bluuebunny/crossref_metadata_2025 huggingface dataset:
Stratified Communities of Active Archaea in Deep Marine Subsurface Sediments
Authors: Sørensen Ketil B. , Teske Andreas
Published: July 2006

Archaeal 16S rRNA was extracted from samples of deep marine subsurface sediments from Peru Margin site 1227, Ocean Drilling Program leg 201. The amounts of archaeal 16S rRNA in each extract were quantified by serial dilution and reverse transcription (RT)-PCR. The results indicated a 1,000-fold variation in rRNA content with depth in the sediment, with the highest concentrations found near the sediment surface and in the sulfate-methane transition zone (SMTZ). The phylogenetic composition of the active archaeal population revealed by cloning and sequencing of RT-PCR products changed with depth. Several phylotypes affiliated with marine benthic group B (MBGB) dominated clone libraries from the upper part of the SMTZ and were detected only in this layer. Members of the miscellaneous crenarchaeotal group (MCG) dominated clone libraries from the other layers. These results demonstrate that archaeal communities change in activity and community composition over short distances in geochemically distinct zones of deep subseafloor sediments and that these changes are traceable in the rRNA pool. It was shown for the first time that members of both the MCG and MBGB
Archaea
are more active in the SMTZ than in layers above and below. This indicates that they benefit either directly or indirectly from the anaerobic oxidation of methane. They also appear to be ecophysiologically flexible, as they have been retrieved from a wide range of marine sediments of various geochemical properties.
Effectiveness of Enterobacterial Repetitive Intergenic Consensus PCR and Random Amplified Polymorphic DNA Fingerprinting for
Helicobacter pylori
Strain Differentiation
Authors: Finger S. Alison , Velapatiño Billie , Kosek Margaret , Santivañez Livia , Dailidiene Daiva , Quino Willi , Balqui Jacqueline , Herrera Phabiola , Berg Douglas E. , Gilman Robert H.
Published: July 2006

We compared the robustness and discriminatory power of the enterobacterial repetitive intergenic consensus (ERIC) and random amplified polymorphic DNA (RAPD) fingerprinting methods for detecting cases of mixed
Helicobacter pylori
infection in Peruvian shantytown residents.
H. pylori
isolates from 63 participants were cultured, and five single colonies and a pool of additional colonies from each participant were analyzed by ERIC-PCR and by RAPD tests with four 10-nucleotide primers (one primer per reaction). There was 94% agreement between the ERIC and RAPD profiles in classifying sets of isolates as uniform versus closely related but not identical versus probably unrelated, indicating a high kappa statistic of 0.8942. Subtle differences in related ERIC or RAPD patterns likely reflect gene transfer between strains, recombination, and/or mutation, whereas markedly different patterns reflect infection by unrelated strains. At least half of infected shantytown residents seemed to carry more than one
H. pylori
strain, although in 19 of 31 persons, the strains were closely related. Three RAPD tests, each with a different primer, were needed to achieve the sensitivity of one ERIC test. ERIC-PCR constitutes a resource- and time-efficient method for
H. pylori
strain differentiation.
Response of
Lactobacillus helveticus
PR4 to Heat Stress during Propagation in Cheese Whey with a Gradient of Decreasing Temperatures
Authors: Di Cagno Raffaella , De Angelis Maria , Limitone Antonio , Fox Patrick F. , Gobbetti Marco
Published: July 2006

The heat stress response was studied in
Lactobacillus helveticus
PR4 during propagation in cheese whey with a gradient of naturally decreasing temperature (55 to 20°C). Growth under a gradient of decreasing temperature was compared to growth at a constant temperature of 42°C. Proteinase, peptidase, and acidification activities of
L. helveticus
PR4 were found to be higher in cells harvested when 40°C was reached by a gradient of decreasing temperature than in cells grown at constant temperature of 42°C. When cells grown under a temperature gradient were harvested after an initial exposure of 35 min to 55°C followed by decreases in temperature to 40 (3 h), 30 (5 h 30 min), or 20°C (13 h 30 min) and were then compared with cells grown for the same time at a constant temperature of 42°C, a frequently transient induction of the levels of expression of 48 proteins was found by two-dimensional electrophoresis analysis. Expression of most of these proteins increased following cooling from 55 to 40°C (3 h). Sixteen of these proteins were subjected to N-terminal and matrix-assisted laser desorption ionization-time of flight mass spectrometry analyses. They were identified as stress proteins (e.g., DnaK and GroEL), glycolysis-related machinery (e.g., enolase and glyceraldehyde-3-phosphate dehydrogenase), and other regulatory proteins or factors (e.g., DNA-binding protein II and ATP-dependent protease). Most of these proteins have been found to play a role in the mechanisms of heat stress adaptation in other bacteria.
Microsporidian Species Known To Infect Humans Are Present in Aquatic Birds: Implications for Transmission via Water?
Authors: Slodkowicz-Kowalska Anna , Graczyk Thaddeus K. , Tamang Leena , Jedrzejewski Szymon , Nowosad Andrzej , Zduniak Piotr , Solarczyk Piotr , Girouard Autumn S. , Majewska Anna C.
Published: July 2006

