bluuebunny/crossref_metadata_2025 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

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

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

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

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

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

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

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

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

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

Two 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

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

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

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

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

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.
Simultaneous Transport of Two Bacterial Strains in Intact Cores from Oyster, Virginia: Biological Effects and Numerical Modeling
Authors: Dong Hailiang , Rothmel Randi , Onstott Tullis C. , Fuller Mark E. , DeFlaun Mary F. , Streger Sheryl H. , Dunlap Robb , Fletcher Madilyn
Published: July 2002

The transport characteristics of two adhesion-deficient, indigenous groundwater strains,
Comamonas
sp. strain DA001 and
Erwinia herbicola
OYS2-A, were studied by using intact sediment cores (7 by 50 cm) from Oyster, Va. Both strains are gram-negative rods (1.10 by 0.56 and 1.56 by 0.46 μm, respectively) with strongly hydrophilic membranes and a slightly negative surface charge. The two strains exhibited markedly different behaviors when they were transported through granular porous sediment. To eliminate any effects of physical and chemical heterogeneity on bacterial transport and thus isolate the biological effect, the two strains were simultaneously injected into the same core. DA001 cells were metabolically labeled with
35
S and tagged with a vital fluorescent stain, while OYS2-A cells were metabolically labeled with
14
C. The fast decay of
35
S allowed deconvolution of the two isotopes (and therefore the two strains). Dramatic differences in the transport behaviors were observed. The breakthrough of DA001 and the breakthrough of OYS2-A both occurred before the breakthrough of a conservative tracer (termed differential advection), with effluent recoveries of 55 and 30%, respectively. The retained bacterial concentration of OYS2-A in the sediment was twofold higher than that of DA001. Among the cell properties analyzed, the statistically significant differences between the two strains were cell length and diameter. The shorter, larger-diameter DA001 cells displayed a higher effluent recovery than the longer, smaller-diameter OYS2-A cells. CXTFIT modeling results indicated that compared to the DA001 cells, the OYS2-A cells experienced lower pore velocity, higher porosity, a higher attachment rate, and a lower detachment rate. All these factors may contribute to the observed differences in transport.
Decreasing the Level of Ethyl Acetate in Ethanolic Fermentation Broths ofEscherichia coliKO11 by Expression ofPseudomonas putida estZEsterase
Authors: Hasona Adnan , York S. W. , Yomano L. P. , Ingram L. O. , Shanmugam K. T.
Published: July 2002

During the fermentation of sugars to ethanol relatively high levels of an undesirable coproduct, ethyl acetate, are also produced. With ethanologenicEscherichia colistrain KO11 as the biocatalyst, the level of ethyl acetate in beer containing 4.8% ethanol was 192 mg liter−1. Although theE. coligenome encodes several proteins with esterase activity, neither wild-type strains nor KO11 contained significant ethyl acetate esterase activity. A simple method was developed to rapidly screen bacterial colonies for the presence of esterases which hydrolyze ethyl acetate based on pH change. This method allowed identification ofPseudomonas putidaNRRL B-18435 as a source of this activity and the cloning of a new esterase gene,estZ. Recombinant EstZ esterase was purified to near homogeneity and characterized. It belongs to family IV of lipolytic enzymes and contains the conserved catalytic triad of serine, aspartic acid, and histidine. As expected, this serine esterase was inhibited by phenylmethylsulfonyl fluoride and the histidine reagent diethylpyrocarbonate. The native and subunit molecular weights of the recombinant protein were 36,000, indicating that the enzyme exists as a monomer. By using α-naphthyl acetate as a model substrate, optimal activity was observed at pH 7.5 and 40°C. TheKmandVmaxfor α-naphthyl acetate were 18 μM and 48.1 μmol · min−1 · mg of protein−1, respectively. Among the aliphatic esters tested, the highest activity was obtained with propyl acetate (96 μmol · min−1 · mg of protein−1), followed by ethyl acetate (66 μmol · min−1 · mg of protein−1). Expression ofestZinE. coliKO11 reduced the concentration of ethyl acetate in fermentation broth (4.8% ethanol) to less than 20 mg liter−1.
Microtiter Plate Assay for Assessment of
Listeria monocytogenes
Biofilm Formation
Authors: Djordjevic D. , Wiedmann M. , McLandsborough L. A.
Published: July 2002

