bluuebunny/crossref_metadata_2025 huggingface dataset:
A 12-Amino-Acid Segment, Present in Type s2 but Not Type s1
Helicobacter pylori
VacA Proteins, Abolishes Cytotoxin Activity and Alters Membrane Channel Formation
Authors: McClain Mark S. , Cao Ping , Iwamoto Hideki , Vinion-Dubiel Arlene D. , Szabo Gabor , Shao Zhifeng , Cover Timothy L.
Published: July 2002

Helicobacter pylori
, a gram-negative bacterium associated with gastritis, peptic ulceration, and gastric adenocarcinoma in humans, secretes a protein toxin, VacA, that causes vacuolar degeneration of epithelial cells. Several different families of
H. pylori vacA
alleles can be distinguished based on sequence diversity in the “middle” region (i.e., m1 and m2) and in the 5′ end of the gene (i.e., s1 and s2). Type s2 VacA toxins contain a 12-amino-acid amino-terminal hydrophilic segment, which is absent from type s1 toxins. To examine the functional properties of VacA toxins containing this 12-amino-acid segment, we analyzed a wild-type s1/m1 VacA and a chimeric s2/m1 VacA protein. Purified s1/m1 VacA from
H. pylori
strain 60190 induced vacuolation in HeLa and Vero cells, whereas the chimeric s2/m1 toxin (in which the s1 sequence of VacA from strain 60190 was replaced with the s2 sequence from strain Tx30a) lacked detectable cytotoxic activity. Type s1/m1 VacA from strain 60190 formed membrane channels in a planar lipid bilayer assay at a significantly higher rate than did s2/m1 VacA. However, membrane channels formed by type s1 VacA and type s2 VacA proteins exhibited similar anion selectivities (permeability ratio, P
Cl
/P
Na
= 5). When an equimolar mixture of the chimeric s2/m1 toxin and the wild-type s1/m1 toxin was added to HeLa cells, the chimeric toxin completely inhibited the activity of the s1/m1 toxin. Thus, the s2/m1 toxin exhibited a dominant-negative phenotype similar to that of a previously described mutant toxin, VacA-(Δ6–27). Immunoprecipitation experiments indicated that both s2/m1 VacA and VacA-(Δ6–27) could physically interact with a c-myc epitope-tagged s1/m1 VacA, which suggests that the dominant-negative phenotype results from the formation of heterooligomeric VacA complexes with defective functional activity. Despite detectable differences in the channel-forming activities and cytotoxic properties of type s1 and type s2 VacA proteins, the conservation of type s2 sequences in many
H. pylori
isolates suggests that type s2 VacA proteins retain an important biological activity.
Transcript Analysis of Multiple Copies of
amo
(Encoding Ammonia Monooxygenase) and
hao
(Encoding Hydroxylamine Oxidoreductase) in
Nitrosomonas europaea
Authors: Hommes Norman G. , Sayavedra-Soto Luis A. , Arp Daniel J.
Published: July 2002

The genes encoding ammonia monooxygenase (
amoCAB
), hydroxylamine oxidoreductase (
hao
), and the
c
-type cytochrome
c
-554 (
hcy
) are present in multiple copies in the genome of
Nitrosomonas europaea.
The upstream regions of the two copies of
amoC
, the three copies of
hao
, and one copy of
hcy
were cloned and sequenced. Primer extension reactions were done to identify transcription start sites for these genes, as well as for
amoA
. Putative ς
70
promoter sequences were found associated with all but one of the mapped transcription start sites. Primer extensions were done with
amoC
primers using RNA harvested from cells incubated with and without ammonium. The experiments suggested that
N. europaea
cells may be able to use different promoters in the presence and absence of ammonium.
Initiation of Biofilm Formation by
Pseudomonas aeruginosa
57RP Correlates with Emergence of Hyperpiliated and Highly Adherent Phenotypic Variants Deficient in Swimming, Swarming, and Twitching Motilities
Authors: Déziel Eric , Comeau Yves , Villemur Richard
Published: July 2002

