bluuebunny/crossref_metadata_2025 huggingface dataset:
Microbial Thiocyanate Utilization under Highly Alkaline Conditions
Authors: Sorokin Dimitry Y. , Tourova Tatyana P. , Lysenko Anatoly M. , Kuenen J. Gijs
Published: July 2002

Three kinds of alkaliphilic bacteria able to utilize thiocyanate (CNS
−
) at pH 10 were found in highly alkaline soda lake sediments and soda soils. The first group included obligate heterotrophs that utilized thiocyanate as a nitrogen source while growing at pH 10 with acetate as carbon and energy sources. Most of the heterotrophic strains were able to oxidize sulfide and thiosulfate to tetrathionate. The second group included obligately autotrophic sulfur-oxidizing alkaliphiles which utilized thiocyanate nitrogen during growth with thiosulfate as the energy source. Genetic analysis demonstrated that both the heterotrophic and autotrophic alkaliphiles that utilized thiocyanate as a nitrogen source were related to the previously described sulfur-oxidizing alkaliphiles belonging to the gamma subdivision of the division
Proteobacteria
(the
Halomonas
group for the heterotrophs and the genus
Thioalkalivibrio
for autotrophs). The third group included obligately autotrophic sulfur-oxidizing alkaliphilic bacteria able to utilize thiocyanate as a sole source of energy. These bacteria could be enriched on mineral medium with thiocyanate at pH 10. Growth with thiocyanate was usually much slower than growth with thiosulfate, although the biomass yield on thiocyanate was higher. Of the four strains isolated, the three vibrio-shaped strains were genetically closely related to the previously described sulfur-oxidizing alkaliphiles belonging to the genus
Thioalkalivibrio
. The rod-shaped isolate differed from the other isolates by its ability to accumulate large amounts of elemental sulfur inside its cells and by its ability to oxidize carbon disulfide. Despite its low DNA homology with and substantial phenotypic differences from the vibrio-shaped strains, this isolate also belonged to the genus
Thioalkalivibrio
according to a phylogenetic analysis. The heterotrophic and autotrophic alkaliphiles that grew with thiocyanate as an N source possessed a relatively high level of cyanase activity which converted cyanate (CNO
−
) to ammonia and CO
2
. On the other hand, cyanase activity either was absent or was present at very low levels in the autotrophic strains grown on thiocyanate as the sole energy and N source. As a result, large amounts of cyanate were found to accumulate in the media during utilization of thiocyanate at pH 10 in batch and thiocyanate-limited continuous cultures. This is a first direct proof of a “cyanate pathway” in pure cultures of thiocyanate-degrading bacteria. Since it is relatively stable under alkaline conditions, cyanate is likely to play a role as an N buffer that keeps the alkaliphilic bacteria safe from inhibition by free ammonia, which otherwise would reach toxic levels during dissimilatory degradation of thiocyanate.
Isolation of a Spotted Fever Group Rickettsia,
Rickettsia peacockii
, in a Rocky Mountain Wood Tick,
Dermacentor andersoni
, Cell Line
Authors: Simser Jason A. , Palmer Ann T. , Munderloh Ulrike G. , Kurtti Timothy J.
Published: July 2002

An embryonic cell line (DAE100) of the Rocky Mountain wood tick,
Dermacentor andersoni
, was observed by microscopy to be chronically infected with a rickettsialike organism. The organism was identified as a spotted fever group (SFG) rickettsia by PCR amplification and sequencing of portions of the 16S rRNA, citrate synthase,
Rickettsia
genus-specific 17-kDa antigen, and SFG-specific 190-kDa outer membrane protein A (rOmpA) genes. Sequence analysis of a partial
rompA
gene PCR fragment and indirect fluorescent antibody data for rOmpA and rOmpB indicated that this rickettsia was a strain (DaE100R) of
Rickettsia peacockii
, an SFG species presumed to be avirulent for both ticks and mammals.
R. peacockii
was successfully maintained in a continuous culture of DAE100 cells without apparent adverse effects on the host cells. Establishing cell lines from embryonic tissues of ticks offers an alternative technique for isolation of rickettsiae that are transovarially transmitted.
Identification of Fecal
Escherichia coli
from Humans and Animals by Ribotyping
Authors: Carson C. Andrew , Shear Brian L. , Ellersieck Mark R. , Asfaw Amha
Published: July 2002

Fecal pollution of water resources is an environmental problem of increasing importance. Identification of individual host sources of fecal
Escherichia coli
, such as humans, pets, production animals, and wild animals, is prerequisite to formulation of remediation plans. Ribotyping has been used to distinguish fecal
E. coli
of human origin from pooled fecal
E. coli
isolates of nonhuman origin. We have extended application of this technique to distinguishing fecal
E. coli
ribotype patterns from human and seven individual nonhuman hosts. Classification accuracy was best when the analysis was limited to three host sources. Application of this technique to identification of host sources of fecal coliforms in water could assist in formulation of pollution reduction plans.
Physiological Properties of
Saccharomyces cerevisiae
from Which Hexokinase II Has Been Deleted
Authors: Diderich Jasper A. , Raamsdonk Léonie M. , Kruckeberg Arthur L. , Berden Jan A. , Van Dam Karel
Published: July 2002