Listeria monocytogenes
has the ability to form biofilms on food-processing surfaces, potentially leading to food product contamination. The objective of this research was to standardize a polyvinyl chloride (PVC) microtiter plate assay to compare the ability of
L. monocytogenes
strains to form biofilms. A total of 31 coded
L. monocytogenes
strains were grown in defined medium (modified Welshimer's broth) at 32°C for 20 and 40 h in PVC microtiter plate wells. Biofilm formation was indirectly assessed by staining with 1% crystal violet and measuring crystal violet absorbance, using destaining solution. Cellular growth rates and final cell densities did not correlate with biofilm formation, indicating that differences in biofilm formation under the same environmental conditions were not due to growth rate differences. The mean biofilm production of lineage I strains was significantly greater than that observed for lineage II and lineage III strains. The results from the standardized microtiter plate biofilm assay were also compared to biofilm formation on PVC and stainless steel as assayed by quantitative epifluorescence microscopy. Results showed similar trends for the microscopic and microtiter plate assays, indicating that the PVC microtiter plate assay can be used as a rapid, simple method to screen for differences in biofilm production between strains or growth conditions prior to performing labor-intensive microscopic analyses.
Differential Induction of Complement Fragment C5a and Inflammatory Cytokines during Intramammary Infections withEscherichia coliandStaphylococcus aureus
Authors: Riollet Céline , Rainard Pascal , Poutrel Bernard
Published: July 2002

The prompt recruitment of neutrophils to the site of infection is essential for the defense of the bovine mammary gland against invading pathogens and is determinant for the outcome of the infection.Escherichia coliis known to induce clinical mastitis, characterized by an intense neutrophil recruitment leading to the eradication of the bacteria, whereasStaphylococcus aureusinduces subclinical mastitis accompanied by a moderate neutrophil recruitment and the establishment of chronic mastitis. To elicit the neutrophil recruitment into the udder, inflammatory mediators must be produced after recognition of the invading pathogen. To our knowledge, those mediators have never been studied duringS. aureusmastitis, although understanding of the neutrophil recruitment mechanisms could allow a better understanding of the differences in the pathogeneses elicited byE. coliandS. aureus. Therefore, we studied, at several time points, the accumulation of neutrophils and the presence of the chemoattractant complement fragment C5a and of the cytokines interleukin-1β (IL-1β), tumor necrosis factor alpha, and IL-8 in milk after inoculation ofE. coliorS. aureusin lactating bovine udders. The low levels of C5a and the absence of cytokines in milk fromS. aureus-infected cows, compared to the high levels found in milk fromE. coli-infected animals, mirror the differences in the severities of the two inflammatory reactions. The cytokine deficit in milk afterS. aureusinoculation in the lactating bovine mammary gland could contribute to the establishment of chronic mastitis. This result could help in the design of preventive or curative strategies against chronic mastitis.
Viability and Functional Activity of Cryopreserved Mononuclear Cells
Authors: Weinberg Adriana , Zhang Li , Brown Darby , Erice Alejo , Polsky Bruce , Hirsch Martin S. , Owens Susan , Lamb Karan
Published: July 2002

