors: Alsan Alper , Atilla Oner M.
Published: June 2003

The use of foresight as a tool in policy and strategic decision making increased especially in the last decade of the twentieth century in order to enhance competitiveness and innovation of nations, regions, corporations and even individuals. Coupled with this development a lot of different definitions which partly include part of the others emerged in the literature. However, it was observed that none of these definitions were capable enough to represent an integrated and holistic view about the impact of foresight on the management of the future. In this article, the integrated foresight management model is introduced which is based on the integrated management model but enriched by a knowledge‐people‐system‐organisation framework. The current list of definitions was tabulated in this new model and the vacancies in the model were filled out. This integrated foresight management model can help practitioners in designing national, regional or corporate programs in developing necessary organisational structures, deliverables and behaviours on policy, strategy and operational levels of management.
Pilin-Like Proteins in the Extremely Thermophilic Bacterium
Thermus thermophilus
HB27: Implication in Competence for Natural Transformation and Links to Type IV Pilus Biogenesis
Authors: Friedrich Alexandra , Rumszauer Judit , Henne Anke , Averhoff Beate
Published: July 2003

The extreme thermophile
Thermus thermophilus
HB27 exhibits high frequencies of natural transformation. Although we recently reported identification of the first competence genes in
Thermus
, the molecular basis of DNA uptake is unknown. A pilus-like structure is assumed to be involved. Twelve genes encoding prepilin-like proteins were identified in three loci in the genome of
T. thermophilus
. Mutational analyses, described in this paper, revealed that one locus, which contains four genes that encode prepilin-like proteins (
pilA1
to
pilA4
), is essential for natural transformation. Additionally,
comZ
, a new competence gene with no similarity to known genes, was identified. Analysis of the piliation phenotype revealed wild-type piliation of a
pilA1-pilA3
Δ
kat
mutant and a
comZ
mutant, whereas a
pilA4
mutant was found to be completely devoid of pilus structures. These findings, together with the significant similarity of PilA4 to prepilins, led to the conclusion that the
T. thermophilus
pilus structures are type IV pili. Furthermore, the loss of the transformation and piliation phenotype in the
pilA4
mutant suggests that type IV pili are implicated in natural transformation of
T. thermophilus
HB27.
Effect of Plasterboard Composition on
Stachybotrys chartarum
Growth and Biological Activity of Spores
Authors: Murtoniemi Timo , Nevalainen Aino , Hirvonen Maija-Riitta
Published: July 2003

The effects of plasterboard composition on the growth and sporulation of
Stachybotrys chartarum
as well as on the inflammatory potential of the spores were studied.
S. chartarum
was grown on 13 modified plasterboards under saturated humidity conditions. The biomass was estimated by measuring the ergosterol content of the
S. chartarum
culture while the spore-induced cytotoxicity and production of nitric oxide (NO), tumor necrosis factor alpha (TNF-α), and interleukin-6 in mouse macrophages was used to illustrate the bioactivity of spores. The ergosterol content of
S. chartarum
correlated with the number of spores collected from plasterboards. The growth and sporulation decreased compared to that of the reference board in those cases where (i) the liner was treated with biocide, (ii) starch was removed from the plasterboard, or (iii) desulfurization gypsum was used in the core. Spores collected from all the plasterboards were toxic to the macrophages. The biocide added to the core did not reduce the growth; in fact, the spores collected from that board evoked the highest cytotoxicity. The conventional additives used in the core had inhibitory effects on growth. Recycled plasterboards used in the core and the board lacking the starch triggered spore-induced TNF-α production in macrophages. In summary, this study shows that the growth of a strain of
S. chartarum
on plasterboard and the subsequent bioactivity of spores were affected by minor changes to the composition of the core or liners, but it could not be totally prevented without resorting to the use of biocides. However, incomplete prevention of microbial growth by biocides even increased the cytotoxic potential of the spores.
The Curing Agent Sodium Nitrite, Used in the Production of Fermented Sausages, Is Less Inhibiting to the Bacteriocin-Producing Meat Starter Culture
Lactobacillus curvatus
LTH 1174 under Anaerobic Conditions
Authors: Verluyten Jurgen , Messens Winy , De Vuyst Luc
Published: July 2003

Curvacin A is a listericidal bacteriocin produced by
Lactobacillus curvatus
LTH 1174, a strain isolated from fermented sausage. The response of this strain to an added curing agent (sodium nitrite) in terms of cell growth and bacteriocin production was investigated in vitro by laboratory fermentations with modified MRS broth. The strain was highly sensitive to nitrite; even a concentration of 10 ppm of curing agent inhibited its growth and both volumetric and specific bacteriocin production. A meat simulation medium containing 5 ppm of sodium nitrite was tested to investigate the influence of the gas phase on the growth and bacteriocin production of
L. curvatus
LTH 1174. Aerating the culture during growth had no effect on biomass formation, but the oxidative stress caused a higher level of specific bacteriocin production and led to a metabolic shift toward acetic acid production. Anaerobic conditions, on the other hand, led to an increased biomass concentration and less growth inhibition. Also, higher maximum volumetric bacteriocin activities and a higher level of specific bacteriocin production were obtained in the presence of sodium nitrite than in fermentations under aerobic conditions or standard conditions of air supply. These results indicate that the inhibitory effect of the curing agent is at least partially masked under anaerobic conditions.
Bacterial Community Dynamics during Production of Registered Designation of Origin Salers Cheese as Evaluated by 16S rRNA Gene Single-Strand Conformation Polymorphism Analysis
Authors: Duthoit Frédérique , Godon Jean-Jacques , Montel Marie-Christine
Published: July 2003

