bluuebunny/crossref_metadata_2025_split huggingface dataset:
Flow Cytometric Assessment of Viability of Lactic Acid Bacteria
Authors: Bunthof Christine J. , Bloemen Karen , Breeuwer Pieter , Rombouts Frank M. , Abee Tjakko 
Published: July 2002

ABSTRACT

The viability of lactic acid bacteria is crucial for their applications as dairy starters and as probiotics. We investigated the usefulness of flow cytometry (FCM) for viability assessment of lactic acid bacteria. The esterase substrate carboxyfluorescein diacetate (cFDA) and the dye exclusion DNA binding probes propidium iodide (PI) and TOTO-1 were tested for live/dead discrimination using a
Lactococcus
, a
Streptococcus
, three
Lactobacillus
, two
Leuconostoc
, an
Enterococcus
, and a
Pediococcus
species. Plate count experiments were performed to validate the results of the FCM assays. The results showed that cFDA was an accurate stain for live cells; in exponential-phase cultures almost all cells were labeled, while 70°C heat-killed cultures were left unstained. PI did not give clear live/dead discrimination for some of the species. TOTO-1, on the other hand, gave clear discrimination between live and dead cells. The combination of cFDA and TOTO-1 gave the best results. Well-separated subpopulations of live and dead cells could be detected with FCM. Cell sorting of the subpopulations and subsequent plating on agar medium provided direct evidence that cFDA labels the culturable subpopulation and that TOTO-1 labels the nonculturable subpopulation. Applied to cultures exposed to deconjugated bile salts or to acid, cFDA and TOTO-1 proved to be accurate indicators of culturability. Our experiments with lactic acid bacteria demonstrated that the combination of cFDA and TOTO-1 makes an excellent live/dead assay with versatile applications.
Elucidation of
Listeria monocytogenes
Contamination Routes in Cold-Smoked Salmon Processing Plants Detected by DNA-Based Typing Methods
Authors: Fonnesbech Vogel Birte , Huss Hans Henrik , Ojeniyi Bente , Ahrens Peter , Gram Lone 
Published: July 2002

ABSTRACT

The contamination routes of
Listeria monocytogenes
in cold-smoked salmon processing plants were investigated by analyzing 3,585 samples from products (produced in 1995, 1996, 1998, and 1999) and processing environments (samples obtained in 1998 and 1999) of two Danish smokehouses. The level of product contamination in plant I varied from 31 to 85%, and no
L. monocytogenes
was found on raw fish (30 fish were sampled). In plant II, the levels of both raw fish and product contamination varied from 0 to 25% (16 of 185 raw fish samples and 59 of 1,000 product samples were positive for
L. monocytogenes
). A total of 429 strains of
L. monocytogenes
were subsequently compared by random amplified polymorphic DNA (RAPD) profiling, and 55 different RAPD types were found. The RAPD types detected on the products were identical to types found on the processing equipment and in the processing environment, suggesting that contamination of the final product (cold-smoked salmon) in both plants (but primarily in plant I) was due to contamination during processing rather than to contamination from raw fish. However, the possibility that raw fish was an important source of contamination of the processing equipment and environment could not be excluded. Contamination of the product occurred in specific areas (the brining and slicing areas). In plant I, the same RAPD type (RAPD type 12) was found over a 4-year period, indicating that an established in-house flora persisted and was not eliminated by routine hygienic procedures. In plant II, where the prevalence of
L. monocytogenes
was much lower, no RAPD type persisted over long periods of time, and several different
L. monocytogenes
RAPD types were isolated. This indicates that persistent strains may be avoided by rigorous cleaning and sanitation; however, due to the ubiquitous nature of the organism, sporadic contamination occurred. A subset of strains was also typed by using pulsed-field gel electrophoresis and amplified fragment length polymorphism profiling, and these methods confirmed the type division obtained by RAPD profiling.
Dual System To Reinforce Biological Containment of Recombinant Bacteria Designed for Rhizoremediation
Authors: Ronchel M. Carmen , Ramos Juan L. 
Published: July 2002

