bluuebunny/crossref_metadata_2025_split huggingface dataset:
Microencapsulation of Bacteriophage Felix O1 into Chitosan-Alginate Microspheres for Oral Delivery
Authors: Ma Yongsheng , Pacan Jennifer C. , Wang Qi , Xu Yongping , Huang Xiaoqing , Korenevsky Anton , Sabour Parviz M. 
Published: May 2008

ABSTRACTThis paper reports the development of microencapsulated bacteriophage Felix O1 for oral delivery using a chitosan-alginate-CaCl2system. In vitro studies were used to determine the effects of simulated gastric fluid (SGF) and bile salts on the viability of free and encapsulated phage. Free phage Felix O1 was found to be extremely sensitive to acidic environments and was not detectable after a 5-min exposure to pHs below 3.7. In contrast, the number of microencapsulated phage decreased by 0.67 log units only, even at pH 2.4, for the same period of incubation. The viable count of microencapsulated phage decreased only 2.58 log units during a 1-h exposure to SGF with pepsin at pH 2.4. After 3 h of incubation in 1 and 2% bile solutions, the free phage count decreased by 1.29 and 1.67 log units, respectively, while the viability of encapsulated phage was fully maintained. Encapsulated phage was completely released from the microspheres upon exposure to simulated intestinal fluid (pH 6.8) within 6 h. The encapsulated phage in wet microspheres retained full viability when stored at 4°C for the duration of the testing period (6 weeks). With the use of trehalose as a stabilizing agent, the microencapsulated phage in dried form had a 12.6% survival rate after storage for 6 weeks. The current encapsulation technique enables a large proportion of bacteriophage Felix O1 to remain bioactive in a simulated gastrointestinal tract environment, which indicates that these microspheres may facilitate delivery of therapeutic phage to the gut.
Differential Interference with
Pythium ultimum
Sporangial Activation and Germination by
Enterobacter cloacae
in the Corn and Cucumber Spermospheres
Authors: Windstam Sofia , Nelson Eric B. 
Published: May 2008

ABSTRACT

Differential protection of plants by
Enterobacter cloacae
was studied by investigating early sensing and response behavior of
Pythium ultimum
sporangia toward seeds in the presence or absence of
E. cloacae
. Ten percent of
P. ultimum
sporangia were activated within the first 30 min of exposure to cucumber seeds. In contrast, 44% of the sporangia were activated as early as 15 min after exposure to corn seeds with over 80% sporangial activation by 30 min. Germ tubes emerged from sporangia after 2.5 and 1.0 h in the cucumber and corn spermospheres, respectively. Seed application of the wild-type strain of
E. cloacae
(EcCT-501R3) reduced sporangial activation by 45% in the cucumber spermosphere, whereas no reduction was observed in the corn spermosphere. Fatty acid transport and degradation mutants of
E. cloacae
(strains EcL1 and Ec31, respectively) did not reduce sporangial activation in either of the spermospheres. Although wild-type or mutant strains of
E. cloacae
failed to reduce seed colonization incidence, pathogen biomass on cucumber seeds was reduced in the presence of
E. cloacae
strains EcCT-501R3 and Ec31 by 4 and 8 h after sowing, respectively. By 12 h, levels of
P. ultimum
on cucumber seeds treated with
E. cloacae
EcCT-501R3 did not differ from levels on noninoculated seeds. On corn seeds,
P. ultimum
biomass was not affected by the presence of any
E. cloacae
strain. When introduced after sporangial activation had occurred,
E. cloacae
failed to reduce
P. ultimum
biomass on cucumber seeds compared with that on nontreated seeds. Also, increasing numbers of sporangia used to inoculate seeds yielded increased pathogen biomass at each sampling time. This indicates a direct link between the level of seed-colonizing biomass of
P. ultimum
and the number of activated and germinated sporangia in the spermosphere, suggesting that
E. cloacae
suppresses
P. ultimum
seed infections by reducing sporangial activation and germination within the first 30 to 90 min after sowing.
Temporal Release of Fatty Acids and Sugars in the Spermosphere: Impacts on
Enterobacter cloacae
-Induced Biological Control
Authors: Windstam Sofia , Nelson Eric B. 
Published: May 2008