Microbial dynamics during processing and ripening of traditional cheeses such as registered designation of origin Salers cheese, an artisanal cheese produced in France, play an important role in the elaboration of sensory qualities. The aim of the present study was to obtain a picture of the dynamics of the microbial ecosystem of RDO Salers cheese by using culture-independent methods. This included DNA extraction, PCR, and single-strand conformation polymorphism (SSCP) analysis. Bacterial and high-GC% gram-positive bacterial primers were used to amplify V2 or V3 regions of the 16S rRNA gene. SSCP patterns revealed changes during the manufacturing of the cheese. Patterns of the ecosystems of cheeses that were provided by three farmers were also quite different. Cloning and sequencing of the 16S rRNA gene revealed sequences related to lactic acid bacteria (
Lactococcus lactis
,
Streptococcus thermophilus
,
Enterococcus faecium
,
Leuconostoc mesenteroides
,
Leuconostoc pseudomesenteroides
,
Lactobacillus plantarum
, and
Lactobacillus pentosus
), which were predominant during manufacturing and ripening. Bacteria belonging to the high-GC% gram-positive group (essentially corynebacteria) were found by using specific primers. The present molecular approach can effectively describe the ecosystem of artisanal dairy products.
Group A Rotavirus in Sewage Samples from Barcelona and Cairo: Emergence of Unusual Genotypes
Authors: Villena Cristina , El-Senousy Waled Morsy , Abad F. Xavier , Pintó Rosa M. , Bosch Albert
Published: July 2003

The presence of rotavirus strains in sewage samples from Cairo, Egypt (November 1998 to October 1999), and Barcelona, Spain (November 1998 to December 2002), was investigated by using a generic molecular detection method based on amplification of a VP6 gene fragment. Overall, 85.7 and 66.9% of the sewage samples from Cairo and Barcelona, respectively, were positive. Positive samples were characterized further, and VP7 and VP4 genotypes were determined. Although 30% of the positive samples from Cairo were G untypeable, the distribution of G types in the positive samples was 69.6% G1, 13% G3, 8.7% G4, and 8.7% G9. The percentage of untypeable samples was much higher for the Barcelona samples (56.5%), and the distribution in the positive samples was 56.4% G1, 31.5% G3, 6% G9, 4% G2, and 2% G5. When the P types were examined, 26.7% of the positive samples from Cairo were untypeable, and the distribution of types in the positive samples was 53.3% P[8], 30% P[6], and 16.6% P[4]. In Barcelona, 27.2% of the samples were P untypeable, and the frequencies of the types detected were 49.7% P[8], 37.2% P[4], 8.8% P[6], and 4.2% P[9]. The distribution for strains from Cairo was 38.5% P[8]G1, 27% P[6]G1, 11.5% P[4]G1, 11.5% P[8]G3, 7.7% P[6]G4, and 3.8% P[8]G9. Strikingly, equivalent frequencies of common and uncommon strains were observed for Barcelona samples, and the distribution was 38.8% P[8]G1, 30.6% P[4]G1, 11.6% P[8]G3, 6.6% P[4]G3, 5.8% P[6]G1, 1.6% P[6]G3, 1.6% P[9]G1, 0.8% P[4]G2, 0.8% P[6]G9, 0.8% P[8]G9, and 0.8% P[8]G5. Additionally, two P[−]G5 strains were isolated in Barcelona, and the porcine or human origin of these strains was unclear. Rotavirus variability exhibited not only a geographic pattern but also a temporal pattern.
Regio- and Stereoselective Metabolism of 7,12-Dimethylbenz[
a
]anthracene by
Mycobacterium vanbaalenii
PYR-1
Authors: Moody Joanna D. , Fu Peter P. , Freeman James P. , Cerniglia Carl E.
Published: July 2003

The degradation of 7,12-dimethylbenz[
a
]anthracene (DMBA), a carcinogenic polycyclic aromatic hydrocarbon, by cultures of
Mycobacterium vanbaalenii
PYR-1 was studied. When
M. vanbaalenii
PYR-1 was grown in the presence of DMBA for 136 h, high-pressure liquid chromatography (HPLC) analysis showed the presence of four ethyl acetate-extractable compounds and unutilized substrate. Characterization of the metabolites by mass and nuclear magnetic resonance spectrometry indicated initial attack at the C-5 and C-6 positions and on the methyl group attached to C-7 of DMBA. The metabolites were identified as
cis
-5,6-dihydro-5,6-dihydroxy-7,12-dimethylbenz[
a
]anthracene (DMBA
cis
-5,6-dihydrodiol),
trans
-5,6-dihydro-5,6-dihydroxy-7,12-dimethylbenz[
a
]anthracene (DMBA
trans
-5,6-dihydrodiol), and 7-hydroxymethyl-12-methylbenz[
a
]anthracene, suggesting dioxygenation and monooxygenation reactions. Chiral stationary-phase HPLC analysis of the dihydrodiols showed that DMBA
cis
-5,6-dihydrodiol had 95% 5
S
,6
R
and 5% 5
R
,6
S
absolute stereochemistry. On the other hand, the DMBA
trans
-5,6-dihydrodiol was a 100% 5
S
,6
S
enantiomer. A minor photooxidation product, 7,12-epidioxy-7,12-dimethylbenz[
a
]anthracene, was also formed. The results demonstrate that
M. vanbaalenii
PYR-1 is highly regio- and stereoselective in the degradation of DMBA.
Salmonella enterica
Serovar Typhimurium and
Listeria monocytogenes
Acid Tolerance Response Induced by Organic Acids at 20°C: Optimization and Modeling
Authors: Greenacre E. J. , Brocklehurst T. F. , Waspe C. R. , Wilson D. R. , Wilson P. D. G.
Published: July 2003