ABSTRACT

Active biological containment (ABC) systems have been designed to control at will the survival or death of a bacterial population. These systems are based on the use of a killing gene, e.g., a porin-inducing protein such as the one encoded by the
Escherichia coli gef
gene, and a regulatory circuit that controls expression of the killing gene in response to the presence or absence of environmental signals. An ABC system for recombinant microorganisms that degrade a model pollutant was designed on the basis of the
Pseudomonas putida
TOL plasmid
meta
-cleavage regulatory circuit. The system consists of a fusion of the Pm promoter to
lacI
, whose expression is controlled by XylS with 3-methylbenzoate, and a fusion of a synthetic P
lac
promoter to
gef
. In the presence of the model pollutant, bacterial cells survived and degraded the target compound, whereas in the absence of the aromatic carboxylic acid cell death was induced. The system had two main drawbacks: (i) the slow death of the bacterial cells in soil versus the fast killing rate in liquid cultures in laboratory assays, and (ii) the appearance of mutants, at a rate of about 10
−8
per cell and generation, that did not die after the pollutant had been exhausted. We reinforced the ABC system by including it in a Δ
asd P. putida
background. A
P. putida
Δ
asd
mutant is viable only in complex medium supplemented with diaminopimelic acid, methionine, lysine, and threonine. We constructed a
P. putida
Δ
asd
strain, called MCR7, with a Pm::
asd
fusion in the host chromosome. This strain was viable in the presence of 3-methylbenzoate because synthesis of the essential metabolites was achieved through XylS-dependent induction. In the
P. putida
MCR7 strain, an ABC system (Pm::
lacI
,
xylS
, P
lac
::
gef
) was incorporated into the host chromosome to yield strain MCR8. The number of MCR8 mutants that escaped killing was below our detection limit (<10
−9
mutants per cell and generation). The MCR8 strain survived and colonized rhizosphere soil with 3-methylbenzoate at a level similar to that of the wild-type strain. However, it disappeared in less than 20 to 25 days in soils without the pollutant, whereas an
asd
+
, biologically contained counterpart such as
P. putida
CMC4 was still detectable in soils after 100 days.
Comparison of Endotoxin Exposure Assessment by Bioaerosol Impinger and Filter-Sampling Methods
Authors: Duchaine Caroline , Thorne Peter S. , Mériaux Anne , Grimard Yan , Whitten Paul , Cormier Yvon 
Published: July 2002

ABSTRACT

Environmental assessment data collected in two prior occupational hygiene studies of swine barns and sawmills allowed the comparison of concurrent, triplicate, side-by-side endotoxin measurements using air sampling filters and bioaerosol impingers. Endotoxin concentrations in impinger solutions and filter eluates were assayed using the
Limulus
amebocyte lysate assay. In sawmills, impinger sampling yielded significantly higher endotoxin concentration measurements and lower variances than filter sampling with IOM inhalable dust samplers. Analysis of variance for repeated measures showed that this association remained after controlling for other factors such as replicate, sawmill, sawmill operation, wood type, and interaction terms. Endotoxin concentrations in the swine barns were 10-fold higher on average than in sawmills. These samples demonstrated comparable endotoxin concentration estimates for impinger and filter methods although the variability was lower using the impinger method. In both occupational settings, side-by-side replicates were more uniform for the impinger samples than for the filter samples. This study demonstrates that impinger sampling is an acceptable method for quantitation of area endotoxin concentrations. Further, when sampling is performed with impingers for airborne microorganism quantitation, these same impinger solutions can yield valid endotoxin exposure estimates, negating the need for additional filter sampling.
Autotrophic Ammonia Oxidation at Low pH through Urea Hydrolysis
Authors: Burton Simon A. Q. , Prosser Jim I. 
Published: July 2002

ABSTRACT

Ammonia oxidation in laboratory liquid batch cultures of autotrophic ammonia oxidizers rarely occurs at pH values less than 7, due to ionization of ammonia and the requirement for ammonium transport rather than diffusion of ammonia. Nevertheless, there is strong evidence for autotrophic nitrification in acid soils, which may be carried out by ammonia oxidizers capable of using urea as a source of ammonia. To determine the mechanism of urea-linked ammonia oxidation, a ureolytic autotrophic ammonia oxidizer,
Nitrosospira
sp. strain NPAV, was grown in liquid batch culture at a range of pH values with either ammonium or urea as the sole nitrogen source. Growth and nitrite production from ammonium did not occur at pH values below 7. Growth on urea occurred at pH values in the range 4 to 7.5 but ceased when urea hydrolysis was complete, even though ammonia, released during urea hydrolysis, remained in the medium. The results support a mechanism whereby urea enters the cells by diffusion and intracellular urea hydrolysis and ammonia oxidation occur independently of extracellular pH in the range 4 to 7.5. A proportion of the ammonia produced during this process diffuses from the cell and is not subsequently available for growth if the extracellular pH is less than 7. Ureolysis therefore provides a mechanism for nitrification in acid soils, but a proportion of the ammonium produced is likely to be released from the cell and may be used by other soil organisms.
Exo-Arabinanase of
Penicillium chrysogenum
Able To Release Arabinobiose from α-1,5-
l
-Arabinan
Authors: Sakamoto Tatsuji , Thibault Jean-François 
Published: July 2002