Factors that influence viability and function of cryopreserved peripheral blood mononuclear cells (PBMC) were identified on 54 samples from 27 AIDS Clinical Trial Units. PBMC viability ranged from 1 to 96% with a median of 70%, was higher in laboratories with experienced staff, and was not significantly associated with CD4 cell number. Function of cryopreserved PBMC, measured by lymphocyte proliferation, was associated with viability. Preparations with viability greater than or equal to 70% had consistent proliferative responses and were suitable for functional analyses.
Defective Neutrophil Degranulation Induced by Interleukin-8 and Complement 5a and Down-Regulation of Associated Receptors in Children Vertically Infected with Human Immunodeficiency Virus Type 1
Authors: Meddows-Taylor Stephen , Kuhn Louise , Meyers Tammy M. , Sherman Gayle , Tiemessen Caroline T.
Published: July 2002

The polymorphonuclear neutrophils (PMNs) of patients infected with human immunodeficiency virus type 1 (HIV-1) show impaired microbicidal responses. The present study assessed the functional integrity of PMN degranulation responses and the expression of specific receptors that mediate these responses in a group of children vertically infected with HIV-1. PMN degranulation in response to interleukin-8 (IL-8) and complement 5a (C5a) was measured in a group of HIV-1-infected children with mild and severe clinical disease and in an uninfected control group. In addition, the expression of CXCR1, CXCR2, and CD88 on whole-blood PMNs was quantified by flow cytometry. Although CXCR1 expression was found to be largely unaltered in the HIV-1-infected children relative to that in the control children, the intensity of CXCR2 expression was significantly reduced in those with severe disease. Furthermore, there was a significant reduction in the percentage of cells expressing CD88 and in the intensity of CD88 fluorescence in the HIV-1-infected children compared to that in control children, with CD88 fluorescence intensity more significantly reduced in the presence of severe disease. PMNs from a large proportion of the HIV-1-infected children either showed reciprocal degranulation responses or were unresponsive to IL-8 and C5a, whereas the PMNs from the uninfected children showed positive responses. Inefficient agonist-induced degranulation may contribute to the increased susceptibility of HIV-1-infected children to secondary microbial infections. Furthermore, reduced expression of CXCR2 and CD88 may be suggestive of defects in other functions of PMNs from HIV-1-infected children.
Western Immunoblotting with FiveTreponema pallidumRecombinant Antigens for Serologic Diagnosis of Syphilis
Authors: Sambri Vittorio , Marangoni Antonella , Eyer Christina , Reichhuber Christine , Soutschek Erwin , Negosanti Massimo , D'Antuono Antonietta , Cevenini Roberto
Published: July 2002

Five immunodominantTreponema pallidumrecombinant polypeptides (rTpN47, rTmpA, rTpN37, rTpN17, and rTpN15) were blotted onto strips, and 450 sera (200 from blood donors, 200 from syphilis patients, and 50 potentially cross-reactive) were tested to evaluate the diagnostic performance of recombinant Western blotting (recWB) in comparison with in-house whole-cell lysate antigen-based immunoblotting (wclWB) andT. pallidumhemagglutination (MHA-TP) for the laboratory diagnosis of syphilis. None of the serum specimens from blood donors or from potential cross-reactors gave a positive result when evaluated by recWB, wclWB, or MHA-TP. The evaluation of the immunoglobulin G immune response by recWB in sera from patients with different stages of syphilis showed that rTmpA was the most frequently identified antigen (95%), whereas only 41% of the specimens were reactive to rTpN37. The remaining recombinant polypeptides were recognized as follows: rTpN47, 92.5%; rTpN17, 89.5%; and rTpN15, 67.5%. The agreement between recWB and MHA-TP was 95.0% (100% with sera from patients with latent and late disease), and the concordance between wclWB and MHA-TP was 92.0%. The overall concordance between recWB and wclWB was 97.5% (100% with sera from patients with secondary and late syphilis and 94.6 and 98.6% with sera from patients with primary and latent syphilis, respectively). The overall sensitivity of recWB was 98.8% and the specificity was 97.1% with MHA-TP as the reference method. These values for sensitivity and specificity were slightly superior to those calculated for wclWB (sensitivity, 97.1%, and specificity, 96.1%). With wclWB as the standard test, the sensitivity and specificity of recWB were 98.9 and 99.3%, respectively. These findings suggest that the five recombinant polypeptides used in this study could be used as substitutes for the whole-cell lysateT. pallidumantigens and that this newly developed recWB test is a good, easy-to-use confirmatory method for the detection of syphilis antibodies in serum.
Bacteroides ovatusas the Predominant Commensal Intestinal Microbe Causing a Systemic Antibody Response in Inflammatory Bowel Disease
Authors: Saitoh Shin , Noda Satoshi , Aiba Yuji , Takagi Atsushi , Sakamoto Mitsuo , Benno Yoshimi , Koga Yasuhiro
Published: July 2002