Hexokinase II is an enzyme central to glucose metabolism and glucose repression in the yeast
Saccharomyces cerevisiae
. Deletion of
HXK2
, the gene which encodes hexokinase II, dramatically changed the physiology of
S. cerevisiae
. The
hxk2
-null mutant strain displayed fully oxidative growth at high glucose concentrations in early exponential batch cultures, resulting in an initial absence of fermentative products such as ethanol, a postponed and shortened diauxic shift, and higher biomass yields. Several intracellular changes were associated with the deletion of hexokinase II. The
hxk2
mutant had a higher mitochondrial H
+
-ATPase activity and a lower pyruvate decarboxylase activity, which coincided with an intracellular accumulation of pyruvate in the
hxk2
mutant. The concentrations of adenine nucleotides, glucose-6-phosphate, and fructose-6-phosphate are comparable in the wild type and the
hxk2
mutant. In contrast, the concentration of fructose-1,6-bisphosphate, an allosteric activator of pyruvate kinase, is clearly lower in the
hxk2
mutant than in the wild type. The results suggest a redirection of carbon flux in the
hxk2
mutant to the production of biomass as a consequence of reduced glucose repression.
Varied Diazotrophies, Morphologies, and Toxicities of Genetically Similar Isolates of
Cylindrospermopsis raciborskii
(Nostocales, Cyanophyceae) from Northern Australia
Authors: Saker Martin L. , Neilan Brett A.
Published: July 2002

The potentially toxic freshwater cyanobacterium
Cylindrospermopsis raciborskii
has become increasingly prevalent in tropical and temperate water bodies worldwide. This paper investigates the effects of different nitrogen sources (NO
3
−
, NH
4
+
, and omission of a fixed form of nitrogen) on the growth rates, morphologies, and cylindrospermopsin (CYL) concentrations (expressed as a percentage of the freeze-dried weight) of seven
C. raciborskii
isolates obtained from a range of water bodies in northern Australia and grown in batch culture. In general, growth rates were lowest in the absence of a fixed-nitrogen source and highest with NH
4
+
as the nitrogen source. Conversely, the highest concentrations of CYL were recorded in cultures grown in the absence of a fixed-nitrogen source and the lowest were found in cultures supplied with NH
4
+
. Cultures supplied with NO
3
−
were intermediate with respect to both CYL concentration and growth rate. Different nitrogen sources resulted in significant differences in the morphology of
C. raciborskii
trichomes. Most notable were the loss of heterocysts and the tapering of end cells in cultures supplied with NH
4
+
and the statistically significant increase in vegetative cell length (nitrogen depleted < NO
3
−
< NH
4
+
). The morphological changes induced by different nitrogen sources were consistent for all isolates, despite measurable differences in vegetative-cell and heterocyst dimensions among isolates. Such induced morphological variation has implications for
Cylindrospermopsis
taxonomy, given that distinctions between species are based on minor and overlapping differences in cell lengths and widths. The close phylogenetic association among all seven isolates was confirmed by the high level (>99.8%) of similarity of their 16S rRNA gene sequences. Another genetic technique, analysis of the HIP1 octameric-palindrome repeated sequence, showed greater heterogeneity among the isolates and appears to be a useful method for distinguishing among isolates of
C. raciborskii
.
Rapid Identification and Differentiation of the Soft Rot Erwinias by 16S-23S Intergenic Transcribed Spacer-PCR and Restriction Fragment Length Polymorphism Analyses
Authors: Toth I. K. , Avrova A. O. , Hyman L. J.
Published: July 2002

Current identification methods for the soft rot erwinias are both imprecise and time-consuming. We have used the 16S-23S rRNA intergenic transcribed spacer (ITS) to aid in their identification. Analysis by ITS-PCR and ITS-restriction fragment length polymorphism was found to be a simple, precise, and rapid method compared to current molecular and phenotypic techniques. The ITS was amplified from
Erwinia
and other genera using universal PCR primers. After PCR, the banding patterns generated allowed the soft rot erwinias to be differentiated from all other
Erwinia
and non-
Erwinia
species and placed into one of three groups (I to III). Group I comprised all
Erwinia carotovora
subsp.
atroseptica
and subsp.
betavasculorum
isolates. Group II comprised all
E. carotovora
subsp.
carotovora,
subsp.
odorifera
, and subsp.
wasabiae
and
E. cacticida
isolates, and group III comprised all
E. chrysanthemi
isolates. To increase the level of discrimination further, the ITS-PCR products were digested with one of two restriction enzymes. Digestion with
Cfo
I identified
E. carotovora
subsp.
atroseptica
and subsp.
betavasculorum
(group I) and
E. chrysanthemi
(group III) isolates, while digestion with
Rsa
I identified
E. carotovora
subsp.
wasabiae, subsp. carotovora
, and subsp.
odorifera/carotovora
and
E. cacticida
isolates (group II). In the latter case, it was necessary to distinguish
E. carotovora
subsp.
odorifera
and subsp.
carotovora
using the α-methyl glucoside test. Sixty suspected soft rot erwinia isolates from Australia were identified as
E. carotovora
subsp.
atroseptica
,
E. chrysanthemi
,
E. carotovora
subsp.
carotovora
, and non-soft rot species. Ten “atypical”
E. carotovora
subsp.
atroseptica
isolates were identified as
E. carotovora
subsp.
atroseptica
, subsp.
carotovora
, and subsp.
betavasculorum
and non-soft rot species, and two “atypical”
E. carotovora
subsp.
carotovora
isolates were identified as
E. carotovora
subsp.
carotovora
and subsp.
atroseptica
.
Characterization of the Single Superoxide Dismutase of
Staphylococcus xylosus
Authors: Barrière Charlotte , Brückner Reinhold , Talon Régine
Published: July 2002