ABSTRACT

The aim of this study was to determine the temporal release of fatty acids and sugars from corn and cucumber seeds during the early stages of seed germination in order to establish whether sugars found in exudate can prevent exudate fatty acid degradation by
Enterobacter cloacae
. Both saturated (long-chain saturated fatty acids [LCSFA]) and unsaturated (long-chain unsaturated fatty acids [LCUFA]) fatty acids were detected in corn and cucumber seed exudates within 15 min after seed sowing. LCSFA and LCUFA were released at a rate of 26.1 and 6.44 ng/min/seed by corn and cucumber seeds, respectively. The unsaturated portion of the total fatty acid pool from both plant species contained primarily oleic and linoleic acids, and these fatty acids were released at a combined rate of 6.6 and 0.67 ng/min/seed from corn and cucumber, respectively. In the absence of seed exudate sugars,
E. cloacae
degraded linoleic acid at rates of 29 to 39 ng/min, exceeding the rate of total fatty acid release from seeds. Sugars constituted a significant percentage of corn seed exudate, accounting for 41% of the total dry seed weight. Only 5% of cucumber seed exudate was comprised of sugars. Glucose, fructose, and sucrose were the most abundant sugars present in seed exudate from both plant species. Corn seeds released a total of 137 μg/seed of these three sugars within 30 min of sowing, whereas cucumber seeds released 0.83 μg/seed within the same time frame. Levels of glucose, fructose, and sucrose found in corn seed exudate (90 to 342 μg) reduced the rate of linoleic acid degradation by
E. cloacae
to 7.5 to 8.8 ng/min in the presence of either sugar, leaving sufficient concentrations of linoleic acid to activate
Pythium ultimum
sporangia Our results demonstrate that elevated levels of sugars in the corn spermosphere can prevent the degradation of LCUFA by
E. cloacae
, leading to its failure to suppress
P. ultimum
sporangial activation, germination, and subsequent disease development.
ThehmsHFRSOperon ofXenorhabdus nematophilaIs Required for Biofilm Attachment toCaenorhabditis elegans
Authors: Drace Kevin , Darby Creg 
Published: May 2008

ABSTRACTThe bacteriumXenorhabdus nematophilais an insect pathogen and an obligate symbiont of the nematodeSteinernema carpocapsae. X. nematophilamakes a biofilm that adheres to the head of the model nematodeCaenorhabditis elegans, a capabilityX. nematophilashares with the biofilms made byYersinia pestisandYersinia pseudotuberculosis. As inYersiniaspp., theX. nematophilabiofilm requires a 4-gene operon,hmsHFRS. Also like itsYersiniacounterparts, theX. nematophilabiofilm is bound by the lectin wheat germ agglutinin, suggesting that β-linkedN-acetyl-d-glucosamine orN-acetylneuraminic acid is a component of the extracellular matrix.C. elegansmutants with aberrant surfaces that do not permitYersiniabiofilm attachment also are resistant toX. nematophilabiofilms. AnX. nematophila hmsHmutant that failed to make biofilms onC. eleganshad no detectable defect in symbiotic association withS. carpocapsae, nor was virulence reduced against the insectManduca sexta.
Short Peptide Induces an “Uncultivable” Microorganism To Grow In Vitro
Authors: Nichols D. , Lewis K. , Orjala J. , Mo S. , Ortenberg R. , O'Connor P. , Zhao C. , Vouros P. , Kaeberlein T. , Epstein S. S. 
Published: May 2008

