bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of Challenge Temperature and Solute Type on Heat Tolerance of
Salmonella
Serovars at Low Water Activity
Authors: Mattick K. L. , Jørgensen F. , Wang P. , Pound J. , Vandeven M. H. , Ward L. R. , Legan J. D. , Lappin-Scott H. M. , Humphrey T. J.
Published: July 2002

Salmonella
spp. are reported to have an increased heat tolerance at low water activity (a
w
; measured by relative vapor pressure [rvp]), achieved either by drying or by incorporating solutes. Much of the published data, however, cover only a narrow treatment range and have been analyzed by assuming first-order death kinetics. In this study, the death of
Salmonella enterica
serovar Typhimurium DT104 when exposed to 54 combinations of temperature (55 to 80°C) and a
w
(rvp 0.65 to 0.90, reduced using glucose-fructose) was investigated. The Weibull model (LogS = −

bt
n

) was used to describe microbial inactivation, and surface response models were developed to predict death rates for serovar Typhimurium at all points within the design surface. The models were evaluated with data generated by using six different
Salmonella
strains in place of serovar Typhimurium DT104 strain 30, two different solutes in place of glucose-fructose to reduce a
w
, or six low-a
w
foods artificially contaminated with
Salmonella
in place of the sugar broths. The data demonstrate that, at temperatures of ≥70°C,
Salmonella
cells at low a
w
were more heat tolerant than those at a higher a
w
but below 65°C the reverse was true. The same patterns were generated when sucrose (rvp 0.80 compared with 0.90) or NaCl (0.75 compared with 0.90) was used to reduce a
w
, but the extent of the protection afforded varied with solute type. The predictions of thermal death rates in the low-a
w
foods were usually fail-safe, but the few exceptions highlight the importance of validating models with specific foods that may have additional factors affecting survival.
Mutagenic Analysis of a Conserved Region of Domain III in the Cry1Ac Toxin of
Bacillus thuringiensis
Authors: Masson Luke , Tabashnik Bruce E. , Mazza Alberto , Préfontaine Gabrielle , Potvin Léna , Brousseau Roland , Schwartz Jean-Louis
Published: July 2002

We used site-directed mutagenesis to probe the function of four alternating arginines located at amino acid positions 525, 527, 529, and 531 in a highly conserved region of domain III in the Cry1Ac toxin of
Bacillus thuringiensis
. We created 10 mutants: eight single mutants, with each arginine replaced by either glycine (G) or aspartic acid (D), and two double mutants (R525G/R527G and R529G/R531G). In lawn assays of the 10 mutants with a cultured
Choristoneura fumiferana
insect cell line (Cf1), replacement of a single arginine by either glycine or aspartic acid at position 525 or 529 decreased toxicity 4- to 12-fold relative to native Cry1Ac toxin, whereas replacement at position 527 or 531 decreased toxicity only 3-fold. The reduction in toxicity seen with double mutants was 8-fold for R525G/R527G and 25-fold for R529G/R531G. Five of the mutants (R525G, R525D, R527G, R529D, and R525G/R527G) were tested in bioassays with
Plutella xylostella
larvae and ion channel formation in planar lipid bilayers. In the bioassays, R525D, R529D, and R525G/R527G showed reduced toxicity. In planar lipid bilayers, the conductance and the selectivity of the mutants were similar to those of native Cry1Ac. Toxins with alteration at position 527 or 529 tended to remain in their subconducting states rather than the maximally conducting state. Our results suggest that the primary role of this conserved region is to maintain both the structural integrity of the native toxin and the full functionality of the formed membrane pore.
Optimization of the Cell Wall Microenvironment Allows Increased Production of Recombinant
Bacillus anthracis
Protective Antigen from
B. subtilis
Authors: Thwaite Joanne E. , Baillie Les W. J. , Carter Noel M. , Stephenson Keith , Rees Mark , Harwood Colin R. , Emmerson Peter T.
Published: July 2002

The stability of heterologous proteins secreted by gram-positive bacteria is greatly influenced by the microenvironment on the
trans
side of the cytoplasmic membrane, and secreted heterologous proteins are susceptible to rapid degradation by host cell proteases. In
Bacillus subtilis,
degradation occurs either as the proteins emerge from the presecretory translocase and prior to folding into their native conformation or after the native conformation has been reached. The former process generally involves membrane- and/or cell wall-bound proteases, while the latter involves proteases that are released into the culture medium. The identification and manipulation of factors that influence the folding of heterologous proteins has the potential to improve the yield of secreted heterologous proteins. Recombinant anthrax protective antigen (rPA) has been used as a model secreted heterologous protein because it is sensitive to proteolytic degradation both before and after folding into its native conformation. This paper describes the influence of the microenvironment on the
trans
side of the cytoplasmic membrane on the stability of rPA. Specifically, we have determined the influence of net cell wall charge and its modulation by the extent to which the anionic polymer teichoic acid is
d
-alanylated on the secretion and stability of rPA. The potential role of the
dlt
operon, responsible for
d
-alanylation, was investigated using a
Bacillus subtilis
strain encoding an inducible
dlt
operon. We show that, in the absence of
d
-alanylation, the yield of secreted rPA is increased 2.5-fold. The function of
d
-alanylation and the use of rPA as a model protein are evaluated with respect to the optimization of
B. subtilis
for the secretion of heterologous proteins.
Characterization ofPseudomonasspp. Associated with Spoilage of Gilt-Head Sea Bream Stored under Various Conditions
Authors: Tryfinopoulou P. , Tsakalidou E. , Nychas G.-J. E.
Published: July 2002