Human microsporidiosis, a serious disease of immunocompetent and immunosuppressed people, can be due to zoonotic and environmental transmission of microsporidian spores. A survey utilizing conventional and molecular techniques for examining feces from 570 free-ranging, captive, and livestock birds demonstrated that 21 animals shed microsporidian spores of species known to infect humans, including
Encephalitozoon hellem
(20 birds; 3.5%) and
Encephalitozoon intestinalis
(1 bird; 0.2%). Of 11 avian species that shed
E. hellem
and
E. intestinalis
, 8 were aquatic birds (i.e., common waterfowl). The prevalence of microsporidian infections in waterfowl (8.6%) was significantly higher than the prevalence of microsporidian infections in other birds (1.1%) (
P
< 0.03); waterfowl fecal droppings contained significantly more spores (mean, 3.6 × 10
5
spores/g) than nonaquatic bird droppings contained (mean, 4.4 × 10
4
spores/g) (
P
< 0.003); and the presence of microsporidian spores of species known to infect humans in fecal samples was statistically associated with the aquatic status of the avian host (
P
< 0.001). We demonstrated that a single visit of a waterfowl flock can introduce into the surface water approximately 9.1 × 10
8
microsporidian spores of species known to infect humans. Our findings demonstrate that waterborne microsporidian spores of species that infect people can originate from common waterfowl, which usually occur in large numbers and have unlimited access to surface waters, including waters used for production of drinking water.
Flow Cytometric Testing of Green Fluorescent Protein-TaggedLactobacillus rhamnosusGG for Response to Defensins
Authors: De Keersmaecker Sigrid C. J. , Braeken Kristien , Verhoeven Tine L. A. , Perea Vélez Mónica , Lebeer Sarah , Vanderleyden Jos , Hols Pascal
Published: July 2006

Lactobacillus rhamnosusGG is of general interest as a probiotic. AlthoughL. rhamnosusGG is often used in clinical trials, there are few genetic tools to further determine its mode of action or to develop it as a vehicle for heterologous gene expression in therapy. Therefore, we developed a reproducible, efficient electroporation procedure forL. rhamnosusGG. The best transformation efficiency obtained was 104transformants per μg of DNA. We validated this protocol by taggingL. rhamnosusGG with green fluorescent protein (GFP) using the nisin-controlled expression (NICE) system. Parameters for overexpression were optimized, which allowed expression ofgfpinL. rhamnosusGG upon induction with nisin. The GFP+strain can be used to monitor the survival and behavior ofL. rhamnosusGG in vivo. Moreover, implementation of the NICE system as a gene expression switch inL. rhamnosusGG opens up possibilities for improving and expanding the performance of this strain. The GFP-labeled strain was used to demonstrate thatL. rhamnosusGG is sensitive to human beta-defensin-2 but not to human beta-defensin-1.
Enhanced Survival of GroESL-Overproducing
Lactobacillus paracasei
NFBC 338 under Stressful Conditions Induced by Drying
Authors: Corcoran B. M. , Ross R. P. , Fitzgerald G. F. , Dockery P. , Stanton C.
Published: July 2006

GroESL-overproducing
Lactobacillus paracasei
NFBC 338 was dried, and its viability was compared with that of controls. Spray- and freeze-dried cultures overproducing GroESL exhibited ∼10-fold and 2-fold better survival, respectively, demonstrating the importance of GroESL in stress tolerance, which can be exploited to enhance the technological performance of sensitive probiotic cultures.
Soft-Agar-Coated Filter Method for Early Detection of Viable and Thermostable Direct Hemolysin (TDH)- or TDH-Related Hemolysin-Producing
Vibrio parahaemolyticus
in Seafood
Authors: Hayashi Sachiko , Okura Masatoshi , Osawa Ro
Published: July 2006

A novel method for detecting viable and thermostable direct hemolysin (TDH)-producing or TDH-related hemolysin (TRH)-producing
Vibrio parahaemolyticus
in seafood was developed. The method involved (i) enrichment culture, selective for viable, motile cells penetrating a soft-agar-coated filter paper, and (ii) a multiplex PCR assay targeting both the TDH gene (
tdh
) and TRH gene (
trh
) following DNase pretreatment on the test culture to eradicate any incidental DNAs that might have been released from dead cells of
tdh
- or
trh
-positive (
tdh
+
trh
+
) strains and penetrated the agar-coated filter. A set of preliminary laboratory tests performed on 190 ml of enrichment culture that had been inoculated simultaneously with ca. 100 viable cells of a strain of
tdh
+
trh
+
V. parahaemolyticus
and dense populations of a viable strain of
tdh-
and
trh-
negative
V. parahaemolyticus
or
Vibrio alginolyticus
indicated that the method detected the presence of viable
tdh
+
trh
+
strains. Another set of preliminary tests on 190 ml of enrichment culture that had been initially inoculated with a large number of dead cells of the
tdh
+
trh
+
strain together with dense populations of the
tdh-
and
trh-
negative strains confirmed that the method did not yield any false-positive results. Subsequent quasi-field tests using various seafood samples (ca. 20 g), each of which was experimentally contaminated with either or both hemolysin-producing strains at an initial density of ca. 5 to 10 viable cells per gram, demonstrated that contamination could be detected within 2 working days.
Microarray-Based Analysis of Subnanogram Quantities of Microbial Community DNAs by Using Whole-Community Genome Amplification
Authors: Wu Liyou , Liu Xueduan , Schadt Christopher W. , Zhou Jizhong
Published: July 2006

Microarray technology provides the opportunity to identify thousands of microbial genes or populations simultaneously, but low microbial biomass often prevents application of this technology to many natural microbial communities. We developed a whole-community genome amplification-assisted microarray detection approach based on multiple displacement amplification. The representativeness of amplification was evaluated using several types of microarrays and quantitative indexes. Representative detection of individual genes or genomes was obtained with 1 to 100 ng DNA from individual or mixed genomes, in equal or unequal abundance, and with 1 to 500 ng community DNAs from groundwater. Lower concentrations of DNA (as low as 10 fg) could be detected, but the lower template concentrations affected the representativeness of amplification. Robust quantitative detection was also observed by significant linear relationships between signal intensities and initial DNA concentrations ranging from (i) 0.04 to 125 ng (
r
2
= 0.65 to 0.99) for DNA from pure cultures as detected by whole-genome open reading frame arrays, (ii) 0.1 to 1,000 ng (
r
2
= 0.91) for genomic DNA using community genome arrays, and (iii) 0.01 to 250 ng (
r
2
= 0.96 to 0.98) for community DNAs from ethanol-amended groundwater using 50-mer functional gene arrays. This method allowed us to investigate the oligotrophic microbial communities in groundwater contaminated with uranium and other metals. The results indicated that microorganisms containing genes involved in contaminant degradation and immobilization are present in these communities, that their spatial distribution is heterogeneous, and that microbial diversity is greatly reduced in the highly contaminated environment.
Expression of the 1-Aminocyclopropane-1-Carboxylic Acid Deaminase Gene Requires Symbiotic Nitrogen-Fixing Regulator GenenifA2inMesorhizobium lotiMAFF303099
Authors: Nukui Noriyuki , Minamisawa Kiwamu , Ayabe Shin-Ichi , Aoki Toshio
Published: July 2006