Pseudomonas aeruginosa
is a ubiquitous environmental bacterium capable of forming biofilms on surfaces as a survival strategy. It exhibits a large variety of competition/virulence factors, such as three types of motilities: flagellum-mediated swimming, flagellum-mediated swarming, and type IV pilus-mediated twitching. A strategy frequently used by bacteria to survive changing environmental conditions is to create a phenotypically heterogeneous population by a mechanism called phase variation. In this report, we describe the characterization of phenotypic variants forming small, rough colonies that spontaneously emerged when
P. aeruginosa
57RP was cultivated as a biofilm or in static liquid cultures. These small-colony (S) variants produced abundant type IV fimbriae, displayed defective swimming, swarming, and twitching motilities, and were impaired in chemotaxis. They also autoaggregated in liquid cultures and rapidly initiated the formation of strongly adherent biofilms. In contrast, the large-colony variant (parent form) was poorly adherent, homogeneously dispersed in liquid cultures, and produced scant polar fimbriae. Further analysis of the S variants demonstrated differences in a variety of other phenotypic traits, including increased production of pyocyanin and pyoverdine and reduced elastase activity. Under appropriate growth conditions, cells of each phenotype switched to the other phenotype at a fairly high frequency. We conclude that these S variants resulted from phase variation and were selectively enriched when
P. aeruginosa
57RP was grown as a biofilm or in static liquid cultures. We propose that phase variation ensures the prior presence of phenotypic forms well adapted to initiate the formation of a biofilm as soon as environmental conditions are favorable.
Adhesion of Type 1-Fimbriated
Escherichia coli
to Abiotic Surfaces Leads to Altered Composition of Outer Membrane Proteins
Authors: Otto Karen , Norbeck Joakim , Larsson Thomas , Karlsson Karl-Anders , Hermansson Malte
Published: July 2002

Phenotypic differences between planktonic bacteria and those attached to abiotic surfaces exist, but the mechanisms involved in the adhesion response of bacteria are not well understood. By the use of two-dimensional (2D) polyacrylamide gel electrophoresis, we have demonstrated that attachment of
Escherichia coli
to abiotic surfaces leads to alteration in the composition of outer membrane proteins. A major decrease in the abundance of resolved proteins was observed during adhesion of type 1-fimbriated
E. coli
strains, which was at least partly caused by proteolysis. Moreover, a study of fimbriated and nonfimbriated mutants revealed that these changes were due mainly to type 1 fimbria-mediated surface contact and that only a few changes occurred in the outer membranes of nonfimbriated mutant strains. Protein synthesis and proteolytic degradation were involved to different extents in adhesion of fimbriated and nonfimbriated cells. While protein synthesis appeared to affect adhesion of only the nonfimbriated strain, proteolytic activity mostly seemed to contribute to adhesion of the fimbriated strain. Using matrix-assisted laser desorption ionization–time of flight mass spectrometry, six of the proteins resolved by 2D analysis were identified as BtuB, EF-Tu, OmpA, OmpX, Slp, and TolC. While the first two proteins were unaffected by adhesion, the levels of the last four were moderately to strongly reduced. Based on the present results, it may be suggested that physical interactions between type 1 fimbriae and the surface are part of a surface-sensing mechanism in which protein turnover may contribute to the observed change in composition of outer membrane proteins. This change alters the surface characteristics of the cell envelope and may thus influence adhesion.
Regulatory RNA as Mediator in GacA/RsmA-Dependent Global Control of Exoproduct Formation inPseudomonas fluorescensCHA0
Authors: Heeb Stephan , Blumer Caroline , Haas Dieter
Published: July 2002

InPseudomonas fluorescensCHA0, an antagonist of root-pathogenic fungi, the GacS/GacA two-component system tightly controls the expression of antifungal secondary metabolites and exoenzymes at a posttranscriptional level, involving the RNA-binding protein and global regulator of secondary metabolism RsmA. This protein was purified fromP. fluorescens, and RNA bound to it was converted to cDNA, which served as a probe to isolate the corresponding chromosomal locus,rsmZ. This gene encoded a regulatory RNA of 127 nucleotides and a truncated form lacking 35 nucleotides at the 3" end. Expression ofrsmZdepended on GacA, increased with increasing population density, and was stimulated by the addition of a solvent-extractable extracellular signal produced by strain CHA0 at the end of exponential growth. This signal appeared to be unrelated toN-acyl-homoserine lactones. A conserved upstream element in thersmZpromoter, but not the stress sigma factor RpoS, was involved inrsmZexpression. Overexpression ofrsmZeffectively suppressed the negative effect ofgacSandgacAmutations on target genes, i.e.,hcnA(for hydrogen cyanide synthase) andaprA(for the major exoprotease). Mutational inactivation ofrsmZresulted in reduced expression of these target genes in the presence of added signal. Overexpression ofrsmAhad a similar, albeit stronger negative effect. These results support a model in which GacA upregulates the expression of regulatory RNAs, such as RsmZ of strain CHA0, in response to a bacterial signal. By a titration effect, RsmZ may then alleviate the repressing activity of RsmA on the expression of target mRNAs.
Filamentous Phage Active on the Gram-Positive BacteriumPropionibacterium freudenreichii
Authors: Chopin Marie-Christine , Rouault Annette , Ehrlich S. Dusko , Gautier Michel
Published: July 2002