To clarify what bacterial species of commensal intestinal microbes are recognized as the antigens that induce a serum antibody response in patients with inflammatory bowel disease (IBD), 72 subjects consisting of 12 Crohn’s disease patients, 30 ulcerative colitis patients, and 30 healthy volunteers were examined for their titers of serum antibody to these intestinal bacteria. In IBD patients, as a result, significant elevations of both the immunoglobulin G (IgG) and IgA titers toBacteroides ovatuswere found. Immunoblotting showed that a definite 19.5-kDa band ofB. ovatuswas bound to the serum antibody raised in IBD patients. It was thus concluded thatB. ovatuscauses serum antibody responses in IBD patients, and a 19.5-kDa molecule of this bacterium appears to be the responsible antigen, although the role of this event in pathogenesis remains unclear.
Dependence of Bacterial Protein Adhesins on Toll-Like Receptors for Proinflammatory Cytokine Induction
Authors: Hajishengallis George , Martin Michael , Sojar Hakimuddin T. , Sharma Ashu , Schifferle Robert E. , DeNardin Ernesto , Russell Michael W. , Genco Robert J.
Published: July 2002

Toll-like receptors (TLRs) are important signal transducers that mediate inflammatory reactions induced by microbes through pattern recognition of virulence molecules such as lipopolysaccharide (LPS) and lipoproteins. We investigated whether proinflammatory cytokine responses induced by certain bacterial protein adhesins may also depend on TLRs. In differentiated THP-1 mononuclear cells stimulated by LPS-free recombinant fimbrillin (rFimA) fromPorphyromonas gingivalis, cytokine release was abrogated by monoclonal antibodies (MAbs) to CD14 and TLR4 but not to TLR2. Similar experiments using anti-β2 integrin MAbs suggested that β2 integrins (CD11/CD18) also play a role in cytokine induction by rFimA or native fimbriae. Minor fimbriae (distinct from thefimA-encoded major fimbriae) ofP. gingivalisinduced proinflammatory cytokine release in a CD14- and TLR2-dependent mode. Cytokine induction by BspA, a leucine-rich repeat protein fromBacteroides forsythus, depended heavily on CD14 and TLR2. We also found that the ability of the streptococcal protein AgI/II to stimulate cytokine release depended partially on CD14 and TLR4, and the AgI/II segment that possibly interacts with these receptors was identified as its N-terminal saliva-binding region. When THP-1 cells were exposed to rFimA for 24 h, surface expression of CD14 and CD18 was decreased and the cells became hyporesponsive to cytokine induction by a second challenge with rFimA. However, tolerance induction was abolished when the THP-1 cells were pretreated with rFimA in the presence of either anti-CD14 MAb or anti-TLR4 MAb. Induction of cross-tolerance between rFimA and LPS correlated with downregulation of the pattern recognition receptors involved. Our data suggest that the CD14-TLR2/4 system is involved in cytokine production and tolerance induction upon interaction with certain proinflammatory bacterial protein adhesins.
Systemic Inflammation in Cardiovascular and Periodontal Disease: Comparative Study
Authors: Glurich Ingrid , Grossi Sara , Albini Boris , Ho Alex , Shah Rashesh , Zeid Mohamed , Baumann Heinz , Genco Robert J. , De Nardin Ernesto
Published: July 2002