Many soil bacteria contain 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, which degrades ACC, a precursor of the phytohormone ethylene. In order to examine the regulation of theacdSgene encoding ACC deaminase inMesorhizobium lotiMAFF303099 during symbiosis with the host legumeLotus japonicus, we introduced the β-glucuronidase (GUS) gene intoacdSso that GUS was expressed under control of theacdSpromoter, and we also generated disruption mutants with mutations in a nitrogen fixation regulator gene,nifA. The histochemical GUS assay showed that there was exclusive expression ofacdSin mature root nodules. Two homologousnifAgenes, mll5857 and mll5837, were found in the symbiosis island ofM. lotiand were designatednifA1andnifA2, respectively. Quantitative reverse transcription-PCR demonstrated thatnifA2disruption resulted in considerably diminished expression ofacdS,nifH, andnifA1in bacteroid cells. In contrast,nifA1disruption slightly enhanced expression of theacdStranscripts and suppressednifHto some extent. These results indicate that theacdSgene and other symbiotic genes are positively regulated by the NifA2 protein, but not by the NifA1 protein, inM. loti. The mode of gene expression suggests thatM. loti acdSparticipates in the establishment and/or maintenance of mature nodules by interfering with the production of ethylene, which induces negative regulation of nodulation.
Molecular and Insecticidal Characterization of a Cry1I Protein Toxic to Insects of the Families Noctuidae, Tortricidae, Plutellidae, and Chrysomelidae
Authors: Ruiz de Escudero Iñigo , Estela Anna , Porcar Manuel , Martínez Clara , Oguiza José A. , Escriche Baltasar , Ferré Juan , Caballero Primitivo
Published: July 2006

The most notable characteristic of
Bacillus thuringiensis
is its ability to produce insecticidal proteins. More than 300 different proteins have been described with specific activity against insect species. We report the molecular and insecticidal characterization of a novel
cry
gene encoding a protein of the Cry1I group with toxic activity towards insects of the families Noctuidae, Tortricidae, Plutellidae, and Chrysomelidae. PCR analysis detected a DNA sequence with an open reading frame of 2.2 kb which encodes a protein with a molecular mass of 80.9 kDa. Trypsin digestion of this protein resulted in a fragment of ca. 60 kDa, typical of activated Cry1 proteins. The deduced sequence of the protein has homologies of 96.1% with Cry1Ia1, 92.8% with Cry1Ib1, and 89.6% with Cry1Ic1. According to the Cry protein classification criteria, this protein was named Cry1Ia7. The expression of the gene in
Escherichia coli
resulted in a protein that was water soluble and toxic to several insect species. The 50% lethal concentrations for larvae of
Earias insulana
,
Lobesia botrana
,
Plutella xylostella
, and
Leptinotarsa decemlineata
were 21.1, 8.6, 12.3, and 10.0 μg/ml, respectively. Binding assays with biotinylated toxins to
E. insulana
and
L. botrana
midgut membrane vesicles revealed that Cry1Ia7 does not share binding sites with Cry1Ab or Cry1Ac proteins, which are commonly present in
B. thuringiensis
-treated crops and commercial
B. thuringiensis
-based bioinsecticides. We discuss the potential of Cry1Ia7 as an active ingredient which can be used in combination with Cry1Ab or Cry1Ac in pest control and the management of resistance to
B. thuringiensis
toxins.
Comparison of Virulence Gene Profiles of
Escherichia coli
Strains Isolated from Healthy and Diarrheic Swine
Authors: Chapman Toni A. , Wu Xi-Yang , Barchia Idris , Bettelheim Karl A. , Driesen Steven , Trott Darren , Wilson Mark , Chin James J.-C.
Published: July 2006

A combination of uni- and multiplex PCR assays targeting 58 virulence genes (VGs) associated with
Escherichia coli
strains causing intestinal and extraintestinal disease in humans and other mammals was used to analyze the VG repertoire of 23 commensal
E. coli
isolates from healthy pigs and 52 clinical isolates associated with porcine neonatal diarrhea (ND) and postweaning diarrhea (PWD). The relationship between the presence and absence of VGs was interrogated using three statistical methods. According to the generalized linear model, 17 of 58 VGs were found to be significant (
P
< 0.05) in distinguishing between commensal and clinical isolates. Nine of the 17 genes represented by
iha
,
hlyA
,
aidA
,
east1
,
aah
,
fimH
,

iroN
E. coli

,
traT
, and
saa
have not been previously identified as important VGs in clinical porcine isolates in Australia. The remaining eight VGs code for fimbriae (F4, F5, F18, and F41) and toxins (STa, STb, LT, and Stx2), normally associated with porcine enterotoxigenic
E. coli
. Agglomerative hierarchical algorithm analysis grouped
E. coli
strains into subclusters based primarily on their serogroup. Multivariate analyses of clonal relationships based on the 17 VGs were collapsed into two-dimensional space by principal coordinate analysis. PWD clones were distributed in two quadrants, separated from ND and commensal clones, which tended to cluster within one quadrant. Clonal subclusters within quadrants were highly correlated with serogroups. These methods of analysis provide different perspectives in our attempts to understand how commensal and clinical porcine enterotoxigenic
E. coli
strains have evolved and are engaged in the dynamic process of losing or acquiring VGs within the pig population.
Identification of an Abscisic Acid Gene Cluster in the Grey Mold
Botrytis cinerea
Authors: Siewers Verena , Kokkelink Leonie , Smedsgaard Jørn , Tudzynski Paul
Published: July 2006