The population dynamics of pseudomonads in gilt-head sea bream Mediterranean fish (Sparusaurata) stored under different conditions were studied. Phenotypic analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins were performed to identify a total of 106Pseudomonasstrains isolated fromS.auratastored under different temperatures (at 0, 10, and 20°C) and packaging conditions (air and a modified atmosphere of 40% CO2-30% N2-30% O2).Pseudomonas lundensiswas the predominant species, followed byPseudomonas fluorescens, whilePseudomonas fragiandPseudomonas putidawere detected less frequently. FluorescentPseudomonasstrains dominated under air conditions, while proteolytic and less lipolytic strains dominated under modified-atmosphere packaging. Different storage conditions appear to govern the selection of pseudomonads in gilt-head sea bream fish.
Molecular Cloning of
XYL3
(
d
-Xylulokinase) from
Pichia stipitis
and Characterization of Its Physiological Function
Authors: Jin Yong-Su , Jones Sharon , Shi Nian-Qing , Jeffries Thomas W.
Published: July 2002

XYL3
, which encodes a
d
-xylulokinase (EC 2.7.1.17), was isolated from
Pichia stipitis
CBS 6054 genomic DNA by using primers designed against conserved motifs. Disruption of
XYL3
eliminated
d
-xylulokinase activity, but
d
-ribulokinase activity was still present. Southern analysis of
P. stipitis
genomic DNA with
XYL3
as a probe confirmed the disruption and did not reveal additional related genes. Disruption of
XYL3
stopped ethanol production from xylose, but the resulting mutant still assimilated xylose slowly and formed xylitol and arabinitol. These results indicate that
XYL3
is critical for ethanol production from xylose but that
P. stipitis
has another pathway for xylose assimilation. Expression of
XYL3
using its
P. stipitis
promoter increased
Saccharomyces cerevisiae
d
-xylulose consumption threefold and enabled the transformants to produce ethanol from a mixture of xylose and xylulose, whereas the parental strain only accumulated xylitol. In vitro,
d
-xylulokinase activity in recombinant
S. cerevisiae
was sixfold higher with a multicopy than with a single-copy
XYL3
plasmid, but ethanol production decreased with increased copy number. These results confirmed the function of
XYL3
in
S. cerevisiae
.
Use of Fe(III) as an Electron Acceptor To Recover Previously Uncultured Hyperthermophiles: Isolation and Characterization of
Geothermobacterium ferrireducens
gen. nov., sp. nov
Authors: Kashefi Kazem , Holmes Dawn E. , Reysenbach Anna-Louise , Lovley Derek R.
Published: July 2002

It has recently been recognized that the ability to use Fe(III) as a terminal electron acceptor is a highly conserved characteristic in hyperthermophilic microorganisms. This suggests that it may be possible to recover as-yet-uncultured hyperthermophiles in pure culture if Fe(III) is used as an electron acceptor. As part of a study of the microbial diversity of the Obsidian Pool area in Yellowstone National Park, Wyo., hot sediment samples were used as the inoculum for enrichment cultures in media containing hydrogen as the sole electron donor and poorly crystalline Fe(III) oxide as the electron acceptor. A pure culture was recovered on solidified, Fe(III) oxide medium. The isolate, designated FW-1a, is a hyperthermophilic anaerobe that grows exclusively by coupling hydrogen oxidation to the reduction of poorly crystalline Fe(III) oxide. Organic carbon is not required for growth. Magnetite is the end product of Fe(III) oxide reduction under the culture conditions evaluated. The cells are rod shaped, about 0.5 μm by 1.0 to 1.2 μm, and motile and have a single flagellum. Strain FW-1a grows at circumneutral pH, at freshwater salinities, and at temperatures of between 65 and 100°C with an optimum of 85 to 90°C. To our knowledge this is the highest temperature optimum of any organism in the
Bacteria
. Analysis of the 16S ribosomal DNA (rDNA) sequence of strain FW-1a places it within the
Bacteria
, most closely related to abundant but uncultured microorganisms whose 16S rDNA sequences have been previously recovered from Obsidian Pool and a terrestrial hot spring in Iceland. While previous studies inferred that the uncultured microorganisms with these 16S rDNA sequences were sulfate-reducing organisms, the physiology of the strain FW-1a, which does not reduce sulfate, indicates that these organisms are just as likely to be Fe(III) reducers. These results further demonstrate that Fe(III) may be helpful for recovering as-yet-uncultured microorganisms from hydrothermal environments and illustrate that caution must be used in inferring the physiological characteristics of at least some thermophilic microorganisms solely from 16S rDNA sequences. Based on both its 16S rDNA sequence and physiological characteristics, strain FW-1a represents a new genus among the
Bacteria.
The name
Geothermobacterium ferrireducens
gen. nov., sp. nov., is proposed (ATCC BAA-426).
Intracellular Carbon Fluxes in Riboflavin-Producing
Bacillus
subtilis
during Growth on Two-Carbon Substrate Mixtures
Authors: Dauner Michael , Sonderegger Marco , Hochuli Michel , Szyperski Thomas , Wüthrich Kurt , Hohmann Hans-Peter , Sauer Uwe , Bailey James E.
Published: July 2002