Epidemiological studies have implicated periodontal disease (PD) as a risk factor for the development of cardiovascular disease (CVD). These studies addressed the premise that local infection may perturb the levels of systemic inflammatory mediators, thereby promoting mechanisms of atherosclerosis. Levels of inflammatory mediators in the sera of subjects with only PD, only CVD, both diseases, or neither condition were compared. Subjects were assessed for levels of C-reactive protein (CRP), serum amyloid A (SAA), ceruloplasmin, α1-acid-glycoprotein (AAG), α1-antichymotrypsin (ACT), and the soluble cellular adhesion molecules sICAM-1 and sVCAM by enzyme-linked immunoabsorbent and/or radial immunodiffusion assays. CRP levels in subjects with either condition alone were elevated twofold above subjects with neither disease, whereas a threefold increase was noted in subjects with both diseases (P= 0.0389). Statistically significant increases in SAA and ACT were noted in subjects with both conditions compared to those with one or neither condition (P= 0.0162 and 0.0408, respectively). Ceruloplasmin levels were increased in subjects with only CVD (P= 0.0001). Increases in sVCAM levels were noted in all subjects with CVD (P= 0.0054). No differences in sICAM levels were noted among subject groups. A trend toward higher levels of AAG was noted in subjects with both conditions and for ACT in subjects with only PD. Immunohistochemical examination of endarterectomy specimens of carotid arteries from subjects with atherosclerosis documented SAA and CRP deposition in association with atheromatous lesions. The data support the hypothesis that localized persistent infection may influence systemic levels of inflammatory mediators. Changes in inflammatory mediator levels potentially impact inflammation-associated atherosclerotic processes.
Increased Numbers of ESAT-6- and Purified Protein Derivative-Specific Gamma Interferon-Producing Cells in Subclinical and Active Tuberculosis Infection
Authors: Ulrichs Timo , Anding Peter , Porcelli Steven , Kaufmann Stefan H. E. , Munk Martin E.
Published: July 2002

Numbers of gamma interferon (IFN-γ)-producing cells reactive to ESAT-6 antigen were increased in recent converters to purified protein derivative positivity and in tuberculosis patients but not in unvaccinated or
Mycobacterium bovis
BCG-vaccinated healthy donors. ESAT-6-reactive IFN-γ-producing cells in recent converters and tuberculosis patients recognized similar synthetic peptides. Thus, ESAT-6 is a potential candidate for use in detection of early, as well as active, tuberculosis and for control of the disease.
Intranasal Immunization of Mice with a Mixture of the Pneumococcal Proteins PsaA and PspA Is Highly Protective against Nasopharyngeal Carriage ofStreptococcus pneumoniae
Authors: Briles David E. , Ades Eddie , Paton James C. , Sampson Jacquelyn S. , Carlone George M. , Huebner Robert C. , Virolainen Anni , Swiatlo Edwin , Hollingshead Susan K.
Published: July 2002

Acquisition of pneumococci is generally from carriers rather than from infected individuals. Therefore, to induce herd immunity againstStreptococcus pneumoniaeit will be necessary to elicit protection against carriage. Capsular polysaccharide-protein conjugates, PspA, and PsaA are known to elicit some protection against nasopharyngeal carriage of pneumococci but do not always completely eliminate carriage. In this study, we observed that PsaA elicited better protection than did PspA against carriage. Pneumolysin elicited no protection against carriage. Immunization with a mixture of PsaA and PspA elicited the best protection against carriage. These results indicate that PspA and PsaA may be useful for the elicitation of herd immunity in humans. As PspA and pneumolysin are known to elicit immunity to bacteremia and pneumonia, their inclusion in a mucosal vaccine may enable such a vaccine to prevent invasive disease as well as carriage.
Direct Quantitative Transcript Analysis of the
agr
Regulon of
Staphylococcus aureus
during Human Infection in Comparison to the Expression Profile In Vitro
Authors: Goerke Christiane , Campana Silvia , Bayer Manfred G. , Döring Gerd , Botzenhart Konrad , Wolz Christiane
Published: July 2002