Staphylococcus xylosus
is a facultative anaerobic bacterium used as a starter culture for fermented meat products. In an attempt to analyze the antioxidant capacities of this organism, the superoxide dismutase (SOD) was characterized.
S. xylosus
contains a single cytoplasmic SOD, which was not inhibited by H
2
O
2
. The SOD activity in crude extracts was completely lost upon metal depletion, but it could be recovered by manganese and very weakly by iron. It is therefore suggested that the
S. xylosus
SOD is a manganese-preferring enzyme. The corresponding gene,
sod
, was isolated from a genomic library of
S. xylosus
DNA and complemented the growth defect of an
Escherichia coli
SOD-deficient mutant. As deduced from the nucleotide sequence,
sod
encodes a protein of 199 amino acids with a molecular mass of 22.5 kDa. Two transcriptional start sites 25 and 120 bp upstream of the
sod
start codon were identified. A terminator-like structure downstream of the gene suggested a monocistronic
sod
mRNA. Regulation of
sod
expression was studied using fusions of the
sod
promoters to a genomic promoterless β-galactosidase gene. The
sod
expression was not affected by manganese and increased slightly with paraquat. It was induced during stationary phase in a complex medium but not in a chemically defined medium. To investigate the physiological role of SOD, a mutant devoid of SOD activity was constructed. Growth experiments showed that
sod
is not essential for aerobic growth in complex medium. However, in chemically defined medium without leucine, isoleucine, and valine, the
sod
mutant hardly grew, in contrast to the wild-type strain. In addition, the mutant was sensitive to hyperbaric oxygen and to paraquat. Therefore,
sod
plays an important role in the protection of
S. xylosus
from oxidative stress.
Lytic and Lysogenic Infection of Diverse
Escherichia coli
and
Shigella
Strains with a Verocytotoxigenic Bacteriophage
Authors: James Chloe E. , Stanley Karen N. , Allison Heather E. , Flint Harry J. , Stewart Colin S. , Sharp Richard J. , Saunders Jon R. , McCarthy Alan J.
Published: July 2002

A verocytotoxigenic bacteriophage isolated from a strain of enterohemorrhagic
Escherichia coli
O157, into which a kanamycin resistance gene (
aph3
) had been inserted to inactivate the verocytotoxin gene (

vt
2

), was used to infect
Enterobacteriaceae
strains. A number of
Shigella
and
E. coli
strains were susceptible to lysogenic infection, and a smooth
E. coli
isolate (O107) was also susceptible to lytic infection. The lysogenized strains included different smooth
E. coli
serotypes of both human and animal origin, indicating that this bacteriophage has a substantial capacity to disseminate verocytotoxin genes. A novel indirect plaque assay utilizing an
E. coli recA441
mutant in which phage-infected cells can enter only the lytic cycle, enabling detection of all infective phage, was developed.
Chimeric
Vitreoscilla
Hemoglobin (VHb) Carrying a Flavoreductase Domain Relieves Nitrosative Stress in
Escherichia coli
: New Insight into the Functional Role of VHb
Authors: Kaur Ramandeep , Pathania Ranjana , Sharma Vishwamitra , Mande Shekhar C. , Dikshit Kanak L.
Published: July 2002

Dimeric hemoglobin (VHb) from the bacterium
Vitreoscilla
sp. strain C1 displays 30 to 53% sequence identity with the heme-binding domain of flavohemoglobins (flavoHbs) and exhibits the presence of potential sites for the interaction with its FAD/NADH reductase partner. The intersubunit contact region of VHb indicates a small interface between two monomers of the homodimer, suggesting that the VHb dimers may dissociate easily. Gel filtration chromatography of VHb exhibited a 25 to 30% monomeric population of VHb, at a low protein concentration (0.05 mg/ml), whereas dimeric VHb remained dominant at a high protein concentration (10 mg/ml). The structural characteristics of VHb suggest that the flavoreductase can also associate and interact with VHb in a manner analogous to flavoHbs and could yield a flavo-VHb complex. To unravel the functional relevance of the VHb-reductase association, the reductase domain of flavoHb from
Ralstonia eutropha
(formerly
Alcaligenes eutrophus
) was genetically engineered to generate a VHb-reductase chimera (VHb-R). The physiological implications of VHb and VHb-R were studied in an
hmp
mutant of
Escherichia coli
, incapable of producing any flavoHb. Cellular respiration the of the
hmp
mutant was instantaneously inhibited in the presence of 10 μM nitric oxide (NO) but remained insensitive to NO inhibition when these cells produced VHb-R. In addition,
E. coli
overproducing VHb-R exhibited NO consumption activity that was two to three times slower in cells overexpressing only VHb and totally undetectable in the control cells. A purified preparation of VHb-R exhibited a three- to fourfold-higher NADH-dependent NO uptake activity than that of VHb alone. Overproduction of VHb-R in the
hmp
mutant of
E. coli
conferred relief from the toxicity of sodium nitroprusside, whereas VHb alone provided only partial benefit under similar condition, suggesting that the association of VHb with reductase improves its capability to relieve the deleterious effect of nitrosative stress. Based on these results, it has been proposed that the unique structural features of VHb may allow it to acquire two functional states in vivo, namely, a single-domain homodimer that may participate in facilitated oxygen transfer or a two-domain heterodimer in association with its partner reductase that may be involved in modulating the cellular response under different environmental conditions. Due to this inherent structural flexibility, it may perform multiple functions in the cellular metabolism of its host. Separation of the oxidoreductase domain from VHb may thus provide a physiological advantage to its host.
Comparison of Soil Bacterial Communities in Rhizospheres of Three Plant Species and the Interspaces in an Arid Grassland
Authors: Kuske Cheryl R. , Ticknor Lawrence O. , Miller Mark E. , Dunbar John M. , Davis Jody A. , Barns Susan M. , Belnap Jayne
Published: July 2002