Metabolic responses to cofeeding of different carbon substrates in carbon-limited chemostat cultures were investigated with riboflavin-producing
Bacillus
subtilis
. Relative to the carbon content (or energy content) of the substrates, the biomass yield was lower in all cofeeding experiments than with glucose alone. The riboflavin yield, in contrast, was significantly increased in the acetoin- and gluconate-cofed cultures. In these two scenarios, unusually high intracellular ATP-to-ADP ratios correlated with improved riboflavin yields. Nuclear magnetic resonance spectra recorded with amino acids obtained from biosynthetically directed fractional
13
C labeling experiments were used in an isotope isomer balancing framework to estimate intracellular carbon fluxes. The glycolysis-to-pentose phosphate (PP) pathway split ratio was almost invariant at about 80% in all experiments, a result that was particularly surprising for the cosubstrate gluconate, which feeds directly into the PP pathway. The in vivo activities of the tricarboxylic acid cycle, in contrast, varied more than twofold. The malic enzyme was active with acetate, gluconate, or acetoin cofeeding but not with citrate cofeeding or with glucose alone. The in vivo activity of the gluconeogenic phosphoenolpyruvate carboxykinase was found to be relatively high in all experiments, with the sole exception of the gluconate-cofed culture.
Synthesis of Pyruvate Dehydrogenase in
Staphylococcus aureus
Is Stimulated by Osmotic Stress
Authors: Vilhelmsson Oddur , Miller Karen J.
Published: July 2002

The pyruvate dehydrogenase multienzyme complex (PDHC) was found to be upregulated by osmotic stress in the osmotolerant pathogen
Staphylococcus aureus
. Upregulation was detectable in the levels of both activity and protein and was judged to be about fourfold when sodium chloride was used to adjust the water activity (a
w
) of the growth medium to 0.94. The upregulation of the PDHC was also found to be humectant dependent and was greatest when impermeant, nonmetabolizable humectants were used to adjust a
w
. Further experiments provided evidence that in addition to osmotic upregulation, the PDHC complex is also subject to catabolite repression, thus providing a possible explanation for the observation that high concentrations of carbohydrates are generally more inhibitory to the growth of this bacterial pathogen than are high concentrations of salts.
Modulation of Gene Expression Made Easy
Authors: Solem Christian , Jensen Peter Ruhdal
Published: July 2002

A new approach for modulating gene expression, based on randomization of promoter (spacer) sequences, was developed. The method was applied to chromosomal genes in
Lactococcus lactis
and shown to generate libraries of clones with broad ranges of expression levels of target genes. In one example, overexpression was achieved by introducing an additional gene copy into a phage attachment site on the chromosome. This resulted in a series of strains with phosphofructokinase activities from 1.4 to 11 times the wild-type activity level. In this example, the
pfk
gene was cloned upstream of a
gusA
gene encoding β-glucuronidase, resulting in an operon structure in which both genes are transcribed from a common promoter. We show that there is a linear correlation between the expressions of the two genes, which facilitates screening for mutants with suitable enzyme activities. In a second example, we show that the method can be applied to modulating the expression of native genes on the chromosome. We constructed a series of strains in which the expression of the
las
operon, containing the genes
pfk
,
pyk
, and
ldh
, was modulated by integrating a truncated copy of the
pfk
gene. Importantly, the modulation affected the activities of all three enzymes to the same extent, and enzyme activities ranging from 0.5 to 3.5 times the wild-type level were obtained.
Biochemical and Genetic Characterization of the Two-Peptide Bacteriocin Enterocin 1071 Produced by
Enterococcus faecalis
FAIR-E 309
Authors: Franz Charles M. A. P. , Grube Alexandra , Herrmann Anette , Abriouel Hikmate , Stärke Joachim , Lombardi Angiolella , Tauscher Bernhard , Holzapfel Wilhelm H.
Published: July 2002

The structural genes for the two-peptide bacteriocin enterocin 1071 (Ent1071) in
Enterococcus faecalis
FAIR-E 309 were cloned. DNA sequence analysis showed that the enterocin 1071A (Ent1071A) peptide of strain FAIR-E 309 differed by two amino acids from the Ent1071A reported for
E. faecalis
BFE 1071 (E. Balla, L. M. T. Dicks, M. Du Toit, M. J. van der Merwe, and W. H. Holzapfel, Appl. Environ. Microbiol. 66:1298-1304, 2000), while the Ent1071B gene encoded identical peptides in these strains. However, resequencing of
ent1071A
from
E. faecalis
BFE 1071 showed that the Ent1071A peptide sequence reported previously was incorrect in two amino acids. Also,
ent1071B
in
E. faecalis
FAIR-E 309 encoded a prepeptide that was three amino acids shorter than that previously reported for
E. faecalis
BFE 1071 Ent1071B. A presumptive immunity gene (
eni1071
) was located downstream of the bacteriocin structural genes. This gene was cloned into the heterologous host
E. faecalis
ATCC 19433 and was shown to confer immunity. A truncated ABC transporter gene was located upstream of the Ent1071 structural genes.
An Antifungal Protein from the Marine Bacterium
Streptomyces
sp. Strain AP77 Is Specific for
Pythium porphyrae
, a Causative Agent of Red Rot Disease in
Porphyra
spp
Authors: Woo Jung-Hee , Kitamura Etsushi , Myouga Hisashi , Kamei Yuto
Published: July 2002

A novel antifungal protein (SAP) was found in the culture supernatant of a marine bacterium,
Streptomyces
sp. strain AP77, and was purified. This protein was characterized by chemical, biochemical, and biological analyses. By using gel filtration, the molecular mass of SAP was estimated to be 160 kDa. Structural analysis of SAP by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization-time of flight mass spectrometry suggested that SAP is composed of three heterologous protein subunits of 41.7 kDa (SAP1), 21.7 kDa (SAP2), and 18.7 kDa (SAP3) at a molar ratio of 1:1:5 (or 1:1:6). N-terminal amino acid sequence analysis and a homology search revealed that SAP1, SAP2, and SAP3 exhibit 64.3, 68.4, and 86.7% similarity to three
Streptomyces coelicolor
polypeptides, puromycin resistance protein (Pur8), a conserved hypothetical protein, and bacterioferritin, respectively. The MIC of purified SAP against
Pythium porphyrae
was determined to be 1.6 μg/disk, whereas no inhibitory effect was observed at concentrations up to 100 μg/disk against most of the fungal and bacterial strains tested; the only exception was relatively strong antifungal activity against
Pythium ultimum
(MIC, 6.3 μg/disk). In vitro and in vivo toxicity tests demonstrated that SAP showed no toxicity against
Porphyra yezoensis
cells, human normal dermal fibroblasts, and mice at doses up to 700 μg/ml (for 24 h), 250 μg/ml (for 12 h), and 75 mg/kg (for 35 days), respectively. SAP was labile when it was subjected to a heated-air drying treatment, which is a great advantage in food production procedures. These results indicated that
Streptomyces
sp. strain AP77 might be useful as a gene source for safe transgenic
Porphyra
breeding for tolerance to
Pythium
infection.
High Prevalence of Vancomycin-Resistant Enterococci in Swedish Sewage
Authors: Iversen Aina , Kühn Inger , Franklin Anders , Möllby Roland
Published: July 2002