An acid tolerance response (ATR) has been demonstrated in
Listeria monocytogenes
and
Salmonella enterica
serovar Typhimurium in response to low pH poised (i.e., adapted) with acetic or lactic acids at 20°C and modeled by using dynamic differential equations. The ATR was not immediate or prolonged, and optimization occurred after exposure of
L. monocytogenes
for 3 h at pH 5.5 poised with acetic acid and for 2 h at pH 5.5 poised with lactic acid and after exposure of
S. enterica
serovar Typhimurium for 2 h at pH 5.5 poised with acetic acid and for 3 h at pH 5.5 poised with lactic acid. An objective mechanistic analysis of the acid inactivation data yielded estimates of the duration of the shoulder (
t

s

), the log-linear decline (
k
max
), and the magnitude of a critical component (
C
). The magnitude of
k
max
gave the best agreement with estimates of conditions for optimum ATR induction made from the raw data.
Presence of Viral Genomes in Mineral Water: a Sufficient Condition To Assume Infectious Risk?
Authors: Gassilloud Benoît , Schwartzbrod Louis , Gantzer Christophe
Published: July 2003

Appropriate interpretation of a positive reverse transcription-PCR is an important issue for virus-related health hazard assessment because viral genomes and infectious viruses exhibit different behavior patterns in water. In this context, using
Poliovirus 1
and
Feline calicivirus
f9 as examples of enteric viruses, first we demonstrated that the stability of infectious viruses is greatly affected by the temperature of mineral water (10, 20, and 35°C) and that, in contrast, temperature has little effect on the corresponding genomes. Second, we demonstrated that infectious particles are degraded more rapidly than viral genomes at all temperatures studied. At 35°C,
Poliovirus 1
infectivity was reduced 4 logs after only 19 days, while an equivalent reduction would have taken 75 years (according to the model applied) for the viral genome. Contradictory conclusions can also be drawn concerning the sensitivity of viral serotypes depending on whether the infectious virus or the viral genome is considered. The
Feline calicivirus
f9 genome is more resistant than the
Poliovirus 1
genome, whereas the opposite is true for the corresponding infectious viruses. Thus, we concluded that a positive test for a viral genome in mineral water must be interpreted with utmost caution because of the lack of a correlation between the presence of viral genomes and viral infectivity. Detection of viral genomes may be necessary to identify infectious risk for the human population, but it cannot be considered sufficient.
Reduction of Norwalk Virus, Poliovirus 1, and Bacteriophage MS2 by Ozone Disinfection of Water
Authors: Shin Gwy-Am , Sobsey Mark D.
Published: July 2003

Norwalk virus and other human caliciviruses (noroviruses) are major agents of gastroenteritis, and water is a major route of their transmission. In an effort to control Norwalk virus in drinking water, Norwalk virus reduction by bench-scale ozone disinfection was determined using quantitative reverse transcription (RT)-PCR for virus assays. Two other enteric viruses, poliovirus 1 and coliphage MS2, were included for comparison, and their reductions were assayed by infectivity assays as well as by RT-PCR. Virus reductions by ozone were determined using a dose of 0.37 mg of ozone/liter at pH 7 and 5°C for up to 5 min. Based on two RT-PCR assays, the reductions of Norwalk virus were >3 log
10
within a contact time of 10 s, and these were similar to the reductions of the other two viruses determined by the same assay methods. Also, the virus reductions detected by RT-PCR assays were similar to those detected by infectivity assays, indicating that the RT-PCR assay is a reliable surrogate assay for both culturable and nonculturable viruses disinfected with ozone. Overall, the results of this study indicate that Norwalk virus as well as other enteric viruses can be reduced rapidly and extensively by ozone disinfection and that RT-PCR is a useful surrogate assay for both culturable and nonculturable viruses disinfected with ozone.
Chlorine Inactivation of Adenovirus Type 40 and Feline Calicivirus
Authors: Thurston-Enriquez Jeanette A. , Haas Charles N. , Jacangelo Joseph , Gerba Charles P.
Published: July 2003

Ct
values, the concentration of free chlorine multiplied by time of contact with virus, were determined for free-chlorine inactivation experiments carried out with chloroform-extracted (dispersed) and non-chloroform-extracted (aggregated) feline calicivirus (FCV), adenovirus type 40 (AD40), and polio virus type 1 (PV-1). Experiments were carried out with high and low pH and temperature conditions.
Ct
values were calculated directly from bench-scale free-chlorine inactivation experiments and from application of the efficiency factor Hom model. For each experimental condition,
Ct
values were higher at pH 8 than at pH 6, higher at 5°C than at 15°C, and higher for dispersed AD40 (dAD40) than for dispersed FCV (dFCV). dFCV and dAD40 were more sensitive to free chlorine than dispersed PV-1 (dPV-1).
Ct
s for 2 log inactivation of aggregated FCV (aFCV) and aggregated PV-1 (aPV-1) were 31.0 and 2.8 orders of magnitude higher than those calculated from experiments carried out with dispersed virus.
Ct
s for 2 log inactivation of dFCV and dAD40 in treated groundwater at 15°C were 1.2 and 13.7 times greater than in buffered-demand-free (BDF) water experiments at 5°C.
Ct
values listed in the U.S. Environmental Protection Agency (EPA) Guidance Manual were close to, or lower than,
Ct
values generated for experiments conducted with dispersed and aggregated viruses suspended in BDF water and for dispersed viruses suspended in treated groundwater. Since the state of viruses in water is most likely to be aggregated and associated with organic or inorganic matter, reevaluation of the EPA Guidance Manual
Ct
values is necessary, since they would not be useful for ensuring inactivation of viruses in these states. Under the tested conditions, dAD40, dFCV, aFCV, dPV-1, and aPV-1 particles would be inactivated by commonly used free chlorine concentrations (1 mg/liter) and contact times (60 to 237 min) applied for drinking water treatment in the United States.
Molecular, Serological, and Virulence Characteristics of
Vibrio parahaemolyticus
Isolated from Environmental, Food, and Clinical Sources in North America and Asia
Authors: DePaola Angelo , Ulaszek Jodie , Kaysner Charles A. , Tenge Bradley J. , Nordstrom Jessica L. , Wells Joy , Puhr Nancy , Gendel Steven M.
Published: July 2003