ABSTRACT

Microorganisms comprise the bulk of biodiversity, but only a small fraction of this diversity grows on artificial media. This phenomenon was noticed almost a century ago, repeatedly confirmed, and termed the “great plate count anomaly.” Advances in microbial cultivation improved microbial recovery but failed to explain why most microbial species do not grow in vitro. Here we show that at least some of such species can form domesticated variants capable of growth on artificial media. We also present evidence that small signaling molecules, such as short peptides, may be essential factors in initiating growth of nongrowing cells. We identified one 5-amino-acid peptide, LQPEV, that at 3.5 nM induces the otherwise “uncultivable” strain
Psychrobacter
sp. strain MSC33 to grow on standard media. This demonstrates that the restriction preventing microbial in vitro growth may be different from those offered to date to explain the “great plate count anomaly,” such as deficiencies in nutrient composition and concentrations in standard media, medium toxicity, and inappropriate incubation time. Growth induction of MSC33 illustrates that some microorganisms do not grow in vitro because they are removed from their native communities and the signals produced therein. “Uncultivable” species represent the largest source of unexplored biodiversity, and provide remarkable opportunities for both basic and applied research. Access to cultures of some of these species should be possible through identification of the signaling compounds necessary for growth, their addition to standard medium formulations, and eventual domestication.
Two-Log Increase in Sensitivity for Detection of Norovirus in Complex Samples by Concentration with Porcine Gastric Mucin Conjugated to Magnetic Beads
Authors: Tian Peng , Engelbrektson Anna , Mandrell Robert 
Published: May 2008

ABSTRACTHuman histo-blood group antigens (HBGA) have been identified previously as candidate receptors for human norovirus (NOR). Type A, type H1, and Lewis HBGA in humans have been identified as major HBGA for NOR binding. We have found that pig stomach (gastric) mucin (PGM) contains blood group A, H1, and Lewis b HBGA and binds to multiple strains of NOR more broadly than do specific antibodies to NOR. Both genogroup I (GGI) and GGII NOR strains were recovered by PGM-conjugated magnetic beads. A fecal sample containing GGII NOR was detected at a dilution of 1:1,000,000 by the standard RNA extraction procedure, whereas NOR in a 1:100,000,000 dilution could be concentrated by PGM-conjugated magnetic beads and NOR in spiked food samples (e.g., oyster extract, strawberry, raspberry, and lettuce) was captured by PGM, thus minimizing the reverse transcription-PCR inhibitors in food and increasing sensitivity.
Gamma Irradiation Can Be Used To Inactivate
Bacillus anthracis
Spores without Compromising the Sensitivity of Diagnostic Assays
Authors: Dauphin Leslie A. , Newton Bruce R. , Rasmussen Max V. , Meyer Richard F. , Bowen Michael D. 
Published: May 2008

ABSTRACT

The use of
Bacillus anthracis
as a biological weapon in 2001 heightened awareness of the need for validated methods for the inactivation of
B. anthracis
spores. This study determined the gamma irradiation dose for inactivating virulent
B. anthracis
spores in suspension and its effects on real-time PCR and antigen detection assays. Strains representing eight genetic groups of
B. anthracis
were exposed to gamma radiation, and it was found that subjecting spores at a concentration of 10
7
CFU/ml to a dose of 2.5 × 10
6
rads resulted in a 6-log-unit reduction of spore viability. TaqMan real-time PCR analysis of untreated versus irradiated Ames strain (K1694) spores showed that treatment significantly enhanced the detection of
B. anthracis
chromosomal DNA targets but had no significant effect on the ability to detect targets on the pXO1 and pXO2 plasmids of
B. anthracis
. When analyzed by an enzyme-linked immunosorbent assay (ELISA), irradiation affected the detection of
B. anthracis
spores in a direct ELISA but had no effect on the limit of detection in a sandwich ELISA. The results of this study showed that gamma irradiation-inactivated spores can be tested by real-time PCR or sandwich ELISA without decreasing the sensitivity of either type of assay. Furthermore, the results suggest that clinical and public health laboratories which test specimens for
B. anthracis
could potentially incorporate gamma irradiation into sample processing protocols without compromising the sensitivity of the
B. anthracis
assays.
In Vitro Study of the Effect of Cationic Biocides on Bacterial Population Dynamics and Susceptibility
Authors: Moore Louise E. , Ledder Ruth G. , Gilbert Peter , McBain Andrew J. 
Published: May 2008