In Europe the use of the growth promoter avoparcin is considered to have selected for vancomycin-resistant enterococci (VRE). Sweden ceased using avoparcin in 1986, and only occasional cases of VRE from hospitals have been reported since 1995. Within the framework of a European study, samples from urban raw sewage, treated sewage, surface water, and hospital sewage in Sweden (
n
= 118) were screened for VRE. Surprisingly, VRE were isolated from 21 of 35 untreated sewage samples (60%), from 5 of 14 hospital sewage samples (36%), from 6 of 32 treated sewage samples (19%), and from 1 of 37 surface water samples. Thirty-five isolates from 33 samples were further characterized by geno- and phenotyping, MIC determination, and PCR analysis. Most isolates (30 of 35) carried the
vanA
gene, and the majority (24 of 35) of the isolates were
Enterococcus faecium
. Most of the VRE were multiresistant. The typing revealed high diversity of the isolates. However, one major cluster with seven identical or similar isolates was found. These isolates came from three different sewage treatment plants and were collected at different occasions during 1 year. All VRE from hospital sewage originated from one of the two hospitals studied. That hospital also had vancomycin consumption that was 10-fold that of the other. We conclude that VRE were commonly found in sewage samples in Sweden. The origin might be both healthy individuals and individuals in hospitals. Possibly, antimicrobial drugs or chemicals released into the sewage system may sustain VRE in the system.
Development of a Flow Cytometric Method To Analyze Subpopulations of Bacteria in Probiotic Products and Dairy Starters
Authors: Bunthof Christine J. , Abee Tjakko
Published: July 2002

Flow cytometry (FCM) is a rapid and sensitive technique that can determine cell numbers and measure various physiological characteristics of individual cells by using appropriate fluorescent probes. Previously, we developed an FCM assay with the viability probes carboxyfluorescein diacetate (cFDA) and TOTO-1 {1′-(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)-bis-4-[3-methyl-2,3dihydro(benzo-1,3-oxazole)-2-methylidene]-1-(3′-trimethylammoniumpropyl)-pyridinium tetraiodide} for (stressed) lactic acid bacteria (C. J. Bunthof, K. Bloemen, P. Breeuwer, F. M. Rombouts, and T. Abee, Appl. Environ. Microbiol.
67:
2326-2335, 2001). cFDA stains intact cells with enzymatic activity, and TOTO-1 stains membrane-permeabilized cells. Here we used this assay to study the viability of bacterial suspensions in milk, dairy fermentation starters, and probiotic products. To facilitate FCM analysis of bacteria in milk, a commercially available milk-clearing solution was used. The procedure was optimized to increase the signal-to-noise ratio. FCM enumerations were accurate down to a concentration of 10
5
cells ml
−1
. The level of retrieval of
Lactobacillus plantarum
WCFS 1 suspended in milk was high, and viability was not affected by the procedure. The plate counts for cleared samples of untreated cell suspensions were nearly as high as the total FCM counts, and the correlation was strong (
r
> 0.99). In dairy fermentation starters and in probiotic products the FCM total cell counts were substantially higher than the numbers of CFU. Three functional populations could be distinguished: culturable cells, cells that are intact and metabolically active but not culturable, and permeabilized cells. The proportions of the populations differed in the products tested. This FCM method provides tools to assess the functionality of different populations in fermentation starters and probiotic products.
Identification and Isolation of Genes Involved in Poly(β-Hydroxybutyrate) Biosynthesis in
Azospirillum brasilense
and Characterization of a
phbC
Mutant
Authors: Kadouri Daniel , Burdman Saul , Jurkevitch Edouard , Okon Yaacov
Published: July 2002

Like many other prokaryotes, rhizobacteria of the genus
Azospirillum
produce high levels of poly(β-hydroxybutyrate) (PHB) under suboptimal growth conditions. Utilization of PHB by bacteria under stress has been proposed as a mechanism that favors their compatible establishment in competitive environments, thus showing great potential for the improvement of bacterial inoculants for plants and soils. The three genes that are considered to be essential in the PHB biosynthetic pathway,
phbA
(β-ketothiolase),
phbB
(acetoacetyl coenzyme A reductase), and
phbC
(PHB synthase), were identified in
Azospirillum brasilense
strain Sp7, cloned, and sequenced. The
phbA
, -
B
, and -
C
genes were found to be linked together and located on the chromosome. An
A. brasilense phbC
mutant was obtained by insertion of a kanamycin resistance cassette within the
phbC
gene. No PHB production was detected in this mutant. The capability of the wild-type strain to endure starvation conditions was higher than that of the mutant strain. However, motility, cell aggregation, root adhesion, and exopolysaccharide (EPS) and capsular polysaccharide (CPS) production were higher in the
phbC
mutant strain than in the wild type.
Immunomagnetic Separation (IMS)-Fluorescent Antibody Detection and IMS-PCR Detection of Seeded
Cryptosporidium parvum
Oocysts in Natural Waters and Their Limitations
Authors: Sturbaum Gregory D. , Klonicki Patricia T. , Marshall Marilyn M. , Jost B. Helen , Clay Brec L. , Sterling Charles R.
Published: July 2002