We present the first description of a single-stranded DNA filamentous phage able to replicate in a gram-positive bacterium. Phage B5 infectsPropionibacterium freudenreichiiand has a genome consisting of 5,806 bases coding for 10 putative open reading frames. The organization of the genome is very similar to the organization of the genomes of filamentous phages active on gram-negative bacteria. The putative coat protein exhibits homology with the coat proteins of phages PH75 and Pf3 active onThermus thermophilusandPseudomonas aeruginosa, respectively. B5 is, therefore, evolutionarily related to the filamentous phages active on gram-negative bacteria.
Measles Virus-Specific Immunoglobulin G Isotype Immune Response in Early and Late Infections
Authors: Isa Marı́a Beatrı́z , Martı́nez Laura , Giordano Miguel , Zapata Marta , Passeggi Carlos , De Wolff Marı́a Cristina , Nates Silvia
Published: July 2002

A total of 154 human serum samples (32 acute-phase and 22 convalescent-phase serum samples obtained within a week and between days 8 and 26 after the onset of rash, respectively, and 100 samples drawn from healthy immune adults) were processed by an immunofluorescence assay for the detection of immunoglobulin M (IgM), total immunoglobulin G (IgG), IgG1, IgG2, IgG3, and IgG4 measles virus-specific antibodies. In the acute phase, IgG1 was seen first, followed by IgG2, IgG3, and IgG4 responses, the mean seropositivity of which gradually increased during convalescence, reaching 100% (standard deviation [SD], 84 to 100%), 57% (SD, 34 to 80%), 86% (SD, 66 to 100%), and 86% (SD, 66 to 100%), respectively. IgG2 rose and fell in connection with IgG3 subclass antibodies, showing a rate of detection of IgG2 and/or IgG3 subclass antibodies of 95.5% (range, 100 to 86.5%) in the convalescent phase of infection. The mean percentage of measles IgG2 and IgG3 seropositivity dropped significantly during the memory phase, to 2% (range, 2 to 6%) and 3% (range, 3 to 7%), respectively (
P
< 0.05); meanwhile IgG1 and IgG4 subclass responses remained relatively unmodified in samples obtained years after infection (mean 100% [SD, 96 to 100%] and 86% [SD, 79 to 93%], respectively). Results obtained defined two highly different immune isotypic response patterns. One pattern is restrictive to IgG2 and/or IgG3 in the convalescent phase and is kinetically similar to the IgM antibody response, so its detection could be referred to as a recent viral activity. On the other hand, IgG1 and IgG4 were detected in both the convalescent and memory phases of the immune response, but their isolated occurrence without IgG2 and IgG3 could be related to the long-lasting immunity.
Characterization of
Salmonella
Associated with Pig Ear Dog Treats in Canada
Authors: Clark Clifford , Cunningham Jane , Ahmed Rafiq , Woodward David , Fonseca Kevin , Isaacs Sandy , Ellis Andrea , Anand Chandar , Ziebell Kim , Muckle Anne , Sockett Paul , Rodgers Frank
Published: July 2002