ABSTRACT

An exo-arabinanase, designated Abnx, was purified from a culture filtrate of
Penicillium chrysogenum
31B by ammonium sulfate precipitation, anion-exchange chromatography, and hydrophobic chromatography. Abnx had an apparent molecular mass of 47 kDa. The enzyme released only arabinobiose from the nonreducing terminus of α-1,5-
l
-arabinan and showed no activity towards
p
-nitrophenyl-α-
l
-arabinofuranoside and α-1,5-
l
-arabinofuranobiose. Abnx is the first enzyme with this mode of action.
Incidence of
Klebsiella
Species in Surface Waters and Their Expression of Virulence Factors
Authors: Podschun R. , Pietsch S. , Höller C. , Ullmann U. 
Published: July 2002

ABSTRACT

To investigate the occurrence of different
Klebsiella
spp. in aquatic environments, a total of 208 samples of natural surface waters was examined. From half (53%) of these samples, 123
Klebsiella
strains were isolated, the most common species being
Klebsiella pneumoniae
. A comparison of these isolates to a group of 207 clinical
K. pneumoniae
isolates demonstrated that water isolates of
K. pneumoniae
, unlike those of
K. oxytoca
and
K. planticola
, are as capable as clinical isolates of expressing putative virulence factors such as serum resistance and capsular polysaccharides, pili, and siderophores.
Relationship between Monokaryotic Growth Rate and Mating Type in the Edible Basidiomycete
Pleurotus ostreatus
Authors: Larraya Luis M. , Pérez Gúmer , Iribarren Iñaki , Blanco Juan A. , Alfonso Mikel , Pisabarro Antonio G. , Ramı́rez Lucı́a 
Published: July 2002

ABSTRACT

The edible fungus
Pleurotus ostreatus
(oyster mushroom) is an industrially produced heterothallic homobasidiomycete whose mating is controlled by a bifactorial tetrapolar genetic system. Two mating loci (
matA
and
matB
) control different steps of hyphal fusion, nuclear migration, and nuclear sorting during the onset and progress of the dikaryotic growth. Previous studies have shown that the segregation of the alleles present at the
matB
locus differs from that expected for a single locus because (i) new nonparental
B
alleles appeared in the progeny and (ii) there was a distortion in the segregation of the genomic regions close to this mating locus. In this study, we pursued these observations by using a genetic approach based on the identification of molecular markers linked to the
matB
locus that allowed us to dissect it into two genetically linked subunits (
matBα
and
matBβ
) and to correlate the presence of specific
matBα
and
matA
alleles with differences in monokaryotic growth rate. The availability of these molecular markers and the mating type dependence of growth rate in monokaryons can be helpful for marker-assisted selection of fast-growing monokaryons to be used in the construction of dikaryons able to colonize the substrate faster than the competitors responsible for reductions in the industrial yield of this fungus.
Potential Role of Fomites in the Vehicular Transmission of Human Astroviruses
Authors: Abad F. Xavier , Villena Cristina , Guix Susana , Caballero Santiago , Pintó Rosa M. , Bosch Albert 
Published: July 2002