Detection and enumeration of
Cryptosporidium parvum
in both treated and untreated waters are important to facilitate prevention of future cryptosporidiosis incidents. Immunomagnetic separation (IMS)-fluorescent antibody (FA) detection and IMS-PCR detection efficiencies were evaluated in two natural waters seeded with nominal seed doses of 5, 10, and 15 oocysts. IMS-FA detected oocysts at concentrations at or below the three nominal oocyst seed doses, illustrating that IMS-FA is sensitive enough to detect low oocyst numbers. However, the species of the oocysts could not be determined with this technique. IMS-PCR, targeting the 18S rRNA gene in this study, yielded positive amplification for 17 of the 18 seeded water samples, and the amplicons were subjected to restriction fragment length polymorphism digestion and DNA sequencing for species identification. Interestingly, the two unseeded, natural water samples were also PCR positive; one amplicon was the same base pair size as the
C. parvum
amplicon, and the other amplicon was larger. These two amplified products were determined to be derived from DNA of
Cryptosporidium muris
and a dinoflagellate. These IMS-PCR results illustrate that (i) IMS-PCR is able to detect low oocyst numbers in natural waters, (ii) PCR amplification alone is not confirmatory for detection of target DNA when environmental samples are used, (ii) PCR primers, especially those designed against the rRNA gene region, need to be evaluated for specificity with organisms closely related to the target organism, and (iv) environmental amplicons should be subjected to appropriate species-specific confirmatory techniques.
Characterization of Cholylglycine Hydrolase from a Bile-Adapted Strain of
Xanthomonas maltophilia
and Its Application for Quantitative Hydrolysis of Conjugated Bile Salts
Authors: Dean Mariangela , Cervellati Carlo , Casanova Elena , Squerzanti Monica , Lanzara Vincenzo , Medici Alessandro , Polverino de Laureto Patrizia , Bergamini Carlo M.
Published: July 2002

Purified bile salt hydrolase from bile-adapted
Xanthomonas maltophilia
displays Michaelis-Menten kinetics on cholylglycine and cholyltaurine and hydrolyzes bile salts also in crude bovine bile. The protein is a dimer and is resistant to proteinases and to heating at 55 to 60°C for up to 60 min, in agreement with calorimetric data.
Comparative Study of Vaginal
Lactobacillus
Phages Isolated from Women in the United States and Turkey: Prevalence, Morphology, Host Range, and DNA Homology
Authors: Kiliç Ali O. , Pavlova Sylvia I. , Alpay Sengul , Kiliç S. Sirri , Tao Lin
Published: July 2002

Lactobacilli play an important role in maintaining vaginal health. However, during bacterial vaginosis lactobacilli decrease for unknown reasons. Our preliminary study showed that phages could infect vaginal lactobacilli. Therefore, the aim of this study was to analyze the distribution, virulence, and types of vaginal
Lactobacillus
phages isolated from women of two countries: the United States and Turkey. A total of 209 vaginal lactobacilli were isolated from reproductive-aged women in the United States (
n
= 107) and Turkey (
n
= 102). By analysis of 16S rRNA gene sequence and by comparison of protein profiles, most lactobacilli were identified as
L. crispatus
,
L. gasseri
, and
L. jensenii
. After mitomycin C induction, 28% of American lactobacilli and 36% of Turkish lactobacilli released phages. A total of 67 phages were isolated and further characterized by their host range, electron microscopy, and DNA homology. All 67 phages were infective against lactobacilli from both collections. The host ranges of most phages were broad, including multiple
Lactobacillus
species. Even though the phages were all temperate, they were able to cause lytic infection in various strains. The electron micrographs of these phages showed a hexagon-shaped head and a long tail with or without a contractile tail sheath. Based on their morphology, these phages belonged to Bradley's phage groups A and B, and could be further classified into four morphotypes. All four types were found among American phages, but only three were found among Turkish isolates. DNA hybridization with labeled probes of the four types of phages revealed that additional genetic types existed within each morphotype among these phages. The phage genomic sizes ranged between 34 and 55 kb. Many of the lysogenic
Lactobacillus
strains released phages spontaneously at a high frequency of 10
−3
to 10
−4
PFU/cell. In conclusion, lysogeny in vaginal lactobacilli is widely spread. Some lysogenic lactobacilli spontaneously release phages with a broad host range, which can be lytic against other vaginal lactobacilli regardless of their geographic origin.
Use of Synthetic Peptides Derived from the Antigens ESAT-6 and CFP-10 for Differential Diagnosis of Bovine Tuberculosis in Cattle
Authors: Vordermeier H. M. , Whelan A. , Cockle P. J. , Farrant L. , Palmer N. , Hewinson R. G.
Published: July 2002