In the summer of 1999, the incidence of
Salmonella enterica
serotype Infantis infections in Alberta rose dramatically. Subsequent laboratory and epidemiological investigations established that an outbreak of human disease caused by this organism was occurring across Canada and was associated with pet treats for dogs produced from processed pig ears. Laboratory investigations using phage typing and pulsed-field gel electrophoresis (PFGE) established that isolates of
Salmonella
serotype Infantis from pig ear pet treats and humans exposed to pig ear pet treats comprised a well-defined subset of all isolates analyzed. Of the 53 subtypes of
Salmonella
serotype Infantis obtained around the time of the outbreak as defined by PFGE and phage typing, only 6 subtypes were associated with both human infection and isolation from pig ears. Together with information from epidemiological studies, these investigations established pig ear pet treats as the cause of the
Salmonella
serotype Infantis outbreak. The results are consistent with a model in which contaminated pig ear pet treats constitute a long-term, continuing vehicle for infection of the human population rather than causing temporally delimited point-source outbreaks. During the course of this outbreak, several other
Salmonella
serotypes were also isolated from pet treats, suggesting these products may be an important source of enteric infection in both humans and dogs. Though isolates of
Salmonella
serotypes other than
Salmonella
serotype Infantis from pet treats were also subjected to PFGE and phage typing, no link with human disease could be definitively established, and the contribution of pig ear pet treats to human disease remains unclear. Elimination of bacterial contamination from pet treats is required to reduce the risk of infection from these products.
Polymorphic Internal Transcribed Spacer Region 1 DNA Sequences Identify Medically Important Yeasts
Authors: Chen Yi-Ching , Eisner Jessica D. , Kattar Mireille M. , Rassoulian-Barrett Sara L. , Lafe Karen , Bui Uyen , Limaye Ajit P. , Cookson Brad T.
Published: July 2002

Species-specific polymorphisms in the noncoding internal transcribed spacer 2 (ITS2) region of the rRNA operon provide accurate identification of clinically significant yeasts. In this study, we tested the hypothesis that ITS1 noncoding regions contain diagnostically useful alleles. The length of ITS1 region PCR products amplified from 40 species (106 clinical strains, 5 reference strains, and 30 type strains) was rapidly determined with single-base precision by automated capillary electrophoresis. Polymorphisms in the PCR product length permitted 19 species to be distinguished by ITS1 alone, compared with 16 species distinguished by using only ITS2. However, combination of both ITS alleles permitted identification of 30 species (98% of clinical isolates). The remaining 10 species with PCR products of similar sizes contained unique ITS alleles distinguishable by restriction enzyme analysis. DNA sequence analysis of amplified ITS1 region DNA from 79 isolates revealed species-specific ITS1 alleles for each of the 40 pathogenic species examined. This provided identification of unusual clinical isolates, and 53 diagnostic ITS1 sequences were deposited in GenBank. Phylogenetic analyses based on ITS sequences showed a similar overall topology to 26S rRNA gene-based trees. However, different species with identical 26S sequences contained distinct ITS alleles that provided species identification with strong statistical support. Together, these data indicate that the analysis of ITS polymorphisms can reliably identify 40 species of clinically significant yeasts and that the capacity for identifying potentially new pathogenic species by using this database holds significant promise.
Differentiation of
Candida albicans
and
Candida dubliniensis
by Fluorescent In Situ Hybridization with Peptide Nucleic Acid Probes
Authors: Oliveira Kenneth , Haase Gerhard , Kurtzman Cletus , Hyldig-Nielsen Jens Jo/rgen , Stender Henrik
Published: July 2002

The recent discovery of
Candida dubliniensis
as a separate species that traditionally has been identified as
Candida albicans
has led to the development of a variety of biochemical and molecular methods for the differentiation of these two pathogenic yeasts. rRNA sequences are well-established phylogenetic markers, and probes targeting species-specific rRNA sequences have been used in diagnostic assays for the detection and identification of microorganisms. Peptide nucleic acid (PNA) is a DNA mimic with improved hybridization characteristics, and the neutral backbone of PNA probes offers significant advantages in whole-cell in situ hybridization assays. In this study, we developed PNA probes targeting the rRNAs of
C. albicans
and
C. dubliniensis
and applied them to a fluorescence in situ hybridization method (PNA FISH) for differentiation between
C. albicans
and
C. dubliniensis
. Liquid cultures were smeared onto microscope slides, heat fixed, and then hybridized for 30 min. Unhybridized PNA probe was removed by washing, and smears were examined by fluorescence microscopy. Evaluation of the PNA FISH method using smears of 79
C. dubliniensis
and 70
C. albicans
strains showed 100% sensitivity and 100% specificity for both PNA probes. We concluded that PNA FISH is a powerful tool for the differentiation of
C. albicans
and
C. dubliniensis
.
Influence of Glucose Supplementation and Inoculum Size on Growth Kinetics and Antifungal Susceptibility Testing of
Candida
spp
Authors: Cuenca-Estrella Manuel , Dı́az-Guerra Teresa M. , Mellado Emilia , Rodrı́guez-Tudela Juan L.
Published: July 2002