Soil bacteria are important contributors to primary productivity and nutrient cycling in arid land ecosystems, and their populations may be greatly affected by changes in environmental conditions. In parallel studies, the composition of the total bacterial community and of members of the
Acidobacterium
division were assessed in arid grassland soils using terminal restriction fragment length polymorphism (TRF, also known as T-RFLP) analysis of 16S rRNA genes amplified from soil DNA. Bacterial communities associated with the rhizospheres of the native bunchgrasses
Stipa hymenoides
and
Hilaria jamesii
, the invading annual grass
Bromus tectorum
, and the interspaces colonized by cyanobacterial soil crusts were compared at three depths. When used in a replicated field-scale study, TRF analysis was useful for identifying broad-scale, consistent differences in the bacterial communities in different soil locations, over the natural microscale heterogeneity of the soil. The compositions of the total bacterial community and
Acidobacterium
division in the soil crust interspaces were significantly different from those of the plant rhizospheres. Major differences were also observed in the rhizospheres of the three plant species and were most apparent with analysis of the
Acidobacterium
division. The total bacterial community and the
Acidobacterium
division bacteria were affected by soil depth in both the interspaces and plant rhizospheres. This study provides a baseline for monitoring bacterial community structure and dynamics with changes in plant cover and environmental conditions in the arid grasslands.
Physiological Function of Alcohol Dehydrogenases and Long-Chain (C
30
) Fatty Acids in Alcohol Tolerance of
Thermoanaerobacter ethanolicus
Authors: Burdette D. S. , Jung S.-H. , Shen G.-J. , Hollingsworth R. I. , Zeikus J. G.
Published: July 2002

A mutant strain (39E H8) of
Thermoanaerobacter ethanolicus
that displayed high (8% [vol/vol]) ethanol tolerance for growth was developed and characterized in comparison to the wild-type strain (39E), which lacks alcohol tolerance (<1.5% [vol/vol]). The mutant strain, unlike the wild type, lacked primary alcohol dehydrogenase and was able to increase the percentage of transmembrane fatty acids (i.e., long-chain C
30
fatty acids) in response to increasing levels of ethanol. The data support the hypothesis that primary alcohol dehydrogenase functions primarily in ethanol consumption, whereas secondary alcohol dehydrogenase functions in ethanol production. These results suggest that improved thermophilic ethanol fermentations at high alcohol levels can be developed by altering both cell membrane composition (e.g., increasing transmembrane fatty acids) and the metabolic machinery (e.g., altering primary alcohol dehydrogenase and lactate dehydrogenase activities).
Propionate Formation by
Opitutus terrae
in Pure Culture and in Mixed Culture with a Hydrogenotrophic Methanogen and Implications for Carbon Fluxes in Anoxic Rice Paddy Soil
Authors: Chin Kuk-Jeong , Janssen Peter H.
Published: July 2002

Propionate-forming bacteria seem to be abundant in anoxic rice paddy soil, but biogeochemical investigations show that propionate is not a correspondingly important intermediate in carbon flux in this system. Mixed cultures of
Opitutus terrae
strain PB90-1, a representative propionate-producing bacterium from rice paddy soil, and the hydrogenotrophic
Methanospirillum hungatei
strain SK maintained hydrogen partial pressures similar to those in the soil. The associated shift away from propionate formation observed in these cultures helps to reconcile the disparity between microbiological and biogeochemical studies.
Apoptosis of Primary-Culture Rat Microglial Cells Induced by PathogenicAcanthamoebaspp
Authors: Shin Ho-Joon , Cho Myung-Soo , Kim Hyung-Il , Lee Millina , Park Sun , Sohn Seonghyang , Im Kyung-Il
Published: July 2002

To determine whether trophozoites and lysates of pathogenicAcanthamoebaspp. induce apoptosis in primary-culture microglial cells, transmission electron microscopic (TEM) examinations, assessment of DNA fragmentation by agarose gel electrophoresis, and the TdT-mediated dUTP nick-end labeling assay were performed. When a trophozoite of pathogenicAcanthamoeba culbertsonicame in contact with a microglial cell, the digipodium was observed by TEM. Nuclear chromatin condensation was observed in 10% of microglial cells, while it was not revealed when they were cocultured with weakly pathogenicAcanthamoeba royrebatrophozoites. DNA fragmentation in microglial cells cocultured with theA. culbertsonilysate was detected by electrophoresis, showing DNA ladder formation, whereas it was hardly observed in microglial cells cocultured withA. royreba. DNA fragmentation of microglial cells was also confirmed by flow cytometry analysis. The fluorescence of TdT-stained apoptotic bodies became intensely visible with microglial cells cocultured with theA. culbertsonilysate. In contrast, with microglial cells cocultured with theA. royrebalysate, only a background level of fluorescence of TdT-stained apoptotic bodies was detected. These results suggest that some rat microglial cells cocultured with pathogenicA. culbertsoniundergo cytopathic changes which show the characteristics of the apoptotic process, such as nuclear condensation and DNA fragmentation.
Prevalence of
Chlamydia pneumoniae
and
Mycoplasma pneumoniae
Immunoglobulin G and A Antibodies in a Healthy Finnish Population as Analyzed by Quantitative Enzyme Immunoassays
Authors: Tuuminen Tamara , Varjo Sirpa , Ingman Heidi , Weber Theodor , Oksi Jarmo , Viljanen Matti
Published: July 2002