Potential virulence attributes, serotypes, and ribotypes were determined for 178 pathogenic
Vibrio parahaemolyticus
isolates from clinical, environmental, and food sources on the Pacific, Atlantic, and Gulf Coasts of the United States and from clinical sources in Asia. The food and environmental isolates were generally from oysters, and they were defined as being pathogenic by using DNA probes to detect the presence of the thermostable direct hemolysin (
tdh
) gene. The clinical isolates from the United States were generally associated with oyster consumption, and most were obtained from outbreaks in Washington, Texas, and New York. Multiplex PCR was used to confirm the species identification and the presence of
tdh
and to test for the
tdh
-related hemolysin
trh
. Most of the environmental, food, and clinical isolates from the United States were positive for
tdh, trh
, and urease production. Outbreak-associated isolates from Texas, New York, and Asia were predominantly serotype O3:K6 and possessed only
tdh
. A total of 27 serotypes and 28 ribogroups were identified among the isolates, but the patterns of strain distribution differed between the serotypes and ribogroups. All but one of the O3:K6 isolates from Texas were in a different ribogroup from the O3:K6 isolates from New York or Asia. The O3:K6 serotype was not detected in any of the environmental and food isolates from the United States, and none of the food or environmental isolates belonged to any of the three ribogroups that contained all of the O3:K6 and related clinical isolates. The combination of serotyping and ribotyping showed that the Pacific Coast
V. parahaemolyticus
population appeared to be distinct from that of either the Atlantic Coast or Gulf Coast. The fact that certain serotypes and ribotypes contained both clinical and environmental isolates while many others contained only environmental isolates implies that certain serotypes or ribotypes are more relevant for human disease.
Analysis of
Vibrio vulnificus
from Market Oysters and Septicemia Cases for Virulence Markers
Authors: DePaola Angelo , Nordstrom Jessica L. , Dalsgaard Anders , Forslund Anita , Oliver James , Bates Tonya , Bourdage Keri L. , Gulig Paul A.
Published: July 2003

Representative encapsulated strains of
Vibrio vulnificus
from market oysters and oyster-associated primary septicemia cases (25 isolates each) were tested in a blinded fashion for potential virulence markers that may distinguish strains from these two sources. These isolates were analyzed for plasmid content, for the presence of a 460-bp amplicon by randomly amplified polymorphic DNA PCR, and for virulence in subcutaneously (s.c.) inoculated, iron-dextran-treated mice. Similar percentages of market oyster and clinical isolates possessed detectable plasmids (24 and 36%, respectively), produced the 460-bp amplicon (45 and 50%, respectively), and were judged to be virulent in the mouse s.c. inoculation-iron-dextran model (88% for each). Therefore, it appears that nearly all
V. vulnificus
strains in oysters are virulent and that genetic tests for plasmids and specific PCR size amplicons cannot distinguish between fully virulent and less virulent strains or between clinical and environmental isolates. The inability of these methods to distinguish food and clinical
V. vulnificus
isolates demonstrates the need for alternative subtyping approaches and virulence assays.
Adaptation ofSaccharomyces cerevisiaeto the Herbicide 2,4-Dichlorophenoxyacetic Acid, Mediated by Msn2p- and Msn4p-Regulated Genes: Important Role ofSPI1
Authors: Simões T. , Teixeira M. C. , Fernandes A. R. , Sá-Correia Isabel
Published: July 2003

The possible roles of 13 Msn2p- and Msn4p-regulated genes in the adaptation ofSaccharomyces cerevisiaeto the herbicide 2,4-d-dichlorophenoxyacetic acid (2,4-D) were examined. Single deletion of genes involved in defense against oxidizing agents (CTT1,GRX1, andGRX2/TTR1) or encoding chaperones of the HSP70 family (SSA1,SSA4, andSSE2) showed a slight effect. A more significant role was observed for the heat shock genesHSP78,HSP26,HSP104,HSP12, andHSP42, most of which encode molecular chaperones. However, theSPI1gene, encoding a member of the glycosylphosphatidylinositol-anchored cell wall protein family, emerged as the major determinant of 2,4-D resistance.SPI1expression reduced the loss of viability of an unadapted yeast population suddenly exposed to the herbicide, allowing earlier growth resumption. Significantly, yeast adaptation to 2,4-D involves the rapid and transient Msn2p- and Msn4p-mediated activation (fivefold) of SPI1transcription.SPI1mRNA levels were reduced to values slightly above those in unstressed cells when the adapted population started duplication in the presence of 2,4-D. SinceSPI1deletion leads to the higher β-1,3-glucanase sensitivity of 2,4-D-stressed cells, it was hypothesized that adaptation may involve an Spi1p-mediated increase in the diffusional restriction of the liposoluble acid form of the herbicide across the cell envelope. Such a cell response would avoid a futile cycle due to acid reentry into the cell counteracting the active export of the anionic form, presumably through an inducible plasma membrane transporter(s). Consistent with this concept, the concentration of14C-labeled 2,4-D in 2,4-D-energized adapted Δspi1mutant cells and the consequent intracellular acidification are higher than in wild-type cells.
Production of 6-Phenylacetylene Picolinic Acid from Diphenylacetylene by a Toluene-Degrading
Acinetobacter
Strain
Authors: Spain Jim C. , Nishino Shirley F. , Witholt Bernard , Tan Loon-Seng , Duetz Wouter A.
Published: July 2003