ABSTRACT

Cationic biocides (CBs) are widely used in domestic and public hygiene and to control biofouling and microbial contamination in industry. The increased use of biocides has led to concern regarding possible reductions in biocide effectiveness. Domestic drain microcosms were stabilized for 5 months and then exposed to polyhexamethylene biguanide (PHMB) at 0.1, 0.2, and 0.4g liter
−1
over 6 months and characterized throughout by differential culture, together with eubacterial-specific PCR-denaturing gradient gel electrophoresis. Additionally, MICs and minimal bactericidal concentrations (MBCs) for bacteria previously isolated from a domestic drain (
n
= 18) and the human skin (
n
= 13) were determined before, during, and after escalating, sublethal exposure (14 passages) to two quaternary ammonium compounds (QAC1 and QAC2), the bisbiguanide chlorhexidine (CHX), and PHMB. Exposure of the drain microcosm to PHMB did not decrease the total viable count although significant (
P
< 0.01) decreases in recovery were observed for the gram-positive cocci with associated clonal expansion of pseudomonads (from ca. 0.1% of the population to ca. 10%). This clonal expansion was also manifested as elevations in bacteria that could grow in the presence of PHMB, CHX, and QAC1. Decreases in susceptibility (greater than twofold) occurred for 10/31 of the test bacteria for QAC1, 14/31 for QAC2, 10/31 for CHX, and 7/31 for PHMB. Exposure of microcosms to PHMB targeted gram-positive species and caused the clonal expansion of pseudomonads. In terms of prolonged-sublethal passage on CBs, exposure to all the biocides tested resulted in susceptibility decreases for a proportion of test bacteria, but refractory clones were not generated.
Role of the Lysozyme Inhibitor Ivy in Growth or Survival ofEscherichia coliandPseudomonas aeruginosaBacteria in Hen Egg White and in Human Saliva and Breast Milk
Authors: Deckers Daphne , Vanlint Dietrich , Callewaert Lien , Aertsen Abram , Michiels Chris W. 
Published: May 2008

ABSTRACTIvy is a lysozyme inhibitor that protectsEscherichia coliagainst lysozyme-mediated cell wall hydrolysis when the outer membrane is permeabilized by mutation or by chemical or physical stress. In the current work, we have investigated whether Ivy is necessary for the survival or growth ofE. coliMG1655 andPseudomonas aeruginosaPAO1 in hen egg white and in human saliva and breast milk, which are naturally rich in lysozyme and in membrane-permeabilizing components. Wild-typeE. coliwas able to grow in saliva and breast milk but showed partial inactivation in egg white. The knockout of Ivy did not affect growth in breast milk but slightly increased sensitivity to egg white and caused hypersensitivity to saliva, resulting in the complete inactivation of 104CFU ml−1of bacteria within less than 5 hours. The depletion of lysozyme from saliva completely restored the ability of theivymutant to grow like the parental strain.P. aeruginosa, in contrast, showed growth in all three substrates, which was not affected by the knockout of Ivy production. These results indicate that lysozyme inhibitors like Ivy promote bacterial survival or growth in particular lysozyme-rich secretions and suggest that they may promote the bacterial colonization of specific niches in the animal host.
Development of an In Vitro Activity Assay as an Alternative to the Mouse Bioassay for
Clostridium botulinum
Neurotoxin Type A
Authors: Rasooly Reuven , Do Paula M. 
Published: May 2008