Like several other phytopathogenic fungi, the ascomycete
Botrytis cinerea
is known to produce the plant hormone abscisic acid (ABA) in axenic culture. Recently,
bcaba1
, the first fungal gene involved in ABA biosynthesis, was identified. Neighborhood analysis of
bcaba1
revealed three further candidate genes of this pathway: a putative P450 monooxygenase-encoding gene (
bcaba2
), an open reading frame without significant similarities (
bcaba3
), and a gene probably coding for a short-chain dehydrogenase/reductase (
bcaba4
). Targeted inactivation of the genes proved the involvement of BcABA2 and BcABA3 in ABA biosynthesis and suggested a contribution of BcABA4. The close linkage of at least three ABA biosynthetic genes is strong evidence for the presence of an abscisic acid gene cluster in
B. cinerea
.
A DNA Microarray Platform Based on Direct Detection of rRNA for Characterization of Freshwater Sediment-Related Prokaryotic Communities
Authors: Peplies Jörg , Lachmund Christine , Glöckner Frank Oliver , Manz Werner
Published: July 2006

A DNA microarray platform for the characterization of bacterial communities in freshwater sediments based on a heterogeneous set of 70 16S rRNA-targeted oligonucleotide probes and directly labeled environmental RNA was developed and evaluated. Application of a simple protocol for the efficient background blocking of aminosilane-coated slides resulted in an improved signal-to-noise ratio and a detection limit of 10 ng for particular 16S rRNA targets. An initial specificity test of the system using RNA from pure cultures of different phylogenetic lineages showed a fraction of false-positive signals of ∼5% after protocol optimization and a marginal loss of correct positive signals. Subsequent microarray analysis of sediment-related community RNA from four different German river sites suggested low diversity for the groups targeted but indicated distinct differences in community composition. The results were supported by parallel fluorescence in situ hybridization in combination with sensitive catalyzed reporter deposition (CARD-FISH). In comparisons of the data of different sampling sites, specific detection of populations with relative cellular abundances down to 2% as well as a correlation of microarray signal intensities and population size is suggested. Our results demonstrate that DNA microarray technology allows for the fast and efficient precharacterization of complex bacterial communities by the use of standard single-cell hybridization probes and the direct detection of environmental rRNA, also in methodological challenging habitats such as heterogeneous lotic freshwater sediments.
13
C-Labeled Gluconate Tracing as a Direct and Accurate Method for Determining the Pentose Phosphate Pathway Split Ratio in
Penicillium chrysogenum
Authors: Kleijn Roelco J. , van Winden Wouter A. , Ras Cor , van Gulik Walter M. , Schipper Dick , Heijnen Joseph J.
Published: July 2006

In this study we developed a new method for accurately determining the pentose phosphate pathway (PPP) split ratio, an important metabolic parameter in the primary metabolism of a cell. This method is based on simultaneous feeding of unlabeled glucose and trace amounts of [U-
13
C]gluconate, followed by measurement of the mass isotopomers of the intracellular metabolites surrounding the 6-phosphogluconate node. The gluconate tracer method was used with a penicillin G-producing chemostat culture of the filamentous fungus
Penicillium chrysogenum
. For comparison, a
13
C-labeling-based metabolic flux analysis (MFA) was performed for glycolysis and the PPP of
P. chrysogenum
. For the first time mass isotopomer measurements of
13
C-labeled primary metabolites are reported for
P. chrysogenum
and used for a
13
C-based MFA. Estimation of the PPP split ratio of
P. chrysogenum
at a growth rate of 0.02 h
−1
yielded comparable values for the gluconate tracer method and the
13
C-based MFA method, 51.8% and 51.1%, respectively. A sensitivity analysis of the estimated PPP split ratios showed that the 95% confidence interval was almost threefold smaller for the gluconate tracer method than for the
13
C-based MFA method (40.0 to 63.5% and 46.0 to 56.5%, respectively). From these results we concluded that the gluconate tracer method permits accurate determination of the PPP split ratio but provides no information about the remaining cellular metabolism, while the
13
C-based MFA method permits estimation of multiple fluxes but provides a less accurate estimate of the PPP split ratio.
Greengenes, a Chimera-Checked 16S rRNA Gene Database and Workbench Compatible with ARB
Authors: DeSantis T. Z. , Hugenholtz P. , Larsen N. , Rojas M. , Brodie E. L. , Keller K. , Huber T. , Dalevi D. , Hu P. , Andersen G. L.
Published: July 2006

A 16S rRNA gene database (
http://greengenes.lbl.gov
) addresses limitations of public repositories by providing chimera screening, standard alignment, and taxonomic classification using multiple published taxonomies. It was found that there is incongruent taxonomic nomenclature among curators even at the phylum level. Putative chimeras were identified in 3% of environmental sequences and in 0.2% of records derived from isolates. Environmental sequences were classified into 100 phylum-level lineages in the
Archaea
and
Bacteria
.
Toll-Like Receptor 2 Polymorphisms and Nontuberculous Mycobacterial Lung Diseases
Authors: Ryu Yon Ju , Kim Eun Joo , Koh Won-Jung , Kim Hojoong , Kwon O Jung , Chang Jung Hyun
Published: July 2006

To investigate the occurrence of the Toll-like receptor 2 (TLR2) polymorphisms in patients with pulmonary disease caused by nontuberculous mycobacteria (NTM), TLR2 Arg677Trp and Arg753Gln polymorphisms were examined. TLR2 polymorphisms do not appear to be responsible for host susceptibility to NTM lung disease, at least in the Korean population.
Epitope-Blocking Enzyme-Linked Immunosorbent Assay for Detection of Antibodies to Ross River Virus in Vertebrate Sera
Authors: Oliveira Nidia M. M. , Broom Annette K. , Mackenzie John S. , Smith David W. , Lindsay Michael D. A. , Kay Brian H. , Hall Roy A.
Published: July 2006