Several strategies for using enzymes to catalyze reactions leading to the synthesis of relatively simple substituted picolinic acids have been described. The goal of the work described here was to synthesize a more complex molecule, 6-phenylacetylene picolinic acid [6-(2-phenylethynyl)pyridine-2-carboxylic acid], for use as a potential endcapping agent for aerospace polymers. We screened 139 toluene-degrading strains that use a variety of catabolic pathways for the ability to catalyze oxidative transformation of diphenylacetylene.
Acinetobacter
sp. strain F4 catalyzed the overall conversion of diphenylacetylene to a yellow metabolite, which was identified as a putative
meta
ring fission product (2-hydroxy-8-phenyl-6-oxoocta-2,4-dien-7-ynoic acid [RFP]). The activity could be sustained by addition of toluene at a flow rate determined empirically so that the transformations were sustained in spite of the fact that toluene is a competitive inhibitor of the enzymes. The overall rate of transformation was limited by the instability of RFP. The RFP was chemically converted to 6-phenylacetylene picolinic acid by treatment with ammonium hydroxide. The results show the potential for using the normal growth substrate to provide energy and to maintain induction of the enzymes involved in biotransformation during preliminary stages of biocatalyst development.
Optimization of a Reusable Hollow-Fiber Ultrafilter for Simultaneous Concentration of Enteric Bacteria, Protozoa, and Viruses from Water
Authors: Morales-Morales Hugo A. , Vidal Guadalupe , Olszewski John , Rock Channah M. , Dasgupta Debanjana , Oshima Kevin H. , Smith Geoffrey B.
Published: July 2003

The detection and identification of pathogens from water samples remain challenging due to variations in recovery rates and the cost of procedures. Ultrafiltration offers the possibility to concentrate viral, bacterial, and protozoan organisms in a single process by using size-exclusion-based filtration. In this study, two hollow-fiber ultrafilters with 50,000-molecular-weight cutoffs were evaluated to concentrate microorganisms from 2- and 10-liter water samples. When known quantities (10
5
to 10
6
CFU/liter) of two species of enteric bacteria were introduced and concentrated from 2 liters of sterile water, the addition of 0.1% Tween 80 increased
Escherichia coli
strain K-12 recoveries from 70 to 84% and
Salmonella enterica
serovar Enteritidis recoveries from 36 to 72%. An
E. coli
antibiotic-resistant strain, XL1-Blue, was recovered at a level (87%) similar to that for strain K-12 (96%) from 10 liters of sterile water. When
E. coli
XL1-Blue was introduced into 10 liters of nonsterile Rio Grande water with higher turbidity levels (23 to 29 nephelometric turbidity units) at two inoculum levels (9 × 10
5
and 2.4 × 10
3
per liter), the recovery efficiencies were 89 and 92%, respectively. The simultaneous addition of
E. coli
XL1-Blue (9 × 10
5
CFU/liter),
Cryptosporidium parvum
oocysts (10 oocysts/liter), phage T1 (10
5
PFU/liter), and phage PP7 (10
5
PFU/liter) to 10 liters of Rio Grande surface water resulted in mean recoveries of 96, 54, 59, and 46%, respectively. Using a variety of surface waters from around the United States, we obtained recovery efficiencies for bacteria and viruses that were similar to those observed with the Rio Grande samples, but recovery of
Cryptosporidium
oocysts was decreased, averaging 32% (the site of collection of these samples had previously been identified as problematic for oocyst recovery). Results indicate that the use of ultrafiltration for simultaneous recovery of bacterial, viral, and protozoan pathogens from variable surface waters is ready for field deployment.
Ingestion of
Salmonella enterica
Serotype Poona by a Free-Living Nematode,
Caenorhabditis elegans
, and Protection against Inactivation by Produce Sanitizers
Authors: Caldwell Krishaun N. , Adler Barbara B. , Anderson Gary L. , Williams Phillip L. , Beuchat Larry R.
Published: July 2003

Free-living nematodes are known to ingest food-borne pathogens and may serve as vectors to contaminate preharvest fruits and vegetables.
Caenorhabditis elegans
was selected as a model to study the effectiveness of sanitizers in killing
Salmonella enterica
serotype Poona ingested by free-living nematodes. Aqueous suspensions of adult worms that had fed on
S. enterica
serotype Poona were treated with produce sanitizers. Treatment with 20 μg of free chlorine/ml significantly (α = 0.05) reduced the population of
S. enterica
serotype Poona compared to results for treating worms with water (control). However, there was no significant difference in the number of
S. enterica
serotype Poona cells surviving treatments with 20 to 500 μg of chlorine/ml, suggesting that reductions caused by treatment with 20 μg of chlorine/ml resulted from inactivation of
S. enterica
serotype Poona on the surface of
C. elegans
but not cells protected by the worm cuticle after ingestion. Treatment with Sanova (850 or 1,200 μg/ml), an acidified sodium chlorite sanitizer, caused reductions of 5.74 and 6.34 log
10
CFU/worm, respectively, compared to reductions from treating worms with water. Treatment with 20 or 40 μg of Tsunami 200/ml, a peroxyacetic acid-based sanitizer, resulted in reductions of 4.83 and 5.34 log
10
CFU/worm, respectively, compared to numbers detected on or in worms treated with water. Among the organic acids evaluated at a concentration of 2%, acetic acid was the least effective in killing
S. enterica
serotype Poona and lactic acid was the most effective. Treatment with up to 500 μg of chlorine/ml, 1% hydrogen peroxide, 2,550 μg of Sanova/ml, 40 μg of Tsunami 200/ml, or 2% acetic, citric, or lactic acid had no effect on the viability or reproductive behavior of
C. elegans
. Treatments were also applied to cantaloupe rind and lettuce inoculated with
S. enterica
serotype Poona or
C. elegan
s that had ingested
S. enterica
serotype Poona. Protection of ingested
S. enterica
serotype Poona against sanitizers applied to cantaloupe was not evident; however, ingestion afforded protection of the pathogen on lettuce. These results indicate that
S. enterica
serotype Poona ingested by
C. elegans
may be protected against treatment with chlorine and other sanitizers, although the basis for this protection remains unclear.
Use of Pulsed-Field Gel Electrophoresis To Characterize the Heterogeneity and Clonality ofSalmonellaIsolates Obtained from the Carcasses and Feces of Swine at Slaughter
Authors: Wonderling Laura , Pearce Rachel , Wallace F. Morgan , Call Jeffrey E. , Feder Ingrid , Tamplin Mark , Luchansky John B.
Published: July 2003