Bacteria possess a repertoire of distinct regulatory systems promoting survival in disparate environments. Under in vitro conditions it was demonstrated for the human pathogen
Staphylococcus aureus
that the expression of most virulence factors is coordinated by the global regulator
agr
. To monitor bacterial gene regulation in the host, we developed a method for direct transcript analysis from clinical specimens. Quantification of specific transcripts was performed by competitive reverse transcription-PCR, and results were normalized against the constitutively expressed gene for gyrase (
gyr
). Using sputum from cystic fibrosis (CF) patients infected with
S. aureus
we examined the transcription of the effector molecule RNAIII of
agr
, of
spa
(protein A), generally repressed by
agr
, and of
hla
(alpha-toxin), generally activated by
agr
. In the CF lung RNAIII was expressed poorly, indicating an inactive
agr
in vivo. Despite the low level of RNAIII expression,
spa
was detectable only in minute amounts and an irregular transcription of
hla
was observed in all sputum samples. After subculturing of patient strains
agr
-deficient isolates and isolates with unusual expression profiles, i.e., not consistent with those obtained from prototypic strains, were observed. In conclusion, the
agr
activity seems to be nonessential in CF, and from the described expression pattern of
spa
and
hla
, other regulatory circuits aside from
agr
are postulated in vivo.
Cytokine Gene Expression in Innately Susceptible BALB/c Mice and Relatively Resistant C57BL/6 Mice during Infection with VirulentBurkholderia pseudomallei
Authors: Ulett Glen C. , Ketheesan Natkunam , Hirst Robert G.
Published: July 2002

Production of cytokines including gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) is an important early-stage host response following infection with intracellular pathogens. Development of immunity to these pathogens is determined to a large extent by the timing and relative level of expression of the cytokines. Numerous studies have shown that early cytokine responses involving interleukin-12 (IL-12) and IFN-γ are important for resistance to intracellular pathogens, whereas responses involving IL-4 and IL-10 increase host susceptibility. These often-indistinct early cytokine responses influence the differentiation of naı̈ve CD4+T helper cells, which later develop into what have commonly been termed Th1- and Th2-type cells. The characterization of CD4+T-helper-cell responses as Th1 or Th2 type is based largely on the cytokine profiles during the specific phase and has been used in recent years to account for the innate resistance and susceptibility of different inbred strains of mice to several intracellular pathogens. Studies investigating cytokine production in terms of CD4+T-helper-cell polarization inBurkholderia pseudomalleiinfection have not been undertaken. In this study, we used semiquantitative reverse transcription-PCR to assess induction of cytokine mRNA in liver and spleen ofB. pseudomallei-susceptible BALB/c and relatively resistant C57BL/6 mice following infection with virulentB. pseudomallei. The levels of mRNA for IFN-γ, TNF-α, IL-1β, IL-6, IL-10, and IL-12 increased in both BALB/c and C57BL/6 mice 24 to 36 h after infection. A comparison of BALB/c and C57BL/6 responses revealed the relative levels of expression of mRNA for several of these cytokines, including IFN-γ, were greater in BALB/c mice, suggesting a role for endotoxic shock and cytokine-mediated immunopathology in the development of acute melioidosis. Early induction of mRNA for the cytokines classically associated with development of Th1- and Th2-type responses was absent or minimal, and induction levels in both strains of mice were similar. During the specific phase, cytokine mRNA profiles occurred as a combination of Th1- and Th2-type patterns. Collectively, these results demonstrate that cytokine mRNA responses in BALB/c and C57BL/6 mice following infection with virulentB. pseudomalleido not develop as polarized Th1- or Th2-type profiles. Considering the role of TNF-α and IFN-γ in the processes of endotoxic shock, these results also indicate that selected cytokines, while important for resistance toB. pseudomalleiinfection, are also potential contributors to immunopathology and the development of acute fulminating disease.