We describe the development of an epitope-blocking enzyme-linked immunosorbent assay (ELISA) for the sensitive and rapid detection of antibodies to Ross River virus (RRV) in human sera and known vertebrate host species. This ELISA provides an alternative method for the serodiagnosis of RRV infections.
Understanding the Immune Responses to the Meningococcal Strain-Specific Vaccine MeNZB Measured in Studies of Infants
Authors: Ruijne N. , Lea R. A. , O'Hallahan J. , Oster P. , Martin D.
Published: July 2006

Vaccine trials with infants enrolled between 6 and 10 weeks of age (young infants) and 6 and 8 months of age (older infants) provided an opportunity to evaluate immunoglobulin G (IgG) isotype distribution and avidity maturation as indicators of antibody function and immunologic memory. Following vaccination with a strain-specific outer membrane vaccine, MeNZB, pre- and postvaccination sera were used to determine IgG isotype responses and avidity indices (AI) in subsets of vaccinated subjects. Measurements of IgG isotypes involved 100 infants from each trial. AI were measured in 50 infants from the young infant trial who received a fourth vaccine dose and in 40 older infants from whom serum was collected 7 months after the primary vaccination course. IgG1 and IgG3 dominated the responses to the vaccine. A modest linear correlation (
P
< 0.001) occurred between serum bactericidal antibody (SBAb) titers and the total IgG or the IgG1 antibody units in older infants. The young infants showed a modest linear correlation between SBAb and total IgG (
P
= 0.005) and a weak linear correlation between SBAb and IgG1 (
P
= 0.003). Increased avidity with age was demonstrated in both groups. The AI in the young infants increased from 51.5% (95% confidence interval [CI], 47.7 to 54.7) postvaccination to 68.7% (95% CI, 65.5 to 71.9%) following the fourth dose of vaccine (
P
< 0.001). The mean avidity of the older infants increased significantly (
P
= 0.00012) from 42.4% (95% CI, 39.1 to 45.3%) postvaccination to 50.4% (95% CI, 47.2 to 53.6%) 4 months later. A fourth dose of MeNZB is now being given to young infants at 10 months of age.
Innate Immune Response toAnaplasma phagocytophilumContributes to Hepatic Injury
Authors: Scorpio Diana G. , von Loewenich Friederike D. , Göbel Heike , Bogdan Christian , Dumler J. Stephen
Published: July 2006

In mice,Anaplasma phagocytophilumcontrol is independent of phagocyte oxidase (phox), inducible NO synthase (NOS2), tumor necrosis factor (TNF), and MyD88 Toll-like receptor signaling. We show that despite evasion of these host responses, phox, NOS2, TNF, and MyD88 are activated and contribute to inflammation and hepatic injury more thanA. phagocytophilumitself.
Quantifying Specific Antibody Concentrations by Enzyme-Linked Immunosorbent Assay Using Slope Correction
Authors: Barrette Roger W. , Urbonas Jessica , Silbart Lawrence K.
Published: July 2006

Assessing the magnitude of an antibody response is important to many research and clinical endeavors; however, there are considerable differences in the experimental approaches used to achieve this end. Although the time-honored approach of end point titration has merit, the titer can often be misleading due to differences in how it is calculated or when samples contain high concentrations of low-avidity antibodies. One frequently employed alternative is to adapt commercially available enzyme-linked immunosorbent assay kits, designed to measure total antibody concentrations, to estimate antigen-specific antibody concentrations. This is accomplished by coating the specific antigen of interest in place of the capture antibody provided with the kit and then using the kit's standard curve to quantify the specific antibody concentration. This approach introduces considerable imprecision, due primarily to its reliance on a single sample dilution. This “single-point” approach fails to address differences in the slope of the sample titration curve compared to that of the standard curve. Here, we describe a general approach for estimating the effective concentration of specific antibodies, using antisera against foot-and-mouth disease virus VP1 peptide. This was accomplished by initially calculating the slope of the sample titration curve and then mathematically correcting the slope to that of a corresponding standard curve. A significantly higher degree of precision was attained using this approach rather than the single-point method.
Sequence-Based Methods for Identifying Epidemiologically Linked Herpes Simplex Virus Type 2 Strains
Authors: Martin Emily Toth , Koelle David M. , Byrd Benjamin , Huang Meei-Li , Vieira Jeffrey , Corey Lawrence , Wald Anna
Published: July 2006

Traditional methods for confirming the identity of herpes simplex virus (HSV) isolates use restriction fragment length polymorphism (RFLP). However, RFLP is less amenable to high-throughput analyses of many samples, and the extent to which small differences in RFLP patterns distinguish between different viral strains remains unclear. Viral HSV type 2 (HSV-2) DNA isolates from 14 persons experiencing a primary HSV-2 infection and from their sexual partners were analyzed by RFLP and heteroduplex mobility assays. We also compared the HSV-2 sequences from seven regions, including noncoding regions between UL19 and UL20, UL24 and UL25, UL37 and UL38, and UL41 and UL42 and coding segments of the gC, gB, and gG genes. Although the resulting RFLP patterns of the couples were almost identical, minor banding differences existed between the source and susceptible partners in five couples. Heteroduplex mobility assays were unable to distinguish between unrelated strains. Overall, 22 sites of sequence variation were found in 1,482 bp of analyzed sequence. The DNA sequences differentiated between all unrelated infections, and epidemiologically related isolates had identical sequences in all but two pairs. Our results suggest that a multilocus assay based on several DNA sequences has the potential to be an informative tool for identifying epidemiologically related HSV-2 strains.
Use of Genotype MTBDR Assay for Molecular Detection of Rifampin and Isoniazid Resistance in
Mycobacterium tuberculosis
Clinical Strains Isolated in Italy
Authors: Miotto Paolo , Piana Federica , Penati Valeria , Canducci Filippo , Migliori Giovanni Battista , Cirillo Daniela Maria
Published: July 2006