Salmonella entericaisolates were recovered from swine at a collaborating processing plant over a 2-month period in the spring of 2000. In the present study, molecular subtyping by pulsed-field gel electrophoresis (PFGE) was performed on the 581 confirmedSalmonellaisolates from the 84Salmonella-positive samples obtained from the previous study. A total of 32 different PFGE pulsotypes were observed visually, and a BioNumerics software analysis clustered those pulsotypes into 12 PFGE groups. The B, F, and G groups predominated throughout the sampling period and were isolated from 39, 22, and 13% of the swine, respectively. In addition, multiple isolates were obtained from 67 of the 84Salmonella-positive samples, and subtyping revealed multiple PFGE profiles in 35 of these 67 (62%) samples. Both carcass and fecal isolates ofSalmonellawere recovered from 13 swine, resulting in “matched” samples. Molecular typing of the 252 isolates recovered from the matched samples revealed that 7 (54%) of the 13 carcasses were contaminated withSalmonellapulsotypes that were not isolated from the feces of the same animal. Conversely, from 6 of the 13 (46%) matched animals,Salmonellaclonal types were isolated from the feces that were not isolated from the carcass of the same animal. These data establish that each lot of swine introduces new contaminants into the plant environment and that swine feces from one animal can contaminate many carcasses. In addition, these results indicate that the examination of multipleSalmonellaisolates from positive samples is necessary to determine the variety of potential contaminants of swine carcasses during slaughter and processing.
Identification ofCryptosporidiumspp. Oocysts in United Kingdom Noncarbonated Natural Mineral Waters and Drinking Waters by Using a Modified Nested PCR-Restriction Fragment Length Polymorphism Assay
Authors: Nichols R. A. B. , Campbell B. M. , Smith H. V.
Published: July 2003

We describe a nested PCR-restriction fragment length polymorphism (RFLP) method for detecting low densities ofCryptosporidiumspp. oocysts in natural mineral waters and drinking waters. Oocysts were recovered from seeded 1-liter volumes of mineral water by filtration through polycarbonate membranes and from drinking waters by filtration, immunomagnetizable separation, and filter entrapment, followed by direct extraction of DNA. The DNA was released from polycarbonate filter-entrapped oocysts by disruption in lysis buffer by using 15 cycles of freeze-thawing (1 min in liquid nitrogen and 1 min at 65°C), followed by proteinase K digestion. Amplicons were readily detected from two to five intact oocysts on ethidium bromide-stained gels. DNA extracted fromCryptosporidium parvumoocysts,C. muris(RN 66),C. baileyi(Belgium strain, LB 19), human-derivedC. meleagridis,C. felis(DNA from oocysts isolated from a cat), andC. andersoniwas used to demonstrate species identity by PCR-RFLP after simultaneous digestion with the restriction enzymesDraI andVspI. Discrimination betweenC. andersoniandC. murisisolates was confirmed by a separate, subsequent digestion withDdeI. Of 14 drinking water samples tested, 12 were found to be positive by microscopy, 8 were found to be positive by direct PCR, and 14 were found to be positive by using a nested PCR. TheCryptosporidiumspecies detected in these finished water samples wasC. parvumgenotype 1. This method consistently and routinely detected >5 oocysts per sample.
A Selective Medium for Quantitative Reisolation of
Trichoderma harzianum
from
Agaricus bisporus
Compost
Authors: Williams Josie , Clarkson John M. , Mills Peter R. , Cooper Richard M.
Published: July 2003

We adapted a selective medium, previously developed for reisolation of
Trichoderma
spp. from soil, for quantitative determination of growth of
T. harzianum
from commercial
Agaricus bisporus
composts. This medium enables comparisons of aggressive (sensu inhibition of
A. bisporus
yield) with nonaggressive
T. harzianum
groups. The resulting medium contains the antimicrobials chloramphenicol, streptomycin, quintozene, and propamocarb and was highly selective, allowing the recovery of
T. harzianum
, as viable conidia and hyphal fragments, in compact colonies with the absence of visible microbial contaminants.
Detection and Diversity Assessment of
Xylella fastidiosa
in Field-Collected Plant and Insect Samples by Using 16S rRNA and
gyrB
Sequences
Authors: Rodrigues Jorge L. M. , Silva-Stenico M. E. , Gomes J. E. , Lopes J. R. S. , Tsai S. M.
Published: July 2003

The causal agent of diseases in many economically important plants is attributed to the xylem-limited bacterium
Xylella fastidiosa.
The detection of this plant pathogen has been hampered due to its difficult isolation and slow growth on plates. Nearly complete nucleotide sequences of the 16S rRNA gene and partial sequences of the
gyrB
gene were determined for 18 strains of
X. fastidiosa
isolated from different plant hosts. A phylogenetic analysis, based on
gyrB
, grouped strains in three clusters; grape-isolated strains formed one cluster, citrus-coffee strains formed another cluster, and a third cluster resulted from all other strains. Primer pairs designed for the 16S rRNA and
gyrB
genes were extensively searched in databases to verify their in silico specificity. Primer pairs were certified with 30 target and 36 nontarget pure cultures of microorganisms, confirming 100% specificity. A multiplex PCR protocol was developed and its sensitivity tested. Sequencing of PCR products confirmed the validity of the multiplex PCR.
Xylella fastidiosa
was detected in field-collected plants, disease vector insects, and nonsymptomatic but infected plants. Specific detection of
X. fastidiosa
may facilitate the understanding of its ecological significance and prevention of spread of the disease.
Mode of Bactericidal Action of Silver Zeolite and Its Comparison with That of Silver Nitrate
Authors: Matsumura Yoshinobu , Yoshikata Kuniaki , Kunisaki Shin-ichi , Tsuchido Tetsuaki
Published: July 2003