Chlamydia pneumoniae
and
Mycoplasma pneumoniae
immunoglobulin G (IgG) and IgA antibody seroprevalence rates and antibody levels related to age and gender were studied. The samples (
n
= 742) were collected during a nonepidemic period and analyzed by quantitative enzyme immunoassays (EIAs). Seroprevalence to
C. pneumoniae
was found to increase sharply in young children, and in the 15- to 19-year-old group it reached levels as high as 70 and 60% for IgG and IgA, respectively. After adolescence, seroprevalence showed a transient decrease and then continued to increase, although less dramatically than in early childhood. In the elderly the seroprevalence of IgG antibodies reached 75 and 100% in women and men, respectively. The corresponding rates of IgA antibodies were 73 and 100%. When a randomly selected subgroup of samples (
n
= 66) was analyzed in parallel by a microimmunofluorescence test and an EIA for
C. pneumoniae
IgA antibodies, similar seroprevalence rates were obtained (36 versus 35%). Seroprevalence to
M. pneumoniae
was already found to increase very sharply in 2- to 4-year-old children, reaching 16% for IgG and 8% for IgA. Seroprevalence to
M. pneumoniae
also continued to increase in adolescence, but in contrast to that to
C. pneumoniae
, the increase leveled off at about 40 to 50% in adulthood. In subjects aged over 65 years, prevalence did not exceed 60% for IgG or 35% for IgA. The seroprevalence patterns as well as the medians and variations of levels of
C. pneumoniae
and
M. pneumoniae
IgG antibodies were similar to those of corresponding IgA antibodies. Compared to IgG antibodies, IgA antibodies do not seem to be of additional value in the diagnosis of infections caused by these pathogens when single serum specimens are studied.
Human Dengue Antibodies against Structural and Nonstructural Proteins
Authors: Valdés Katia , Alvarez Mayling , Pupo Maritza , Vázquez Susana , Rodríguez Rayner , Guzmán María G.
Published: July 2002

Antibodies against dengue virus type 2 and 4 proteins in acute-phase sera of 10 primary and 10 secondary dengue fever and dengue hemorrhagic fever patients were studied by Western blotting. In the first group the immune response was barely detectable, while in the second group more proteins were detected, with a very strong reaction. Anti-NS1 and -NS3 antibodies were detected mainly in secondary cases. Anti-E, -NS3, and -NS5 antibodies were detected in a high number of cases. The possibility of implementing early diagnostic assays for antigen detection is suggested.
Genomic Characterization of Porcine Rotaviruses in Italy
Authors: Martella Vito , Pratelli Annamaria , Greco Grazia , Tempesta Maria , Ferrari Maura , Losio Marina Nadia , Buonavoglia Canio
Published: July 2002

A total of 23 rotavirus strains isolated from pigs were analyzed. Twenty strains had been isolated from diarrheic piglets from an outbreak that occurred in northern Italy in 1983. Three strains had been isolated in 1984 from swine herds located in distinct areas of northern Italy. All 23 strains were characterized as type G6P[5] by PCR. The isolation from piglets of rotaviruses displaying typical bovine G- and P-type specificities points out the high frequency of rotavirus transmission between cattle and pigs.
Validation of a Modified Commercial Enzyme-Linked Immunoassay for Detection of Human Immunodeficiency Virus Type 1 Immunoglobulin G Antibodies in Saliva
Authors: Chohan Bhavna H. , Lavreys Ludo , Mandaliya Kishorchandra N. , Kreiss Joan K. , Bwayo Job J. , Ndinya-Achola Jeckoniah O. , Martin Harold L.
Published: July 2002

This study was performed to evaluate the performance of a saliva collection device (OmniSal) and an enzyme-linked immunoassay (EIA) designed for use on serum samples (Detect HIV1/2) to detect human immunodeficiency virus type 1 (HIV-1) antibodies in the saliva of high-risk women in Mombasa, Kenya. The results of the saliva assay were compared to a “gold standard” of a double-EIA testing algorithm performed on serum. Individuals were considered HIV-1 seropositive if their serum tested positive for antibodies to HIV-1 by two different EIAs. The commercial serum-based EIA was modified to test the saliva samples by altering the dilution and lowering the cutoff point of the assay. Using the saliva sample, the EIA correctly identified 102 of the 103 seropositive individuals, yielding a sensitivity of 99% (95% confidence interval [CI], 94 to 100%), and 96 of the 96 seronegative individuals, yielding a specificity of 100% (95% CI, 95 to 100%). In this high-risk population, the positive predictive value of the assay was 100% and the negative predictive value was 99%. We conclude that HIV-1 antibody testing of saliva samples collected with this device and tested by this EIA is of sufficient sensitivity and specificity to make this protocol useful in epidemiological studies.
Late Hematogenous Infection of Subcutaneous Implants in Rats
Authors: Gottenbos B. , Klatter F. , Van Der Mei H. C. , Busscher H. J. , Nieuwenhuis P.
Published: July 2002

Late biomaterial-centered infection is a major complication associated with the use of biomaterial implants. In this study biomaterials that had been implanted subcutaneously in rats were hematogenously challenged with bacteria 4 weeks after implantation. Bacteria were spread either by intravenous injection or by stimulation of bacterial translocation. It was found that none of the biomaterials was infected by hematogenous spread, whereas 5% of the implants were infected by perioperative contamination. We conclude that late hematogenous infection of subcutaneous biomaterials does not occur in the rat. For humans as well, there are growing doubts whether implants actually become infected through hematogenous routes; it is thought that late infections may be caused by delayed appearance of perioperatively introduced bacteria.
Multiplex Assay for Detection of Strain-Specific Antibodies against the Two Variable Regions of the G Protein of Respiratory Syncytial Virus
Authors: Jones Les P. , Zheng Hao-Qiang , Karron Ruth A. , Peret Teresa C. T. , Tsou Cecelia , Anderson Larry J.
Published: July 2002