The influences of inoculum size and glucose supplementation on the growth kinetics of 60
Candida
spp. clinical isolates (
Candida albicans, Candida tropicalis, Candida parapsilosis, Candida glabrata, Candida krusei
, and
Candida lusitaniae
[10 isolates each]) are assessed. The combined influence of growth and reading method (visual or spectrophotometric) on the determination of the MICs of amphotericin B, flucytosine, fluconazole, itraconazole, ketoconazole, and voriconazole is also analyzed, and the MICs are compared with those determined by the National Committee for Clinical Laboratory Standards standard microdilution method (NCCLS document M27-A). Glucose supplementation and inoculum size had a significant influence on the growth cycles of these yeasts, and a statistically significant denser growth (optical density at 540 nm) was seen for both incubation periods, 24 and 48 h (
P
< 0.01). A longer exponential phase and shorter lag phase were also observed. The
A
540
values at 24 h of incubation with medium containing glucose and an inoculum of 10
5
CFU/ml were >0.4 U for all species, with the exception of that for
C. parapsilosis
(
A
540
= 0.26 ± 0.025). The MICs at 24 h determined by testing with 2% glucose and an inoculum of 10
5
CFU/ml showed the strongest agreement (96.83%) with MICs determined by the reference method. MICs were not falsely elevated, and good correlation indexes were obtained. The reproducibility of results with this medium-inoculum combination was high (intraclass correlation coefficient, 0.955). The best agreement and reproducibility of results for spectrophotometric readings were achieved with endpoints of 50% growth inhibition for flucytosine and azoles and 95% for amphotericin B. Supplementation of test media with glucose and an inoculum size of 10
5
CFU/ml yielded a reproducible technique that shows elevated agreement with the reference procedures and a shorter incubation period for obtaining reliable MIC determinations. The spectrophotometric method offers an advantage over the visual method by providing a more objective and automated MIC determination.
Quantitative Detection of Hepatitis C Virus RNA by Light Cycler PCR and Comparison with Two Different PCR Assays
Authors: Schröter Matthias , Zöllner Bernhard , Schäfer Peter , Laufs Rainer , Feucht Heinz-Hubert
Published: July 2002

The new Light Cycler technology was adapted to the detection of hepatitis C virus (HCV) RNA in clinical samples. Sera from 81 patients were tested by Light Cycler PCR, AMPLICOR HCV Monitor assay, and in-house PCR. Our data demonstrate that Light Cycler is a fast and reliable method for the detection and quantitation of HCV RNA.
Relationship between Susceptibility to Hemolytic-Uremic Syndrome and Levels of Globotriaosylceramide in Human Sera
Authors: Watarai Shinobu , Yokota Kenji , Tana  , Kishimoto Takumi , Kanadani Tomohosa , Taketa Kazuhisa , Oguma Keiji
Published: July 2002

The relationship between differential susceptibility to hemolytic-uremic syndrome (HUS) and levels of globotriaosylceramide (Gb3) in serum was studied in patients infected with verotoxin-producing
Escherichia coli
(VTEC). The serum Gb3 levels in patients with HUS were lower than these in diarrheal patients without subsequent HUS or in patients without clinical symptoms, indicating that individuals with a lower content of serum Gb3 show a higher incidence of HUS following VTEC infection.
Phenotypic Methods for Determining Genomovar Status of the
Burkholderia cepacia
Complex
Authors: Henry Deborah A. , Mahenthiralingam Eshwar , Vandamme Peter , Coenye Tom , Speert David P.
Published: July 2002