Mycobacterium tuberculosis
is one of the leading causes of death worldwide, and multidrug-resistant tuberculosis (MDR-TB) is associated with a high case fatality rate. Rapid identification of resistant strains is crucial for the early administration of appropriate therapy, for prevention of development of further resistance, and to curtail the spread of MDR strains. The Genotype MTBDR (Hain Lifescience, Nehren, Germany) is a reverse hybridization line probe assay designed for the rapid detection of
rpoB
and
katG
gene mutations in clinical isolates. The ability of this technique to correctly identify resistant and MDR-TB strains was tested on 206 isolates from the Italian drug resistance surveillance system. This panel included the majority of MDR strains isolated in Italy in the past 3 years. The results of the test were compared to conventional drug susceptibility test performed on isolated strains and verified by sequencing the regions of interest of the bacterial genome. The rate of concordance between the results of the MTBDR and those obtained with “in vitro” sensitivity was 91.5% (130 of 142) for rifampin and 67.1% (116 of 173) for isoniazid. We also applied this test directly to a panel of 36 clinical specimens collected from patients with active TB. The MTBDR correctly identified the two cases of MDR-TB included in the panel. These results show that the MTBDR test is useful in the detection and management of tuberculosis when MDR disease is suspected.
Sensitivities of Four New Commercial Hepatitis B Virus Surface Antigen (HBsAg) Assays in Detection of HBsAg Mutant Forms
Authors: Ly Thoai Duong , Servant-Delmas Annabelle , Bagot Sébastien , Gonzalo Sylvie , Férey Marie-Pierre , Ebel Anne , Dussaix Elisabeth , Laperche Syria , Roque-Afonso Anne-Marie
Published: July 2006

Mutations in hepatitis B virus surface antigen (HBsAg) involving amino acid substitution within the immunodominant “a” determinant may affect the performance of commercial HBsAg assays. The performances of four HBsAg assays that recently received Conformité Européene marking, Advia Centaur HBsAg (Bayer), Monolisa HBsAg Ultra (Bio-Rad), Liaison HBsAg (Dia Sorin), and Vidas HBsAg Ultra (bioMérieux), were compared with that of the routinely used HBsAg assay AxSYM HBsAg V2 (Abbott). Assays were evaluated for (i) analytical sensitivity performance with a national reference HBsAg panel (including 10 samples with calibrated HBsAg concentrations from 0.04 to 2.24 ng/ml) and (ii) the detection of HBsAg mutants by studying a panel of 35 HBsAg mutants (23 collected from patients and 12 recombinant mutants). The limits of detection of these assays were <0.15 ng/ml (from 0.089 to 0.121 ng/ml). The sensitivity performances for mutant virus detection varied, ranging from 37.1% to 91.4%. The lack of detection of these mutants by commercial assays was probably due to the epitope recognition of the anti-HBs assay reagents in the capture phase and in the conjugates. The prevalence and clinical impact of HBsAg mutants are under investigation. However, the manufacturers must be vigilant in the design of the assays in order to reduce the risk of missing a broad range of described S gene mutants.
Amplification of the Hyphal Wall Protein 1 Gene To Distinguish
Candida albicans
from
Candida dubliniensis
Authors: Romeo Orazio , Racco Cosimo , Criseo Giuseppe
Published: July 2006

The authors developed a new, simple, and reliable PCR/restriction fragment length polymorphism technique, using amplification of the hyphal wall protein 1 gene of
Candida albicans
and its gene homologue in
Candida dubliniensis
, to differentiate the two species of
Candida
. Performed with a new primer set, CRR-f/CRR-r, PCR produced two different fragments: one of 1,180 bp for
C. albicans
, and one of 930 bp for
C. dubliniensis
.
Bacillus anthracis
Virulent Plasmid pX02 Genes Found in Large Plasmids of Two Other
Bacillus
Species
Authors: Luna Vicki A. , King Debra S. , Peak K. Kealy , Reeves Frank , Heberlein-Larson Lea , Veguilla William , Heller L. , Duncan Kathleen E. , Cannons Andrew C. , Amuso Philip , Cattani Jacqueline
Published: July 2006

In order to cause the disease anthrax,
Bacillus anthracis
requires two plasmids, pX01 and pX02, which carry toxin and capsule genes, respectively, that are used as genetic targets in the laboratory detection of the bacterium. Clinical, forensic, and environmental samples that test positive by PCR protocols established by the Centers for Disease Control and Prevention for
B. anthracis
are considered to be potentially
B. anthracis
until confirmed by culture and a secondary battery of tests. We report the presence of 10 genes (
acpA
,
capA
,
capB
,
capC
,
capR
,
capD
, IS
1627
, ORF 48, ORF 61, and
repA
) and the sequence for the capsule promoter normally found on pX02 in
Bacillus circulans
and a
Bacillus
species closely related to
Bacillus luciferensis
. Tests revealed these sequences to be present on a large plasmid in each isolate. The 11 sequences consistently matched to
B. anthracis
plasmid pX02, GenBank accession numbers AF188935.1, AE011191.1, and AE017335.3. The percent nucleotide identities for
capD
and the capsule promoter were 99.9% and 99.7%, respectively, and for the remaining nine genes, the nucleotide identity was 100% for both isolates. The presence of these genes, which are usually associated with the pX02 plasmid, in two soil
Bacillus
species unrelated to
B. anthracis
alerts us to the necessity of identifying additional sequences that will signal the presence of
B. anthracis
in clinical, forensic, and environmental samples.
Detection of Multidrug-Resistant
Salmonella enterica
Serovar Typhimurium Phage Types DT102, DT104, and U302 by Multiplex PCR
Authors: Chiu Cheng-Hsun , Su Lin-Hui , Chu Chi-Hong , Wang Mei-Hwei , Yeh Chia-Ming , Weill Francois-Xavier , Chu Chishih
Published: July 2006