The role of strain differences in respiratory syncytial virus (RSV) disease has not been clearly defined. To investigate the possibility that strain differences contribute to susceptibility to repeat infections, we developed assays to detect antibodies to the two variable regions of the RSV G protein by cloning and expressing the internal variable region at amino acids (aa) 60 to 172 (g1) and the carboxy-terminal variable region at aa 193 to the carboxy terminus (g2) from different genotypes of RSV. The purified proteins were covalently linked to beads with different proportions of red and orange fluorescent dyes and reacted against serum specimens. Antibody reacting against the differently colored beads, and thus against different G polypeptides, was detected by use of flow cytometry and the Luminex system. This assay system detected group- and, to some extent, genotype-specific responses to RSV infection and can be used to investigate the role of strain differences in RSV disease.
Assignment of Additional Anticapsular Antibody Concentrations to the
Neisseria meningitidis
Group A, C, Y, and W-135 Meningococcal Standard Reference Serum CDC1992
Authors: Elie Cheryl M. , Holder Patricia K. , Romero-Steiner Sandra , Carlone George M.
Published: July 2002

We assigned additional enzyme-linked immunosorbent assay antibody concentrations (immunoglobulin G [IgG], IgM, and IgA, and total) to the
Neisseria meningitidis
standard reference serum CDC1992 for groups Y and W-135 to 12 Centers for Disease Control and Prevention quality control sera. These assignments will supplement previous assignments and will aid in the evaluation of present and developing vaccines.
Antifungal Susceptibility Testing: Practical Aspects and Current Challenges
Authors: Rex John H. , Pfaller Michael A. , Walsh Thomas J. , Chaturvedi Vishnu , Espinel-Ingroff Ana , Ghannoum Mahmoud A. , Gosey Linda L. , Odds Frank C. , Rinaldi Michael G. , Sheehan Daniel J. , Warnock David W.
Published: July 2002

Development of standardized antifungal susceptibility testing methods has been the focus of intensive research for the last 15 years. Reference methods for yeasts (NCCLS M27-A) and molds (M38-P) are now available. The development of these methods provides researchers not only with standardized methods for testing but also with an understanding of the variables that affect interlaboratory reproducibility. With this knowledge, we have now moved into the phase of (i) demonstrating the clinical value (or lack thereof) of standardized methods, (ii) developing modifications to these reference methods that address specific problems, and (iii) developing reliable commercial test kits. Clinically relevant testing is now available for selected fungi and drugs: Candida spp. against fluconazole, itraconazole, flucytosine, and (perhaps) amphotericin B; Cryptococcus neoformans against (perhaps) fluconazole and amphotericin B; and Aspergillus spp. against (perhaps) itraconazole. Expanding the range of useful testing procedures is the current focus of research in this area.
NO Contributes to Proliferative Suppression in a Murine Model of Filariasis
Authors: O'Connor Richard A. , Jenson Jessica S. , Devaney Eileen
Published: July 2002

Infection of BALB/c mice with microfilariae (mf) ofBrugia pahangileads to the suppression of antigen (Ag)-specific proliferative responses in the spleen. The proliferative defect is dependent on inducible nitric oxide synthase (iNOS) activity, since inhibition of iNOS with eitherl-N-monomethyl arginine (l-NMMA) or aminoguanidine reversed defective proliferation. Splenocytes from mf-infected animals produce high levels of gamma interferon (IFN-γ) upon in vitro restimulation with Ag, and experiments in IFN-γ receptor-deficient (IFN-γR−/−) mice demonstrated that signaling via the IFN-γR is essential in the induction of NO production and subsequent proliferative suppression. Restimulation of splenocytes from mf-infected animals with an extract ofAcanthocheilonema viteae, a related filarial worm which lacks endosymbiotic bacteria, also resulted in NO production and proliferative suppression, demonstrating that lipopolysaccharide of bacterial origin is not essential to the induction of iNOS activity. These results extend previous observations that infection with different life cycle stages ofBrugialeads to the development of differentially polarized immune responses and demonstrate one method by which these differences may exert their effects on the proliferative potential of cells from infected animals.
Role of Neutrophil Leukocytes in Cutaneous Infection Caused byStaphylococcus aureus
Authors: Mölne Lena , Verdrengh Margareta , Tarkowski Andrzej
Published: July 2002

Despite the high prevalence of cutaneous infections, little is known about the role of host immune responsiveness duringStaphylococcus aureusdermatitis. We have recently described a murine model of infectious dermatitis induced by superantigen-producingS. aureus. To assess the role of neutrophils in staphylococcal dermatitis, mice were given granulocyte-depleting monoclonal antibody prior to and on several occasions following intracutaneous inoculation with staphylococci. The granulocyte-depleted mice that had been intradermally inoculated withS. aureusdeveloped crusted ulcerations which tended not to heal, whereas animals injected with control monoclonal antibody displayed only minor and transient skin lesions. The finding of severe ulcerations in neutropenic mice correlated with a significantly higher burden of bacteria in the blood and skin during the early phase of the infection. Importantly, while mice with an intact granulocyte population showed only limited skin infection, bacteremia occurred in the great majority of the neutrophil-depleted animals. As a consequence, the latter individuals exhibited significantly increased levels of the proinflammatory cytokine interleukin-6 and specific antibodies to staphylococcal cell wall components and toxic shock syndrome toxin-1 in the serum. Our data point to a crucial protective role of granulocytes inS. aureusdermatitis.
Detection and Characterization of Autoagglutination Activity by
Campylobacter jejuni
Authors: Misawa Naoaki , Blaser Martin J.
Published: July 2002