Recent taxonomic advances have demonstrated that
Burkholderia cepacia
is a cluster of at least seven closely related genomic species (or genomovars) collectively referred to as the
B. cepacia
complex, all of which may cause infections among cystic fibrosis patients and other vulnerable individuals. Thus, it is important for clinical microbiologists to be able to differentiate genomovars. Prior to this study, 361
B. cepacia
complex isolates and 51 isolates easily confused with
B. cepacia
complex previously had been identified using a polyphasic approach, and in this study, a comparison of phenotypic and biochemical tests was carried out. It was determined that
Burkholderia multivorans
and
Burkholderia stabilis
could reliably be separated from other members of the
B. cepacia
complex by phenotypic methods. A combination of phenotypic and molecular tests such as
recA
PCR and 16S rRNA RFLP are recommended for differentiation among the genomovars of the
B. cepacia
complex. A biochemical reaction scheme for the identification of
B. gladioli
, Pandoraea species, and
Ralstonia pickettii
and the differentiation of these species from the
B. cepacia
complex is also presented.
Resistance of
Pseudomonas aeruginosa
Isolates to Hydrogel Contact Lens Disinfection Correlates with Cytotoxic Activity
Authors: Lakkis Carol , Fleiszig Suzanne M. J.
Published: July 2002

One of the most common pathogens in infection of hydrogel contact lens wearers is
Pseudomonas aeruginosa
, which can gain access to the eye via contamination of the lens, lens case, and lens care solutions. Only one strain per species is used in current regulatory testing for the marketing of chemical contact lens disinfectants. The aim of this study was to determine whether
P. aeruginosa
strains vary in their susceptibility to hydrogel contact lens disinfectants. A method for rapidly screening bacterial susceptibility to contact lens disinfectants was developed, based on measurement of the MIC. The susceptibility of 35
P. aeruginosa
isolates to two chemical disinfectants was found to vary among strains. MICs ranged from 6.25 to 100% for both disinfectants at 37°C, and a number of strains were not inhibited by a 100% disinfectant concentration in the lens case environment at room temperature (22°C). Resistance to disinfection appeared to be an inherent rather than acquired trait, since some resistant strains had been isolated prior to the introduction of the disinfectants and some susceptible
P. aeruginosa
strains could not be made more resistant by repeated disinfectant exposure. A number of
P. aeruginosa
strains which were comparatively more resistant to short-term disinfectant exposure also demonstrated the ability to grow to levels above the initial inoculum in one chemical disinfectant after long-term (24 to 48 h) disinfectant exposure. Resistance was correlated with acute cytotoxic activity toward corneal epithelial cells and with
exsA
, which encodes a protein that regulates cytotoxicity via a complex type III secretion system. These results suggest that chemical disinfection solutions may select for contamination with cytotoxic strains. Further investigation of the mechanisms and factors responsible for resistance may also lead to strategies for reducing adverse responses to contact lens wear.
Fluconazole Disk Diffusion Test with Methylene Blue- and Glucose-Enriched Mueller-Hinton Agar for Determining Susceptibility of
Candida
Species
Authors: Lee Sai-Cheong , Fung Chang-Phone , Lee Ning , See Lai-Chu , Huang Jen-Seng , Tsai Chi-Jen , Chen Kuo-Su , Shieh Wen-Ben
Published: July 2002

A 25-μg fluconazole disk diffusion test using a Mueller-Hinton agar plate containing 2% glucose and 5 μg of methylene blue/ml (GM-MH) was compared to the macrodilution reference method for 210
Candida
species. The GM-MH agar plate was read at 24 h. The predictive values of disks with susceptible, intermediate, and resistant results on the GM-MH agar plate at 24 h were 97.1, 56.3, and 76.5%, respectively.
Monoclonal Antibody-Based Competitive Enzyme-Linked Immunosorbent Assay for Detection of Morbillivirus Antibody in Marine Mammal Sera
Authors: Saliki Jeremiah T. , Lehenbauer Terry W.
Published: July 2002