In Great Britain an independent scientific review for the government has concluded that the development of a cattle vaccine againstMycobacterium bovisinfection holds the best long-term prospect for tuberculosis control in British herds. A precondition for vaccination is the development of a complementary diagnostic test to differentiate between vaccinated animals and those infected withM. bovisso that testing and slaughter-based control strategies can continue alongside vaccination. To date bacillus Calmette-Guérin (BCG), an attenuated strain ofM. bovis, is the only available vaccine for the prevention of tuberculosis. However, tests based on tuberculin purified protein derivative cannot distinguish betweenM. bovisinfection and BCG vaccination. Therefore, specific antigens expressed byM. bovisbut absent from BCG constitute prime candidates for differential diagnostic reagents. Recently, two such antigens, ESAT-6 and CFP-10, have been reported to be promising candidates as diagnostic reagents for the detection ofM. bovisinfection in cattle. Here we report the identification of promiscuous peptides of CFP-10 that were recognized byM. bovis-infected cattle. Five of these peptides were formulated into a peptide cocktail together with five peptides derived from ESAT-6. Using this peptide cocktail in T-cell assays,M. bovis-infected animals were detected, while BCG-vaccinated orMycobacterium avium-sensitized animals did not respond. The sensitivity of the peptide cocktail as an antigen in a whole-blood gamma interferon assay was determined using naturally infected field reactor cattle, and the specificity was determined using blood from BCG-vaccinated and noninfected, nonvaccinated animals. The sensitivity of the assay in cattle with confirmed tuberculosis was found to be 77.9%, with a specificity of 100% in BCG-vaccinated or nonvaccinated animals. This compares favorably with the specificity of tuberculin when tested in noninfected or vaccinated animals. In summary, our results demonstrate that this peptide cocktail can discriminate betweenM. bovisinfection and BCG vaccination with a high degree of sensitivity and specificity.
Anemia and Interleukin-10, Tumor Necrosis Factor Alpha, and Erythropoietin Levels among Children with Acute, UncomplicatedPlasmodium falciparumMalaria
Authors: Nussenblatt Veronique , Mukasa Gelasius , Metzger Amy , Ndeezi Grace , Garrett Elizabeth , Semba Richard D.
Published: July 2002

Anemia is an important complication of malaria, and its pathogenesis is not well understood. To gain insight into potential age-related relationships between tumor necrosis factor alpha (TNF-α), interleukin 10 (IL-10), erythropoietin, and anemia during acute malaria, 273 children of ages 12 to 120 months presenting with acute, uncomplicated malaria in Kampala, Uganda, were monitored at enrollment and 3 and 7 days later. Younger children had higher geometric mean erythropoietin, TNF-α, and α1-acid glycoprotein (AGP) concentrations than older children. Univariate regression analysis revealed that age, log10erythropoietin levels, IL-10/TNF-α ratio, and AGP levels were each significantly associated with hemoglobin levels at baseline. Hemoglobin concentrations were inversely correlated with the log10erythropoietin level at all three visits. For the older age groups, higher levels of TNF-α were significantly associated with higher IL-10 levels at all three visits, but this relationship was significant only at baseline for younger children. These data suggest that younger children do not maintain IL-10 production in response to the inflammatory process, and this mechanism may contribute to the more severe anemia found in younger children. Acute malaria is an illness whose incidence and severity are largely age dependent. Further studies are needed to understand the relationships between age-related immune responses to malaria and their role in the pathogenesis of malarial anemia.
Development of Internal Controls for the Luminex Instrument as Part of a Multiplex Seven-Analyte Viral Respiratory Antibody Profile
Authors: Martins Thomas B.
Published: July 2002

The ability of the Luminex system to simultaneously quantitate multiple analytes from a single sample source has proven to be a feasible and cost-effective technology for assay development. In previous studies, my colleagues and I introduced two multiplex profiles consisting of 20 individual assays into the clinical laboratory. With the Luminex instrument’s ability to classify up to 100 distinct microspheres, however, we have only begun to realize the enormous potential of this technology. By utilizing additional microspheres, it is now possible to add true internal controls to each individual sample. During the development of a seven-analyte serologic viral respiratory antibody profile, internal controls for detecting sample addition and interfering rheumatoid factor (RF) were investigated. To determine if the correct sample was added, distinct microspheres were developed for measuring the presence of sufficient quantities of immunoglobulin G (IgG) or IgM in the diluted patient sample. In a multiplex assay of 82 samples, the IgM verification control correctly identified 23 out of 23 samples with low levels (<20 mg/dl) of this antibody isotype. An internal control microsphere for RF detected 30 out of 30 samples with significant levels (>10 IU/ml) of IgM RF. Additionally, RF-positive samples causing false-positive adenovirus and influenza A virus IgM results were correctly identified. By exploiting the Luminex instrument’s multiplexing capabilities, I have developed true internal controls to ensure correct sample addition and identify interfering RF as part of a respiratory viral serologic profile that includes influenza A and B viruses, adenovirus, parainfluenza viruses 1, 2, and 3, and respiratory syncytial virus. Since these controls are not assay specific, they can be incorporated into any serologic multiplex assay.
Diagnostic Usefulness of Antibodies against Ribosome Recycling Factor from
Brucella melitensis
in Human or Canine Brucellosis
Authors: Cassataro Juliana , Delpino M. Victoria , Velikovsky Carlos A. , Bruno Laura , Fossati Carlos A. , Baldi Pablo C.
Published: July 2002