In several gram-negative bacterial pathogens, autoagglutination (AAG) activity is a marker for interaction with host cells and virulence.
Campylobacter jejuni
strains also show AAG, but this property varies considerably among strains. To examine the characteristics of
C. jejuni
AAG, we developed a quantitative in vitro assay. For strain 81-176, which shows high AAG, activity was optimal for cells grown for ≤24 h, was independent of growth temperature, and was best measured for cells suspended in phosphate-buffered saline at 25°C for 24 h. AAG activity was heat labile and was abolished by pronase or acid-glycine (pH 2.2) treatment but not by lipase, DNase, or sodium metaperiodate. Strain 4182 has low AAG activity, but extraction with water increased AAG, suggesting the loss of an inhibitor. Strain 6960 has weak AAG with no effect due to water extraction. Our study with clinical isolates suggests that
C. jejuni
strains may be grouped into three AAG phenotypes. A variant derived from strain 81116 that is flagellate but immotile showed the strong AAG exhibited by the parent strain, suggesting that motility per se is not necessary for the AAG activity. AAG correlated with both bacterial hydrophobicity and adherence to INT407 cells. Mutants which lack flagella (
flaA
,
flaB
, and
flbA
) or common cell surface antigen (
peb1A
) were constructed in strain 81-176 by natural transformation-mediated allelic exchange. Both AAG activity and bacterial hydrophobicity were abolished in the aflagellate mutants but not the
peb1A
mutant. In total, these findings indicate that
C. jejuni
AAG is highly associated with flagellar expression.
Genetic Diversity of the Capsular Polysaccharide C Biosynthesis Region of
Bacteroides fragilis
Authors: Comstock Laurie E. , Pantosti Annalisa , Kasper Dennis L.
Published: July 2002

A genetic approach was used to assess the heterogeneity of the capsular polysaccharide C (PS C) biosynthesis locus of
Bacteroides fragilis
and to determine whether distinct loci contain genes whose products are likely to be involved in conferring charged groups that enable the
B. fragilis
capsular polysaccharides to induce abscesses. A collection of 50
B. fragilis
strains was examined. PCR analysis demonstrated that the genes flanking the PS C biosynthesis region are conserved, whereas the genes within the loci are heterogeneous. Only
cfiA
+
B. fragilis
strains, which represent 3% of the clinical isolates of
B. fragilis
, displayed heterogeneity in the regions flanking the polysaccharide biosynthesis genes. Primers were designed in the conserved regions upstream and downstream of the PS C locus and were used to amplify the region from 45 of the 50
B. fragilis
strains studied. Fourteen PS C genetic loci could be differentiated by a combination of PCR and extended PCR. These loci ranged in size from 14 to 26 kb. Hybridization analysis with genes from the PS C loci of strains 9343 and 638R revealed that the majority of strains contain homologs of
wcgC
(
N
-acetylmannosamine dehydrogenase),
wcfF
(putative dehydrogenase), and
wcgP
(putative aminotransferase). The data suggest that the synthesis of polysaccharides that have zwitterionic characteristics rendering them able to induce abscesses is common in
B. fragilis
.
The
Cryptococcus neoformans
Gene
DHA1
Encodes an Antigen That Elicits a Delayed-Type Hypersensitivity Reaction in Immune Mice
Authors: Mandel M. Alejandra , Grace Greg G. , Orsborn Kris I. , Schafer Fredda , Murphy Juneann W. , Orbach Marc J. , Galgiani John N.
Published: July 2002

When mice are vaccinated with a culture filtrate from
Cryptococcus neoformans
(CneF), they mount a protective cell-mediated immune response as detected by dermal delayed-type hypersensitivity (DTH) to CneF. We have identified a gene (
DHA1
) whose product accounts at least in part for the DTH reactivity. Using an acapsular mutant (Cap-67) of
C. neoformans
strain B3501, we prepared a culture filtrate (CneF-Cap67) similar to that used for preparing the commonly used skin test antigen made with
C. neoformans
184A (CneF-184A). CneF-Cap67 elicited DTH in mice immunized with CneF-184A. Deglycosylation of CneF-Cap67 did not diminish its DTH activity. Furthermore, size separation by either chromatography or differential centrifugation identified the major DTH activity of CneF-Cap67 to be present in fractions that contained proteins of approximately 19 to 20 kDa. Using N-terminal and internal amino acid sequences derived from the 20-kDa band, oligonucleotide primers were designed, two of which produced a 776-bp amplimer by reverse transcription-PCR (RT-PCR) using RNA from Cap-67 to prepare cDNA for the template. The amplimer was used as a probe to isolate clones containing the full-length
DHA1
gene from a phage genomic library prepared from strain B3501. The full-length cDNA was obtained by 5′ rapid amplification of cDNA ends and RT-PCR. Analysis of
DHA1
revealed a similarity between the deduced open reading frame and that of a developmentally regulated gene from
Lentinus edodes
(shiitake mushroom) associated with fruiting-body formation. Also, the gene product contained several amino acid sequences identical to those determined biochemically from the purified 20-kDa peptide encoded by
DHA1
. Recombinant DHA1 protein expressed in
Escherichia coli
was shown to elicit DTH reactions similar to those elicited by CneF-Cap67 in mice immunized against
C. neoformans
. Thus,
DHA1
is the first gene to be cloned from
C. neoformans
whose product has been shown to possess immunologic activity.
Preparation and Preclinical Evaluation of a Novel Liposomal Complete-Core Lipopolysaccharide Vaccine
Authors: Bennett-Guerrero Elliott , McIntosh Thomas J. , Barclay G. Robin , Snyder D. Scott , Gibbs Richard J. , Mythen Michael G. , Poxton Ian R.
Published: July 2002