ABSTRACT
The persistence of human astroviruses dried on representative porous (paper) and nonporous (china) surfaces was investigated. Long-term astrovirus survival on fomites was monitored by an integrated cell culture-reverse transcription-PCR procedure. Viruses were applied to inanimate surfaces in the presence and absence of fecal material, and their survival was assayed at 4 and 20°C with high relative humidity. Astroviruses exhibited a notable persistence when dried on porous and nonporous materials, particularly at low temperature. Short-term survival of astroviruses on fomites was compared to that of other enteric viruses significant for health, such as rotavirus, adenovirus, poliovirus, and hepatitis A virus. Overall, astroviruses persisted better than poliovirus and adenovirus, although they exhibited a shorter survival than rotavirus and hepatitis A virus. Astroviruses show a high level of persistence at the desiccation step, which is of major significance in determining the chance of subsequent virus survival dried on fomites. Astroviruses are able to survive on inert surfaces long enough to suggest that fomites may play a relevant role in the secondary transmission of astrovirus diarrhea.
Molecular and Biochemical Analysis of Two β-Galactosidases fromBifidobacterium infantisHL96
Authors: Hung Ming-Ni , Xia Zhicheng , Hu Nien-Tai , Lee Byong H. 
Published: July 2002

ABSTRACTTwo genes encoding β-galactosidase isoenzymes,β-galIandβ-galIII, fromBifidobacterium infantisHL96 were revealed on 3.6- and 2.4-kb DNA fragments, respectively, by nucleotide sequence analysis of the two fragments.β-galI(3,069 bp) encodes a 1,022-amino-acid (aa) polypeptide with a predicted molecular mass of 113 kDa. A putative ribosome binding site and a promoter sequence were recognized at the 5′ flanking region ofβ-galI. Further upstream a partial sequence of an open reading frame revealed a putative lactose permease gene transcribing divergently fromβ-galI. Theβ-galIIIgene (2,076 bp) encodes a 691-aa polypeptide with a calculated molecular mass of 76 kDa. A rho-independent transcription terminator-like sequence was found 25 bp downstream of the termination codon. The amino acid sequences of β-GalI and β-GalIII are homologous to those found in the LacZ and the LacG families, respectively. The acid-base, nucleophilic, and substrate recognition sites conserved in the LacZ family were found in β-GalI, and a possible acid-base site proposed for the LacG family was located in β-GalIII, which featured a glutamate at residue 160. The coding regions of theβ-galIandβ-galIIIgenes were each cloned downstream of a T7 promoter for overexpression inEscherichia coli. The molecular masses of the overexpressed proteins, as estimated by polyacrylamide gel electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, agree with their predicted molecular weights. β-GalI and β-GalIII were specific for β-d-anomer-linked galactoside substrates. Both are more active in response to ONPG (o-nitrophenyl-β-d-galactopyranoside) than in response to lactose, particularly β-GalIII. The galacto-oligosaccharide yield in the reaction catalyzed by β-GalI at 37°C in 20% (wt/vol) lactose solution was 130 mg/ml, which is more than six times higher than the maximum yield obtained with β-GalIII. The structure of the major trisaccharide produced by β-GalI catalysis was characterized asO-β-d-galactopyranosyl-(1-3)-O-β-d-galactopyranosyl-(1-4)-d-glucopyranose (3′-galactosyl-lactose).
Transmission of
Escherichia coli
O157:H7 from Contaminated Manure and Irrigation Water to Lettuce Plant Tissue and Its Subsequent Internalization
Authors: Solomon Ethan B. , Yaron Sima , Matthews Karl R. 
Published: July 2002

ABSTRACT

The transmission of
Escherichia coli
O157:H7 from manure-contaminated soil and irrigation water to lettuce plants was demonstrated using laser scanning confocal microscopy, epifluorescence microscopy, and recovery of viable cells from the inner tissues of plants.
E. coli
O157:H7 migrated to internal locations in plant tissue and was thus protected from the action of sanitizing agents by virtue of its inaccessibility. Experiments demonstrate that
E. coli
O157:H7 can enter the lettuce plant through the root system and migrate throughout the edible portion of the plant.
Cloning and Characterization of the Crystal Protein-Encoding Gene of
Bacillus thuringiensis
subsp.
yunnanensis
Authors: Balasubramanian Periasamy , Jayakumar R. , Shambharkar Prashant , Unnamalai N. , Pandian S. Karutha , Kumaraswami N. Selvamuthu , Ilangovan R. , Sekar Vaithilingam 
Published: July 2002