The diagnostic usefulness of an enzyme-linked immunosorbent assay (ELISA) using a purified recombinant ribosome recycling factor from
Brucella melitensis
(CP24 antigen) was tested in human and canine infections caused by smooth and rough
Brucella
species, respectively. Anti-CP24 antibodies were detected in 9 (43%) of 21 consecutive cases of canine brucellosis and in 8 (53%) of 15 dogs followed for 60 days after the diagnosis of acute brucellosis. Among eight patients with acute brucellosis, anti-CP24 antibodies were detected in four in the 10 weeks following diagnosis, but the remaining four were negative during the whole follow-up (22 weeks). The frequency of anti-CP24 antibodies was also low among 24 patients with subacute brucellosis and 23 patients with chronic illness (29 and 26%, respectively). While all patients positive for anti-CP24 antibodies were also positive for antibodies to total cytoplasmic proteins of
Brucella
(CP), five were negative for antibodies to another cytoplasmic protein, the
Brucella
lumazine synthase (BLS). When a larger sample of 35 human sera negative for anti-BLS antibodies was assayed, 85.7% were positive for anti-CP24 antibodies, suggesting that the combined measurement of both reactivities could yield a higher sensitivity than any test alone. To test this hypothesis, an ELISA combining both antigens was designed. The percentage of positive results among chronic cases was higher for this assay than for the individual measurement of anti-CP24 or anti-BLS antibodies (83 versus 26 and 65%, respectively) and was closer to the value obtained for anti-CP antibodies (91%). The frequency of anti-CP24 antibodies is low in both canine and human brucellosis. In the latter case, however, an ELISA combining CP24 and BLS is more sensitive than assays measuring anti-CP24 or anti-BLS antibodies separately and almost as sensitive as the ELISA using CP.
Effect of Contaminating Red Blood Cells on OKT3-Mediated Polyclonal Activation of Peripheral Blood Mononuclear Cells
Authors: Song Samuel , Goodwin Joseph , Zhang Jenny , Babbitt Bruce , Lathey Janet L.
Published: July 2002

Erythrocytes are typically present as impurities in the majority of peripheral blood mononuclear cell (PBMC) preparations. This study was undertaken to investigate the effects of contaminating red blood cells (RBC) on the ability of OKT3 to activate CD4
+
and CD8
+
T cells. Surprisingly, the levels of gamma interferon, tumor necrosis factor alpha, and interleukin-1β (IL-1β) produced by PBMC upon stimulation by OKT3 were increased (
P
< 0.05) in a dose-dependent manner when increasing amounts of autologous RBC (RBC-to-PBMC ratios of 2:1, 10:1, and 50:1) were spiked into PBMC preparations. The OKT3-driven induction of the IL-2 receptor (CD25) and the proliferation of T lymphocytes in response to phorbol myristate acetate were not affected by the addition of RBC.
Antibodies to Malaria Vaccine Candidates Pvs25 and Pvs28 Completely Block the Ability ofPlasmodium vivaxTo Infect Mosquitoes
Authors: Hisaeda Hajime , Stowers Anthony W. , Tsuboi Takafumi , Collins William E. , Sattabongkot Jetsumon S. , Suwanabun Natavadee , Torii Motomi , Kaslow David C.
Published: July 2002

Transmission-blocking vaccines are one strategy for controlling malaria, whereby sexual-stage parasites are inhibited from infecting mosquitoes by human antibodies. To evaluate whether the recently clonedPlasmodium vivaxproteins Pvs25 and Pvs28 are candidates for a transmission-blocking vaccine, the molecules were expressed in yeast as secreted recombinant proteins. Mice vaccinated with these proteins adsorbed to aluminum hydroxide developed strong antibody responses against the immunogens, although for Pvs28, this response was genetically restricted. Antisera against both recombinant Pvs25 and Pvs28 recognized the corresponding molecules expressed by cultured sexual-stage parasites isolated from patients withP. vivaxmalaria. The development of malaria parasites in mosquitoes was completely inhibited when these antisera were ingested with the infected blood meal. Pvs25 and Pvs28, expressed inSaccharomyces cerevisiae, are as yet the only fully characterized transmission-blocking vaccine candidates againstP. vivaxthat induce such a potent antiparasite response.
Human CD8+T Cells Specific forMycobacterium tuberculosisSecreted Antigens in Tuberculosis Patients and Healthy BCG-Vaccinated Controls in The Gambia
Authors: Smith Steven M. , Klein Michèl R. , Malin Adam S. , Sillah Jackson , Huygen Kris , Andersen Peter , McAdam Keith P. W. J. , Dockrell Hazel M.
Published: July 2002

Intracellular flow cytometry analysis of perforin production by CD8+T cells showed levels were greatly reduced in tuberculosis (TB) patients compared to healthy controls. Reduced cytotoxic-T-lymphocyte activity was also obtained with CD8+T cells from TB patients compared to healthy controls in The Gambia. A change in antigen recognition was noted between the two groups of donors: in addition to recognition of Ag85A and Ag85B, as seen in healthy donors, a prominent ESAT-6 response was found in TB patients.
Construction and Characterization of an Effector Strain of
Streptococcus mutans
for Replacement Therapy of Dental Caries
Authors: Hillman J. D. , Brooks T. A. , Michalek S. M. , Harmon C. C. , Snoep J. L. , van der Weijden C. C.
Published: July 2002

An effector strain has been constructed for use in the replacement therapy of dental caries. Recombinant DNA methods were used to make the
Streptococcus mutans
supercolonizing strain, JH1140, lactate dehydrogenase deficient by deleting virtually all of the
ldh
open reading frame (ORF). To compensate for the resulting metabolic imbalance, a supplemental alcohol dehydrogenase activity was introduced by substituting the
adhB
ORF from
Zymomonas mobilis
in place of the deleted
ldh
ORF. The resulting clone, BCS3-L1, was found to produce no detectable lactic acid during growth on a variety of carbon sources, and it produced significantly less total acid due to its increased production of ethanol and acetoin. BCS3-L1 was significantly less cariogenic than JH1140 in both gnotobiotic- and conventional-rodent models. It colonized the teeth of conventional rats as well as JH1140 in both aggressive-displacement and preemptive-colonization models. No gross or microscopic abnormalities of major organs were associated with oral colonization of rats with BCS3-L1 for 6 months. Acid-producing revertants of BCS3-L1 were not observed in samples taken from infected animals (reversion frequency, <10
−3
) or by screening cultures grown in vitro, where no revertants were observed among 10
5
colonies examined on pH indicator medium. The reduced pathogenic potential of BCS3-L1, its strong colonization potential, and its genetic stability suggest that this strain is well suited to serve as an effector strain in the replacement therapy of dental caries in humans.
AMycobacterium ulceransToxin, Mycolactone, Causes Apoptosis in Guinea Pig Ulcers and Tissue Culture Cells
Authors: George Kathleen M. , Pascopella Lisa , Welty Diane M. , Small P. L. C.
Published: July 2002