Our objective is to develop a prophylactic vaccine strategy that can be evaluated for surgical and other high-risk hospitalized patients. In this paper, we describe the preparation and preclinical evaluation of a liposomal complete-core lipopolysaccharide (LPS) vaccine that is nontoxic and broadly antigenic. Complete-core (Ra-chemotype) LPSs were isolated from four gram-negative bacterial strains (
Escherichia coli
K-12,
E. coli
R1,
Pseudomonas aeruginosa
PAC608, and
Bacteroides fragilis
), mixed together to form a cocktail of complete-core LPSs, and then incorporated into multilamellar liposomes consisting of dimyristoyl phosphatidyl choline, dimyristoyl phosphatidylglycerol, and cholesterol in a 4:1:4 molar ratio. The endotoxic activities of these LPS-containing liposomes were less than 0.1% of the endotoxicities of the original free LPSs as measured by the
Limulus
amoebocyte lysate assay. In vivo administration of liposomal complete-core LPS mixed with Al(OH)
3
to rabbits resulted in no pyrogenicity or overt toxicity over a 7-day period. In immunoblots, sera from rabbits following active immunization elicited cross-reactive antibodies to a large panel of rough and smooth LPSs from numerous clinically relevant gram-negative bacteria, including
E. coli
(serotypes O1, O4, O6, O8, O12, O15, O18, O75, O86, O157, and O111),
P. aeruginosa
(Fisher-Devlin serotypes 1, 2, and 3, which correspond to International Antigenic Typing Scheme types 6, 11, and 2, respectively),
Klebsiella pneumoniae
(serotypes O1, O2ab, and O3),
B. fragilis
, and
Bacteroides vulgatus
. Active immunization of mice with liposomal complete-core LPS provided protection against a lethal challenge with
E. coli
O18 LPS. The vaccine tested was nontoxic, nonpyrogenic, and immunogenic against a wide variety of pathogens found in clinical settings.
Distribution of Open Reading Frames of Plasticity Region of Strain J99 in
Helicobacter pylori
Strains Isolated from Gastric Carcinoma and Gastritis Patients in Costa Rica
Authors: Occhialini Alessandra , Marais Armelle , Alm Richard , Garcia Fernando , Sierra Rafaela , Mégraud Francis
Published: July 2002

The plasticity region of
Helicobacter pylori
strain J99 is a large chromosomal segment containing 33 strain-specific open reading frames (ORFs) with characteristics of a pathogenicity island. To study the diversity of the plasticity region, 22 probes corresponding to 20 ORFs inside the plasticity region and two ORFs on its boundaries were hybridized to genomic DNA isolated from clinical strains of
H. pylori
from patients with gastritis or gastric adenocarcinoma. Highly variable hybridization patterns were observed. The majority of the clinical strains presented a hybridization profile similar to that of J99; thus, these ORFs are not J99 strain specific. No association was found between a particular hybridization pattern and the clinical origin of the strain. Nevertheless, two single ORFs (JHP940 and JHP947) were more likely to be found in gastric cancer strains. They may be new pathogenicity markers. An in vitro expression study of these ORFs was also performed for the J99 strain, under different conditions. Thirteen ORFs were consistently expressed, six were consistently shut off, and three were expressed differentially. Most of the constitutionally expressed genes were located on the 3′ part of the plasticity region. Our results show that the plasticity region, rather than being considered a pathogenicity island per se, should be considered a genomic island, which represents a large fragment of foreign DNA integrated into the genome and not necessarily implicated in the pathogenic capacity of the strain.
Antigenic Structure of Outer Membrane Protein E of
Moraxella catarrhalis
and Construction and Characterization of Mutants
Authors: Murphy Timothy F. , Brauer Aimee L. , Yuskiw Norine , Hiltke Thomas J.
Published: July 2002

Outer membrane protein E (OMP E) is a 50-kDa protein of
Moraxella catarrhalis
which possesses several characteristics indicating that the protein will be an effective vaccine antigen. To study the antigenic structure of OMP E, eight monoclonal antibodies were developed and characterized. Three of the antibodies recognized epitopes which are present on the bacterial surface. Fusion peptides corresponding to overlapping regions of OMP E were constructed, and immunoblot assays were performed to localize the areas of the molecule bound by the monoclonal antibodies. These studies identified a surface-exposed epitope in the region of amino acids 80 through 180. To further study the protein, two mutants which lack OMP E were constructed. In bactericidal assays, the mutants were more readily killed by normal human serum compared to the isogenic parent strains. These results indicate that OMP E is involved in the expression of serum resistance of
M. catarrhalis
.
Modulation of Innate Cytokine Responses by Products ofHelicobacter pylori
Authors: Meyer Frank , Wilson Keith T. , James Stephen P.
Published: July 2002

The gastric inflammatory and immune response inHelicobacter pyloriinfection may be due to the effect of differentH. pyloriproducts on innate immune mechanisms. The aim of this study was to determine whether bacterial components could modulate cytokine production in vitro and thus contribute to Th1 polarization of the gastric immune response observed in vivo. The effect ofH. pylorirecombinant urease, bacterial lysate, intact bacteria, and bacterial DNA on proliferation and cytokine production by peripheral blood mononuclear cells (PBMCs) fromH. pylori-negative donors was examined as a model for innate cytokine responses. Each of the differentH. pyloripreparations induced gamma interferon (IFN-γ) and interleukin-12p40 (IL-12p40), but not IL-2 or IL-5, production, and all butH. pyloriDNA stimulated release of IL-10. Addition of anti-IL-12 antibody to cultures partially inhibited IFN-γ production. In addition, each bacterial product inhibited mitogen-stimulated IL-2 production by PBMCs and Jurkat T cells. The inhibitory effect of bacterial products on IL-2 production correlated with inhibition of mitogen-stimulated lymphocyte proliferation, although urease inhibited IL-2 production without inhibiting proliferation, suggesting that inhibition of IL-2 production alone is not sufficient to inhibit lymphocyte proliferation. The results of these studies demonstrate that Th1 polarization of the gastric immune response may be due in part to the direct effects of multiple differentH. pyloricomponents that enhance IFN-γ and IL-12 production while inhibiting both IL-2 production and cell proliferation that may be necessary for Th2 responses.