Salmonella enterica
serovar Typhimurium is a common cause of nontyphoidal salmonellosis in humans and animals. Multidrug-resistant serovar Typhimurium phage type DT104, which emerged in the 1990s, has become widely distributed in many countries. A total of 104 clinical isolates of
Salmonella
serogroup B were collected from three major hospitals in Taiwan during 1997 to 2003 and were examined by a multiplex PCR targeting the resistance genes and the
spv
gene of the virulence plasmid. A total of 51 isolates (49%) were resistant to all drugs (ACSSuT [resistance to ampicillin, chloramphenicol, streptomycin, sulfonamide, and tetracycline]), and all contained a 1.25-kb PCR fragment of integron that is part of the 43-kb
Salmonella
genomic island 1 (SGI1). The second group was resistant to SSu (28%), and the third was susceptible to all five drugs (13%). Fifty-nine isolates were serotyped to be serovar Typhimurium by the tube agglutination method using H antisera. The virulence plasmid was found in 54 (91.5%) of the 59 serovar Typhimurium isolates. A majority (94.1%) of the
Salmonella
serogroup B isolates with the ACSSuT resistance pattern harbored a virulence plasmid. Phage typing identified three major phage types: DT104, DT120, and U302. Analysis of the isolates by pulsed-field gel electrophoresis showed six genotypes. We found two genotypes in DT104 strains, two in DT120, and the other two in U302. The presence of a monophasic serovar (4,5,12:i:−) has added difficulty in the determination of the serovars of multidrug-resistant
Salmonella
serogroup B isolates. Nevertheless, the multiplex PCR devised in the present study appears to be efficient and useful in the rapid identification of ACSSuT-type serovar Typhimurium with SGI1, irrespective of their phage types.
Comparison of Real-Time PCR Assays with Fluorescent-Antibody Assays for Diagnosis of Respiratory Virus Infections in Children
Authors: Kuypers Jane , Wright Nancy , Ferrenberg James , Huang Meei-Li , Cent Anne , Corey Lawrence , Morrow Rhoda
Published: July 2006

Conventional fluorescent-antibody (FA) methods were compared to real-time PCR assays for detection of respiratory syncytial virus (RSV), influenza virus type A (FluA), parainfluenza virus types 1, 2, and 3 (PIV1, PIV2, and PIV3), human metapneumovirus (MPV), and adenovirus (AdV) in 1,138 specimens from children with respiratory illnesses collected over a 1-year period. At least one virus was detected in 436 (38.3%) specimens by FA and in 608 (53.4%) specimens by PCR (
P
< 0.001). Specimen quality was inadequate for FA in 52 (4.6%) specimens; 13 of these (25%) were positive by PCR. In contrast, 18 (1.6%) specimens could not be analyzed by PCR; 1 of these was positive by FA. The number of specimens positive only by PCR among specimens positive by PCR and/or FA was 18 (7.0%) of 257 for RSV, 18 (13.4%) of 134 for FluA, 25 (64.1%) of 39 for PIV1, 8 (88.9%) of 9 for PIV2, 17 (30.1%) of 55 for PIV3, and 101 (76.5%) of 132 for AdV. MPV was detected in 6.6% of all specimens and in 9.5% of the 702 specimens negative by FA. The mean number of virus copies per milliliter in specimens positive by both PCR and FA was significantly higher, at 6.7 × 10
7
, than that in specimens positive only by PCR, at 4.1 × 10
4
(
P
< 0.001). The PCR assays were significantly more sensitive than FA assays for detecting respiratory viruses, especially parainfluenza virus and adenovirus. Use of real-time PCR to identify viral respiratory pathogens in children will lead to improved diagnosis of respiratory illness.
Rapid Increase in Total Torquetenovirus (TTV) Plasma Viremia Load Reveals an Apparently Transient Superinfection by a TTV of a Novel Group 2 Genotype
Authors: Maggi Fabrizio , Andreoli Elisabetta , Lanini Letizia , Meschi Silvia , Rocchi Jara , Fornai Claudia , Vatteroni Maria Linda , Pistello Mauro , Bendinelli Mauro
Published: July 2006

An apparently transient infection by a superimposed torquetenovirus (TTV) in a subject who already carried three different genotypes of the virus is described. The superinfection induced a rapid increase in the plasma TTV load and a decline in immunocomplexed virus. The superinfecting TTV was a novel group 2 genotype.
First Case Report of
Neisseria lactamica
Causing Cavitary Lung Disease in an Adult Organ Transplant Recipient
Authors: Zavascki Alexandre Prehn , Fritscher Leandro , Superti Silvana , Dias Cícero , Kroth Leonardo , Traesel Moacir Alexandre , Antonello Ivan Carlos Ferreira , Saitovitch David
Published: July 2006

We describe a case of an adult organ recipient patient with a pulmonary cavitary lesion due to
Neisseria lactamica
, a harmless commensal organism that rarely causes human infection. To our knowledge, this is the first report of pulmonary disease caused by this organism and the second case of
N. lactamica
infection in an adult patient.
Etiological Misidentification by Routine Biochemical Tests of Bacteremia Caused by
Gordonia terrae
Infection in the Course of an Episode of Acute Cholecystitis
Authors: Gil-Sande E. , Brun-Otero M. , Campo-Cerecedo F. , Esteban E. , Aguilar L. , García-de-Lomas J.
Published: July 2006

Gordonia terrae
has been reported to be a rare cause of bacteremia. We report the first case of bacteremia associated with acute cholecystitis. Commercial biochemical testing was not able to identify the strain at the genus level, classifying it instead as
Rhodococcus
sp. Definitive identification was obtained by sequencing of the 16S rRNA gene.
Blinded, Multicenter Comparison of Methods To Detect a Drug-Resistant Mutant of Human Immunodeficiency Virus Type 1 at Low Frequency
Authors: Halvas Elias K. , Aldrovandi Grace M. , Balfe Peter , Beck Ingrid A. , Boltz Valerie F. , Coffin John M. , Frenkel Lisa M. , Hazelwood J. Darren , Johnson Victoria A. , Kearney Mary , Kovacs Andrea , Kuritzkes Daniel R. , Metzner Karin J. , Nissley Dwight V. , Nowicki Marek , Palmer Sarah , Ziermann Rainer , Zhao Richard Y. , Jennings Cheryl L. , Bremer James , Brambilla Don , Mellors John W.
Published: July 2006