A competitive enzyme-linked immunosorbent assay (cELISA), using two monoclonal antibodies (MAbs), was developed and compared with the standard virus neutralization test (VNT) for detecting antibodies against canine distemper virus (CDV) and phocine distemper virus (PDV) in sera from dogs and various species of marine mammals. The test depends on the blocking of MAb binding to solid-phase antigen in the presence of positive serum. Test conditions were optimized by using control VNT-negative and -positive sera specific for CDV and PDV. A positive cutoff value of 30% inhibition, which represents the mean cutoff of a VNT-negative population (
n
= 623) plus 2 standard deviations, was adopted for the test. A total of 736 serum samples were tested by the new cELISA and by the VNT as the “gold standard.” An unexpected but useful finding was the ability of this CDV- and PDV-specific cELISA to also detect antibodies against the related pair dolphin morbillivirus and porpoise morbillivirus. Based on a subpopulation of 625 sera used in statistical analyses, the overall sensitivity and specificity of cELISA relative to those of the VNT were 94.9 and 97.7%, respectively. Because the cELISA proved to be nearly as sensitive and specific as the VNT while being simpler and more rapid, it would be an adequate screening test for suspect CDV or PDV cases and would also be useful for epidemiological surveillance of morbilliviral infections in marine mammal populations.
Phylogenetic Characterization of Genotype 4 Hepatitis C Virus Isolates from Argentina
Authors: Alfonso Victoria , Flichman Diego , Sookoian Silvia , Mbayed Viviana Andrea , Campos Rodolfo Héctor
Published: July 2002

Among 114 patients infected with hepatitis C virus, three genotype 4 isolates, unusual in Argentina, were detected by phylogenetic analysis over different genomic regions. The patients were not related. One sample was associated with Egyptian sequences, and the others were associated with a Zairean isolate, a fact which reinforces the idea that they are from independent sources.
Development of a Rapid and Sensitive Test for Identification of Major Pathogens in Bovine Mastitis by PCR
Authors: Riffon Renée , Sayasith Khampoune , Khalil Hayssam , Dubreuil Pascal , Drolet Marc , Lagacé Jacqueline
Published: July 2002

Bovine mastitis is the most important source of loss for the dairy industry. A rapid and specific test for the detection of the main pathogens of bovine mastitis is not actually available. Molecular probes reacting in PCR with bacterial DNA from bovine milk, providing direct and rapid detection of
Escherichia coli
,
Staphylococcus aureus
,
Streptococcus agalactiae
,
Streptococcus dysgalactiae
,
Streptococcus parauberis
, and
Streptococcus uberis
, have been developed. Two sets of specific primers were designed for each of these microorganisms and appeared to discriminate close phylogenic bacterial species (e.g.,
S. agalactiae
and
S. dysgalactiae
). In addition, two sets of universal primers were designed to react as positive controls with all major pathogens of bovine mastitis. The sensitivities of the test using
S. aureus
DNA extracted from milk with and without a pre-PCR enzymatic lysis step of bacterial cells were compared. The detection limit of the assay was 3.125 × 10
2
CFU/ml of milk when
S. aureus
DNA was extracted with the pre-PCR enzymatic step compared to 5 × 10
3
CFU/ml of milk in the absence of the pre-PCR enzymatic step. This latter threshold of sensitivity is still compatible with its use as an efficient tool of diagnosis in bovine mastitis, allowing the elimination of expensive reagents. The two PCR tests avoid cumbersome and lengthy cultivation steps, can be performed within hours, and are sensitive, specific, and reliable for the direct detection in milk of the six most prevalent bacteria causing bovine mastitis.
Comparison of a Multiplex Reverse Transcription-PCR-Enzyme Hybridization Assay with Conventional Viral Culture and Immunofluorescence Techniques for the Detection of Seven Viral Respiratory Pathogens
Authors: Liolios Lisa , Jenney Adam , Spelman Denis , Kotsimbos Tom , Catton Michael , Wesselingh Steve
Published: July 2002