ABSTRACT

Currently, the only accepted assay with which to detect active
Clostridium botulinum
neurotoxin is an in vivo mouse bioassay. The mouse bioassay is sensitive and robust and does not require specialized equipment. However, the mouse bioassay is slow and not practical in many settings, and it results in the death of animals. Here, we describe an in vitro cleavage assay for SNAP-25 (synaptosome-associated proteins of 25 kDa) for measuring the toxin activity with the same sensitivity as that of the mouse bioassay. Moreover, this assay is far more rapid, can be automated and adapted to many laboratory settings, and has the potential to be used for toxin typing. The assay has two main steps. The first step consists of immunoseparation and concentration of the toxin, using immunomagnetic beads with monoclonal antibodies directed against the 100-kDa heavy chain subunit, and the second step consists of a cleavage assay targeting the SNAP-25 peptide of the toxin, labeled with fluorescent dyes and detected as a fluorescence resonance energy transfer assay. Our results suggest that the sensitivity of this assay is 10 pg/ml, which is similar to the sensitivity of the mouse bioassay, and this test can detect the activity of the toxin in carrot juice and beef. These results suggest that the assay has a potential use as an alternative to the mouse bioassay for analysis of
C. botulinum
type A neurotoxin.
Evaluation of Major Types of Shiga Toxin 2e-Producing
Escherichia coli
Bacteria Present in Food, Pigs, and the Environment as Potential Pathogens for Humans
Authors: Beutin Lothar , Krüger Ulrike , Krause Gladys , Miko Angelika , Martin Annett , Strauch Eckhard 
Published: May 2008

ABSTRACT

Shiga toxin 2e (Stx2e)-producing strains from food (
n
= 36), slaughtered pigs (
n
= 25), the environment (
n
= 21), diseased pigs (
n
= 19), and humans (
n
= 9) were investigated for production of Stx2e by enzyme-linked immunosorbent assay, for virulence markers by PCR, and for their serotypes to evaluate their role as potential human pathogens. Stx2e production was low in 64% of all 110 strains. Stx2e production was inducible by mitomycin C but differed considerably between strains. Analysis by nucleotide sequencing and transcription of
stx
2e
genes in high- and low-Stx2e-producing strains showed that toxin production correlated with transcription rates of
stx
2e
genes. DNA sequences specific for the
int
,
Q
,
dam
, and
S
genes of the
stx
2e
bacteriophage P27 were found in 109 strains, indicating cryptic P27-like prophages, although 102 of these were not complete for all genes tested. Genes encoding intimin (
eae
), enterohemorrhagic
Escherichia coli
hemolysin (
ehx
), or other
stx
1
or
stx
2
variants were not found, whereas genes for heat-stable enterotoxins STI, STII, or EAST1 were present in 54.5% of the strains. Seven major serotypes that were associated with diseased pigs (O138:H14, O139:H1, and O141:H4) or with slaughter pigs, food, and the environment (O8:H4, O8:H9, O100:H30, and O101:H9) accounted for 60% of all Stx2e strains. The human Stx2e isolates did not belong to these major serotypes of Stx2e strains, and high production of Stx2e in human strains was not related to diarrheal disease. The results from this study and other studies do not point to Stx2e as a pathogenicity factor for diarrhea and hemolytic uremic syndrome in humans.
Presence of a Family of Plasmids (29 to 65 Kilobases) with a 26-Kilobase Common Region in Different Strains of the Sulfur-Oxidizing BacteriumAcidithiobacillus caldus
Authors: van Zyl Leonard J. , Deane Shelly M. , Louw Lilly-Ann , Rawlings Douglas E. 
Published: May 2008