We determined the abilities of 10 technologies to detect and quantify a common drug-resistant mutant of human immunodeficiency virus type 1 (lysine to asparagine at codon 103 of the reverse transcriptase) using a blinded test panel containing mutant-wild-type mixtures ranging from 0.01% to 100% mutant. Two technologies, allele-specific reverse transcriptase PCR and a Ty1HRT yeast system, could quantify the mutant down to 0.1 to 0.4%. These technologies should help define the impact of low-frequency drug-resistant mutants on response to antiretroviral therapy.
Comparison of the Gen-Probe APTIMA Combo 2 Assay to the AMPLICOR CT/NG Assay for Detection of
Chlamydia trachomatis
and
Neisseria gonorrhoeae
in Urine Samples from Australian Men and Women
Authors: Lowe Peter , O'Loughlin Peter , Evans Kathryn , White Michael , Bartley Paul B. , Vohra Renu
Published: July 2006

The performance of the APTIMA Combo 2 assay (AC2) for the detection of
Chlamydia trachomatis
and
Neisseria gonorrhoeae
infections in urine samples was compared to that of the AMPLICOR CT/NG assay (AMP). The AC2 performance was superior to that of AMP for both organisms in this study.
Multispacer Typing To Study the Genotypic Distribution ofBartonella henselaePopulations
Authors: Li Wenjun , Chomel Bruno B. , Maruyama Soichi , Guptil Lynn , Sander Anna , Raoult Didier , Fournier Pierre-Edouard
Published: July 2006

Bartonella henselae, a worldwide fastidious bacterium, has a feline reservoir and is pathogenic for humans. However, the relationship between human and cat isolates ofB. henselae, as well as its population dynamics and geographic heterogeneity, is not fully understood, in part because of the absence of appropriate typing methods. Multilocus sequence typing (MLST), the most discriminatory genotyping method forB. henselae, identified seven genotypes and suggested that human isolates arose from a limited number of cat isolates. Herein, we estimated the discriminatory power of multispacer typing (MST) by studying 126B. henselaecat isolates from various areas of Europe, Asia, and the United States. We identified the nine most variable intergenic spacers conserved by bothB. henselaeandBartonella quintanagenomes. By comparing the sequences obtained from these nine spacers for each studied isolate, we identified 39 MST genotypes. The distribution of isolates into MST genotypes matched their phylogenetic organization into four clusters. MST showed that European and Asian isolates were different, in contrast with American isolates, but failed to identify pandemic strains. Our study demonstrated that MST is a powerful method for genotypingB. henselaeat the strain level and may serve in studying the population dynamics of this bacterium and understanding the relationships between cat and human isolates. Finally, we provide a free-access MST-Rick online software program (http://ifr48.timone.univ-mrs.fr/MST_BHenselae/mst) that investigators may use to compare their own MST sequences to our database.
Rapid Genotypic Detection of Rifampin- and Isoniazid-Resistant
Mycobacterium tuberculosis
Directly in Clinical Specimens
Authors: Bang Didi , Bengård Andersen Åse , Thomsen Vibeke Østergaard
Published: July 2006

A multiplex PCR DNA strip assay (Genotype MTBDR) designed to detect rifampin (
rpoB
) and high-level isoniazid (
katG
) resistance mutations in
Mycobacterium tuberculosis
isolates was optimized for clinical specimens. Successful genotypic results were achieved with 36 of 38 (95%) smear-positive respiratory specimens, allowing rapid therapeutic adjustments in transmittable drug-resistant tuberculosis.
Progression Toward an Improved DNA Amplification-Based Typing Technique in the Study of
Mycobacterium tuberculosis
Epidemiology
Authors: Gopaul Krishna K. , Brown Timothy J. , Gibson Andrea L. , Yates Malcolm D. , Drobniewski Francis A.
Published: July 2006

While high-copy-number IS
6110
-based restriction fragment length polymorphism (HCN-RFLP) is the gold standard for typing most
Mycobacterium tuberculosis
strains, the time taken for culturing and low throughput make it impractical for large-scale prospective typing of large numbers of isolates. The development of a new method, mycobacterial interspersed repetitive units (MIRU), a variation of the original variable-number tandem repeat (VNTR) technique, may provide a viable alternative. Panels based on the original 12-loci MIRU (12MIRU), a combination of 12MIRU and remaining ETR loci (15MIRU-VNTR), and an extended panel with an additional 10 novel regions (25VNTR) were used to study three populations with varying degrees of epidemiological data. MIRU discrimination increased with panel size and the addition of spoligotyping. Combining these two techniques enabled a reduction in the panel size from 25 to 14 loci without a significant loss in discrimination. However, 25VNTR alone or in combination with spoligotyping still possessed weaker discrimination than RFLP for high-copy-number isolates.
Use of 16S rRNA Gene Profiling by Terminal Restriction Fragment Length Polymorphism Analysis To Compare Bacterial Communities in Sputum and Mouthwash Samples from Patients with Cystic Fibrosis
Authors: Rogers G. B. , Carroll M. P. , Serisier D. J. , Hockey P. M. , Jones G. , Kehagia V. , Connett G. J. , Bruce K. D.
Published: July 2006

The bacterial communities present in the oral cavity and the lungs of 19 adult cystic fibrosis (CF) patients were compared by using terminal restriction fragment length polymorphism analysis of 16S rRNA gene PCR products amplified from nucleic acids extracted directly from bacteria in clinical samples. Sputum samples were not found to be subject to profound contamination by oral cavity bacteria. Evidence of colonization of the CF lung by certain oral bacterial species was found.