The properties of the bactericidal action of silver zeolite as affected by inorganic salts and ion chelators were similar to those of silver nitrate. The results suggest that the contact of the bacterial cell with silver zeolite, the consequent transfer of silver ion to the cell, and the generation of reactive oxygen species in the cell are involved in the bactericidal activity of silver zeolite.
Motility of
Colwellia psychrerythraea
Strain 34H at Subzero Temperatures
Authors: Junge Karen , Eicken Hajo , Deming Jody W.
Published: July 2003

We examined the Arctic bacterium
Colwellia psychrerythraea
strain 34H for motility at temperatures from −1 to −15°C by using transmitted-light microscopy in a temperature-controlled laboratory. The results, showing motility to −10°C, indicate much lower temperatures to be permissive of motility than previously reported (5°C), with implications for microbial activity in frozen environments.
Identification of Novel
Cryptosporidium
Genotypes from the Czech Republic
Authors: Ryan Una , Xiao Lihua , Read Carolyn , Zhou Ling , Lal Altaf A. , Pavlasek Ivan
Published: July 2003

Isolates of
Cryptosporidium
from the Czech Republic were characterized from a variety of different hosts using sequence and phylogenetic analysis of the 18S ribosomal DNA and the heat-shock (HSP-70) gene. Analysis expanded the host range of accepted species and identified several novel genotypes, including horse, Eurasian woodcock, rabbit, and cervid genotypes.
Inhibitory Effect of Maillard Reaction Products on Growth of the Aerobic Marine Hyperthermophilic Archaeon
Aeropyrum pernix
Authors: Kim Kee Woung , Lee Sun Bok
Published: July 2003

It was found that the growth of
Aeropyrum pernix
was severely inhibited in a medium containing reducing sugars and tryptone due to the formation of Maillard reaction products. The rate of the Maillard browning reaction was markedly enhanced under aerobic conditions, and the addition of Maillard reaction products to the culture medium caused fatal growth inhibition.
Molecular Phylogenetic Exploration of Bacterial Diversity in a Bakreshwar (India) Hot Spring and Culture of
Shewanella
-Related Thermophiles
Authors: Ghosh Dhritiman , Bal Bijay , Kashyap V. K. , Pal Subrata
Published: July 2003

The bacterial diversity of a hot spring in Bakreshwar, India, was investigated by a culture-independent approach. 16S ribosomal DNA clones derived from the sediment samples were found to be associated with gamma-
Proteobacteria
, cyanobacteria, and green nonsulfur and low-GC gram-positive bacteria. The first of the above phylotypes cobranches with
Shewanella
, a well-known iron reducer. This phylogenetic correlation has been exploited to develop culture conditions for thermophilic iron-reducing microorganisms.
Large-Restriction-Fragment Polymorphism Analysis of
Mycobacterium chelonae
and
Mycobacterium terrae
Isolates
Authors: Vanitha J. Daisy , Venkatasubramani R. , Dharmalingam K. , Paramasivan C. N.
Published: July 2003

Mycobacterium chelonae
and
Mycobacterium terrae
were reported to be frequently present in the environment of the
Mycobacterium bovis
BCG trial area in south India. Six isolates of
M. chelonae
and four isolates of
M. terrae
obtained from different sources in this area were analyzed by pulsed-field gel electrophoresis (PFGE) to examine large-restriction-fragment (LRF) polymorphism using the chromosomal DNA digested with
Dra
I and
Xba
I restriction enzymes. With the exception of one isolate of
M. terrae
, DNA from all other isolates could be digested with
Dra
I and
Xba
I and resulted in separable fragments. Visual comparison of the LRFs showed a unique pattern for each of the isolates tested. A computer-assisted dendrogram of the percent similarity demonstrated a high degree of genetic diversity in this group of isolates. This study demonstrates that species of nontuberculous mycobacteria, particularly
M. chelonae
and
M. terrae
, can be successfully typed by their LRF pattern using PFGE, which does not require species-specific DNA probes.
The Human Cytomegalovirus UL44 Protein Is a Substrate for the UL97 Protein Kinase
Authors: Krosky Paula M. , Baek Moon-Chang , Jahng Wan Jin , Barrera Imma , Harvey Robert J. , Biron Karen K. , Coen Donald M. , Sethna Phiroze B.
Published: June 2003

The human cytomegalovirus UL97 protein is an unusual protein kinase that is able to autophosphorylate and to phosphorylate certain exogenous substrates, including nucleoside analogs such as ganciclovir. However, no natural substrate of UL97 in infected cells has been identified. We report here that recombinant UL44 protein became radiolabeled when incubated with recombinant UL97 and [
32
P]ATP and that both proteins could be coimmunoprecipitated by an antibody that recognizes either protein. Subsequent studies showed that highly purified, recombinant UL97 phosphorylated purified, recombinant UL44. This phosphorylation occurred on serine and threonine residues and was sensitive to inhibition by maribavir and to a mutation that inactivates UL97 catalytic activity. Two-dimensional gel electrophoresis revealed the absence of specific phosphorylated forms of UL44 in immunoprecipitates from lysates of cells infected with a
UL97
null mutant virus or with wild-type virus in the presence of maribavir. The results indicate that UL97 is sufficient to phosphorylate UL44 in vitro and is necessary for the normal phosphorylation of UL44 in infected cells. This strongly suggests that UL44 is a natural substrate of UL97.
Murine Cytomegalovirus with a Transposon Insertional Mutation at Open Reading Frame M35 Is Defective in Growth In Vivo
Authors: Tam Ada , Zhu Jiaming , Hai Rong , Haghjoo Erik , Tong Tuong , Zhan Xiaoyan , Lu Sangwei , Liu Fenyong
Published: June 2003