Mycobacterium ulceransis the causative agent of Buruli ulcer, a tropical ulcerative skin disease. One of the most intriguing aspects of this disease is the presence of extensive tissue damage in the absence of an acute inflammatory response. We recently purified and characterized a macrolide toxin, mycolactone, fromM. ulcerans. Injection of this molecule into guinea pig skin reproduced cell death and lack of acute inflammatory response similar to that seen following the injection of viable bacteria. We also showed that mycolactone causes a cytopathic effect on mouse fibroblast L929 cells that is characterized by cytoskeletal rearrangements and growth arrest within 48 h. However, these results could not account for the extensive cell death which occurs in Buruli ulcer. The results presented here demonstrate that L929 and J774 mouse macrophage cells die via apoptosis after 3 to 5 days of exposure to mycolactone. Treatment of cells with a pan-caspase inhibitor can inhibit mycolactone-induced apoptosis. We demonstrate that injection of mycolactone into guinea pig skin results in cell death via apoptosis and that the extent of apoptosis increases as the lesion progresses. These results may help to explain why tissue damage in Buruli ulcer is not accompanied by an acute inflammatory response.
Serum Amyloid P Component Bound to Gram-Negative Bacteria Prevents Lipopolysaccharide-Mediated Classical Pathway Complement Activation
Authors: de Haas Carla J. C. , van Leeuwen Ester M. M. , van Bommel Toon , Verhoef Jan , van Kessel Kok P. M. , van Strijp Jos A. G.
Published: July 2002

Although serum amyloid P component (SAP) is known to bind many ligands, its biological function is not yet clear. Recently, it was demonstrated that SAP binds to lipopolysaccharide (LPS). In the present study, SAP was shown to bind to gram-negative bacteria expressing short types of LPS or lipo-oligosaccharide (LOS), such asSalmonella entericaserovar Copenhagen Re andEscherichia coliJ5, and also to clinical isolates ofHaemophilus influenzae. It was hypothesized that SAP binds to the bacteria via the lipid A part of LPS or LOS, since thehtrBmutant of the nontypeableH. influenzaestrain NTHi 2019-B29-3, which expresses a nonacetylated lipid A, did not bind SAP. This was in contrast to the parental strain NTHi 2019. The binding of SAP resulted in a clear inhibition of the deposition of complement component C3 on the bacteria. SAP inhibited only the activation of the classical complement pathway; the alternative route remained unaffected. In the classical route, SAP prevented the deposition of the first complement component, Clq, probably by interfering with the binding of Clq to LPS. Since antibody-mediated Clq activation was not inhibited by SAP, SAP seems to inhibit only the LPS-induced classical complement pathway activation. The SAP-induced inhibition of C3 deposition strongly diminished the complement-mediated lysis as well as the phagocytosis of the bacteria. The binding of SAP to gram-negative bacteria, therefore, might influence the pathophysiology of an infection with such bacteria.
γδ T Cells Are a Component of Early Immunity against Preerythrocytic Malaria Parasites
Authors: McKenna Kyle C. , Tsuji Moriya , Sarzotti Marcella , Sacci John B. , Witney Adam A. , Azad Abdu F.
Published: July 2002

We tested the hypothesis that γδ T cells are a component of an early immune response directed against preerythrocytic malaria parasites that are required for the induction of an effector αβ T-cell immune response generated by irradiated-sporozoite (irr-spz) immunization. γδ T-cell-deficient (TCRδ−/−) mice on a C57BL/6 background were challenged withPlasmodium yoelii(17XNL strain) sporozoites, and then liver parasite burden was measured at 42 h postchallenge. Liver parasite burden was measured by quantification of parasite-specific 18S rRNA in total liver RNA by quantitative-competitive reverse transcription-PCR and by an automated 5′ exonuclease PCR. Sporozoite-challenged TCRδ−/−mice showed a significant (P< 0.01) increase in liver parasite burden compared to similarly challenged immunocompetent mice. In support of this result, TCRδ−/−mice were also found to be more susceptible than immunocompetent mice to a sporozoite challenge when blood-stage parasitemia was used as a readout. A greater percentage of TCRδ−/−mice than of immunocompetent mice progressed to a blood-stage infection when challenged with five or fewer sporozoites (odds ratio = 2.35,P= 0.06). TCRδ−/−mice receiving a single irr-spz immunization showed percent inhibition of liver parasites comparable to that of immunized immunocompetent mice following a sporozoite challenge. These data support the hypothesis that γδ T cells are a component of early immunity directed against malaria preerythrocytic parasites and suggest that γδ T cells are not required for the induction of an effector αβ T-cell immune response generated by irr-spz immunization.
Escherichia coli
O157:H7 Causes More-Severe Systemic Disease in Suckling Piglets than in Colostrum-Deprived Neonatal Piglets
Authors: Dean-Nystrom Evelyn A. , Pohlenz Joachim F. L. , Moon Harley W. , O'Brien Alison D.
Published: July 2002

Our objective was to determine if suckling neonatal piglets are susceptible to enterohemorrhagic
Escherichia coli
(EHEC) O157:H7 disease. Surprisingly, EHEC O157:H7 caused more-rapid and more-severe neurological disease in suckling neonates than in those fed an artificial diet. Shiga toxin-negative O157:H7 did not cause neurological disease but colonized and caused attaching-and-effacing intestinal lesions.