ABSTRACT

Molecular cloning and characterization of a novel
cry
gene,
cry32Aa
, of
Bacillus thuringiensis
subsp.
yunnanensis
was carried out. The Cry32Aa protein was predicted to have a molecular mass of 139.2 kDa and was found to have an unusual 42-amino-acid-long tail at the C terminus. The
cry32Aa
gene was localized on the 103-MDa plasmid of the organism. Bioassays showed no toxicity against several moths and mosquitoes. However, it exhibited weak toxicity against larvae of the diamondback moth,
Plutella xylostella
.
Cryptosporidium parvum
Mixed Genotypes Detected by PCR-Restriction Fragment Length Polymorphism Analysis
Authors: Reed Carrie , Sturbaum Gregory D. , Hoover Paul J. , Sterling Charles R. 
Published: July 2002

ABSTRACT

Combinations of 10
Cryptosporidium parvum
oocysts, with various ratios of genotype I to genotype II, were isolated and subjected to PCR-restriction fragment length polymorphism analysis. Amplification of both genotypes in these samples ranged from 31 to 74% and yielded no information about the genotype proportions. In addition, since both genotypes were not always detected, amplification of a single genotype is not conclusive evidence that the sample contains only a single genotype.
Development and Evaluation of a 16S Ribosomal DNA Array-Based Approach for Describing Complex Microbial Communities in Ready-To-Eat Vegetable Salads Packed in a Modified Atmosphere
Authors: Rudi Knut , Flateland Signe L. , Hanssen Jon Fredrik , Bengtsson Gunnar , Nissen Hilde 
Published: July 2002

ABSTRACT

There is a clear need for new approaches in the field of microbial community analyses, since the methods used can be severely biased. We have developed a DNA array-based method that targets16S ribosomal DNA (rDNA), enabling the direct detection and quantification of microorganisms from complex communities without cultivation. The approach is based on the construction of specific probes from the 16S rDNA sequence data retrieved directly from the communities. The specificity of the assay is obtained through a combination of DNA array hybridization and enzymatic labeling of the constructed probes. Cultivation-dependent assays (enrichment and plating) and cultivation-independent assays (direct fluorescence microscopy and scanning electron microscopy) were used as reference methods in the development and evaluation of the method. The description of microbial communities in ready-to-eat vegetable salads in a modified atmosphere was used as the experimental model. Comparisons were made with respect to the effect of storage at different temperatures for up to 12 days and with respect to the geographic origin of the crisphead lettuce (Spanish or Norwegian), the main salad component. The conclusion drawn from the method comparison was that the DNA array-based method gave an accurate description of the microbial communities.
Pseudomonas
spp. dominated both of the salad batches, containing either Norwegian or Spanish lettuce, before storage and after storage at 4°C. The
Pseudomonas
population also dominated the batch containing Norwegian lettuce after storage at 10°C. On the contrary,
Enterobacteriaceae
and lactic acid bacteria dominated the microbial community of the batch containing Spanish lettuce after storage at 10°C. In that batch, the
Enterobacteriaceae
also were abundant after storage at 4°C as well as before storage. The practical implications of these results are that microbial communities in ready-to-eat vegetable salads can be diverse and that microbial composition is dependent both on the origin of the raw material and on the storage conditions.
The Phenolic Hydroxyl Group of Carvacrol Is Essential for Action against the Food-Borne Pathogen
Bacillus cereus
Authors: Ultee A. , Bennik M. H. J. , Moezelaar R. 
Published: July 2002

ABSTRACT

The natural antimicrobial compound carvacrol shows a high preference for hydrophobic phases. The partition coefficients of carvacrol in both octanol-water and liposome-buffer phases were determined (3.64 and 3.26, respectively). Addition of carvacrol to a liposomal suspension resulted in an expansion of the liposomal membrane. Maximum expansion was observed after the addition of 0.50 μmol of carvacrol/mg of
l
-α-phosphatidylethanolamine. Cymene, a biological precursor of carvacrol which lacks a hydroxyl group, was found to have a higher preference for liposomal membranes, thereby causing more expansion. The effect of cymene on the membrane potential was less pronounced than the effect of carvacrol. The pH gradient and ATP pools were not affected by cymene. Measurement of the antimicrobial activities of compounds similar to carvacrol (e.g., thymol, cymene, menthol, and carvacrol methyl ester) showed that the hydroxyl group of this compound and the presence of a system of delocalized electrons are important for the antimicrobial activity of carvacrol. Based on this study, we hypothesize that carvacrol destabilizes the cytoplasmic membrane and, in addition, acts as a proton exchanger, thereby reducing the pH gradient across the cytoplasmic membrane. The resulting collapse of the proton motive force and depletion of the ATP pool eventually lead to cell death.