We had previously constructed a pool of murine cytomegalovirus (MCMV) mutants that contained a Tn
3
-based transposon sequence randomly inserted in the viral genome. In the study reported here, one of the mutants, RvM35, which contains the transposon insertion at open reading frame M35, was characterized both in vitro in tissue cultures and in immunocompetent Balb/c and immunodeficient SCID mice. Our results provide the first direct evidence to suggest that M35 is not essential for viral replication in vitro in NIH 3T3 cells. Compared to the wild-type strain and a rescued virus that restored the M35 region, the viral mutant was attenuated in growth in both the intraperitoneally infected Balb/c and SCID mice. At 21 days postinfection, the titers of the mutant in the salivary glands, lungs, spleens, livers, and kidneys of the SCID mice were lower than the titers of the wild-type Smith strain and the rescued virus by 50,000-, 100-, 10-, 100-, and 50-fold, respectively. Moreover, the growth of RvM35 is severely attenuated in the salivary glands. The virulence of the mutant virus also appears to be attenuated, because no death was observed in SCID mice infected with RvM35 until 35 days postinfection, while all the animals infected with the wild-type and rescued viruses died 27 days postinfection. Our results suggest that M35 is important for MCMV virulence in killing SCID mice and is required for optimal viral growth in vivo, including in the salivary glands.
Variable Sensitivity to Substitutions in the N-Terminal Heptad Repeat of Mason-Pfizer Monkey Virus Transmembrane Protein
Authors: Song Chisu , Hunter Eric
Published: June 2003

The transmembrane protein of Mason-Pfizer monkey virus contains two heptad repeats that are predicted to form amphipathic α-helices that mediate the conformational change necessary for membrane fusion. To analyze the relative sensitivity of the predicted hydrophobic face of the N-terminal heptad repeat to the insertion of uncharged, polar, and charged substitutions, mutations that introduced alanine, serine, or glutamic acid into positions 436, 443, 450, and 457 of the envelope protein were examined. Novel systems using Tat protein and the GHOST cell line were developed to test and quantitate the effects of the mutations on Env-mediated fusion and infectivity of the virus. While no single amino acid change at any of the positions interfered significantly with the synthesis, processing, or transport to the plasma membrane of glycoprotein complexes, 9 of the 12 nonconservative mutations in these residues completely abolished fusion activity and virus infectivity. Mutations in the central positions (443 and 450) of the heptad repeat region were the most detrimental to Env function, and even single alanine substitutions in these positions dramatically altered the fusogenicity of the protein. These results demonstrate that this N-terminal heptad repeat plays a critical role in Env-mediated membrane fusion and highlight the key function of central hydrophobic residues in this process and the sensitivity of all positions to charge substitutions.
Differential Inhibition of RNA Editing in Hepatitis Delta Virus Genotype III by the Short and Long Forms of Hepatitis Delta Antigen
Authors: Cheng Qiufang , Jayan Geetha C. , Casey John L.
Published: June 2003

Hepatitis delta virus (HDV) produces two essential forms of the sole viral protein from the same open reading frame by using host RNA editing activity at the amber/W site in the antigenomic RNA. The roles of these two forms, HDAg-S and HDAg-L, are opposed. HDAg-S is required for viral RNA replication, whereas HDAg-L, which is produced as a result of editing, inhibits viral RNA replication and is required for virion packaging. Both the rate and amount of editing are important because excessive editing will inhibit viral RNA replication, whereas insufficient editing will reduce virus secretion. Here we show that for HDV genotype III, which is associated with severe HDV disease, HDAg-L strongly inhibits editing of a nonreplicating genotype III reporter RNA, while HDAg-S inhibits only when expressed at much higher levels. The different inhibitory efficiencies are due to RNA structural elements located ca. 25 bp 3′ of the editing site in the double-hairpin RNA structure required for editing at the amber/W site in HDV genotype III RNA. These results are consistent with regulation of amber/W editing in HDV genotype III by a negative-feedback mechanism due to differential interactions between structural elements in the HDV genotype III RNA and the two forms of HDAg.
Application of Chimeric Feline Foamy Virus-Based Retroviral Vectors for the Induction of Antiviral Immunity in Cats
Authors: Schwantes Astrid , Truyen Uwe , Weikel Joachim , Weiss Christian , Löchelt Martin
Published: June 2003

In order to define the potential and applicability of replication-competent foamy virus-based vaccine vectors, recombinant feline foamy virus (FFV) vectors encoding defined segments of the feline calicivirus (FCV) capsid protein E domain were constructed. In cell cultures, these FFV-FCV vectors efficiently transduced and expressed a hybrid fusion protein consisting of the essential FFV Bet protein and the attached FCV E domains. The stability of the vectors in vitro was inversely correlated to the size of the heterologous insert. The deletion of a part of the FFV U3 sequence in these FFV-FCV vectors did not interfere with replication and titer in cell cultures but increased the genetic stability of the hybrid vectors. Selected chimeric vectors were injected into immunocompetent cats and persisted in the transduced host concomitant with a strong and specific humoral immune response against vector components. In a substantial number of cats, antibodies directed against the FCV E domain were induced by the FFV-FCV vectors, but no FCV-neutralizing activities were detectable in vitro. When the vaccinated cats were challenged with a high-titer FCV dose, sterile immunity was not induced by any of the hybrid FFV-FCV vectors. However, the FFV-FCV vector with a truncated U3 region of the long terminal repeat promoter significantly reduced the duration of FCV shedding after challenge and suppressed the appearance of FCV-specific ulcers. Possible mechanisms contributing to the partial protection will be discussed.