ABSTRACTThree large cryptic plasmids from different isolates ofAcidithiobacillus calduswere rescued by using an in vitro transposition system that delivers a kanamycin-selectable marker and anEscherichia coliplasmid origin of replication. The largest of the plasmids, the 65-kb plasmid pTcM1, was isolated from a South AfricanA. caldusstrain, MNG. This plasmid was sequenced and compared to that of pTcF1 (39 kb, from strain “f,” South Africa) and pC-SH12 (29 kb, from strain C-SH12, Australia). With the exception of a 2.7-kb insertion sequence, pC-SH12 appears to represent the DNA common to all three plasmids and includes a number of accessory genes plus the plasmid “backbone” containing the replication region. The two larger plasmids carry, in addition, a number of insertion sequences of the ISL3 family and a composite transposon related to the Tn21subfamily containing a highly mosaic region within the borders of the inverted repeats. Genes coding for arsenic resistance, plasmid mobilization, plasmid stability, and a putative restriction-modification system occur within these mosaic regions.
Esterase Autodisplay: Enzyme Engineering and Whole-Cell Activity Determination in Microplates with pH Sensors
Authors: Schultheiss Eva , Weiss Svenja , Winterer Elisa , Maas Ruth , Heinzle Elmar , Jose Joachim 
Published: May 2008

ABSTRACTAmong the GDSL family of serine esterases/lipases is a group of bacterial enzymes that posses C-terminal extensions involved in outer membrane anchoring or translocation. ApeE fromSalmonella entericaserovar Typhimurium, a member of this group, has been expressed inEscherichia coliand was resistant to protease digestion when the protease was added to whole cells, indicating a periplasmic localization. The five consensus blocks conserved within all GDSL esterases were identified in ApeE by multiple sequence alignment and separated from the C-terminal extension. The DNA sequence spanning the four invariant residues Ser, Gly, Asn, and His, and hence representing the catalytic domains of ApeE, was amplified by PCR and fused in frame to the transport domains of the autodisplay system. The resulting artificial esterase, called EsjA, was overexpressed in the cell envelope ofE. coliand was shown to be active by the use of α-naphthyl acetate (α-NA) as a substrate in an in-gel activity stain after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Surface exposure of EsjA was indicated by its accessibility to protease added to whole cells. The esterase activity of whole cells displaying EsjA was determined by a pH agar assay and by the use of microplates with integrated pH-dependent optical sensors. α-NA, α-naphthyl butyrate, and α-naphthyl caproate were used as substrates, and it turned out that the substrate preferences of artificial EsjA were altered in comparison to original ApeE. Our results indicate that autodisplay of esterase in combination with pH sensor microplates can provide a new platform technology for the screening of tailor-made hydrolase activities.
Listeria monocytogenes
Biofilm-Associated Protein (BapL) May Contribute to Surface Attachment of
L. monocytogenes
but Is Absent from Many Field Isolates
Authors: Jordan Suzanne J. , Perni Stefano , Glenn Sarah , Fernandes Isabel , Barbosa Manuela , Sol Manuela , Tenreiro Rogerio P. , Chambel Lelia , Barata Belarmino , Zilhao Isabel , Aldsworth Timothy G. , Adriao Andreia , Faleiro M. Leonor , Shama Gilbert , Andrew Peter W. 
Published: May 2008

ABSTRACT

Listeria monocytogenes
is a food-borne pathogen capable of adhering to a range of surfaces utilized within the food industry, including stainless steel. The factors required for the attachment of this ubiquitous organism to abiotic surfaces are still relatively unknown. In silico analysis of the
L. monocytogenes
EGD genome identified a putative cell wall-anchored protein (Lmo0435 [BapL]), which had similarity to proteins involved in biofilm formation by staphylococci. An insertion mutation was constructed in
L. monocytogenes
to determine the influence of this protein on attachment to abiotic surfaces. The results show that the protein may contribute to the surface adherence of strains that possess BapL, but it is not an essential requirement for all
L. monocytogenes
strains. Several BapL-negative field isolates demonstrated an ability to adhere to abiotic surfaces equivalent to that of BapL-positive strains. BapL is not required for the virulence of
L. monocytogenes
in mice.