A multiplex reverse transcription-PCR-enzyme hybridization assay (RT-PCR-EHA; Hexaplex; Prodesse Inc., Waukesha, Wis.) was used for the simultaneous detection of human parainfluenza virus types 1, 2, and 3, influenza virus types A and B, and respiratory syncytial virus types A and B. One hundred forty-three respiratory specimens from 126 patients were analyzed by RT-PCR-EHA, and the results were compared to those obtained by conventional viral culture and immunofluorescence (IF) methods. RT-PCR-EHA proved to be positive for 17 of 143 (11.9%) specimens, whereas 8 of 143 (5.6%) samples were positive by viral culture and/or IF. Eight samples were positive by both RT-PCR-EHA and conventional methods, while nine samples were RT-PCR-EHA positive and viral culture and IF negative. Eight of the nine samples with discordant results were then independently tested by a different multiplex RT-PCR assay for influenza virus types A and B, and all eight proved to be positive. In comparison to viral culture and IF methods, RT-PCR-EHA gave a sensitivity and a specificity of 100 and 93%, respectively. Since RT-PCR-EHA was able to detect more positive samples, which would otherwise have been missed by routine methods, we suggest that this multiplex RT-PCR-EHA provides a highly sensitive and specific means of diagnostic detection of major respiratory viruses.
Automated Multiplex Assay System for Simultaneous Detection of Hepatitis B Virus DNA, Hepatitis C Virus RNA, and Human Immunodeficiency Virus Type 1 RNA
Authors: Meng Q. , Wong C. , Rangachari A. , Tamatsukuri S. , Sasaki M. , Fiss E. , Cheng L. , Ramankutty T. , Clarke D. , Yawata H. , Sakakura Y. , Hirose T. , Impraim C.
Published: July 2002

We have developed an automated multiplex system for simultaneously screening hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type 1 (HIV-1) in blood donations. The assay, designated AMPLINAT MPX HBV/HCV/HIV-1 Test (AMPLINAT MPX), consists of virus extraction and target sequence-specific probe capture on specimen preparation workstation GT-X (Roche Diagnostics K.K., Tokyo, Japan) and amplification and detection by TaqMan PCR on the ABI PRISM 7700 Analyzer (Perkin-Elmer Applied Biosystems, Foster City, Calif.). An internal control (IC) is incorporated in the assay to monitor the extraction, target amplification, and detection processes. The assay yields qualitative results without discrimination of the three targets. Detection limits (95% confidence interval) are 22 to 60 copies/ml for HBV, 61 to 112 IU/ml for HCV, and 33 to 66 copies/ml for HIV-1, using a specimen input volume of 0.2 ml. The AMPLINAT MPX assay detects a broad range of genotypes or subtypes for all three viruses and has a specificity of 99.6% for all three viruses with seronegative specimens. In an evaluation of seroconversion panels, the AMPLINAT MPX assay detects HBV infection an average of 24 days before the detection of HBsAg by enzyme immunoassay. HCV RNA was detected an average of 31 days before HCV antibody. HIV-1 RNA was detected an average of 14 days before HIV-1 antibody and an average of 9 days before p24 antigen. The Japanese Red Cross has been evaluating the AMPLINAT MPX system since October 1999. The clinical performance indicates that the AMPLINAT MPX system is robust, sensitive, and reproducible, with a high percentage of valid assay runs (96.8%), a low false-positive rate (0.34%), and a low IC failure rate (0.24%).
Mutations in the
rpoB
Gene of Multidrug-Resistant
Mycobacterium tuberculosis
Clinical Isolates from India
Authors: Mani Cheruvu , Selvakumar N. , Narayanan Sujatha , Narayanan P. R.
Published: July 2002

Mutations in the 81-bp rifampin resistance-determining region (RRDR) of the
rpoB
gene were analyzed by DNA sequencing of 50
Mycobacterium tuberculosis
clinical isolates (44 resistant and 6 sensitive) from various parts of India. Fifty-three mutations of 18 different kinds, 17 point mutations and one deletion, were observed in 43 of 44 resistant isolates. Three novel mutations and three new alleles within the RRDR, along with two novel mutations outside the RRDR, are reported in this study.
Identification of Novel
Helicobacter
Species in Pig Stomachs by PCR and Partial Sequencing
Authors: Choi Young K. , Han Jeong H. , Joo Han S.
Published: July 2002

Evidence of infection with
Helicobacter
species in pig stomach was investigated by the use of a PCR with
Helicobacter
genus-specific primers. Forty pig stomachs, each of four different ulcer lesion grades, 0, 1, 2, and 3 in the
pars esophagea
area, were collected from a slaughterhouse in Minnesota. Of 160 stomach samples examined, 102 (63.8%) were positive by the PCR assay. The 40 samples each of lesion grades 0, 1, 2, and 3 showed 22.5, 52.5, 85.0, and 95.0% PCR-positive results, respectively. There was a significant trend (
P
≤ 0.01) in the proportions of PCR-positive cases relative to severity of the lesion. About 80% of the 16S rRNA gene was amplified, and PCR-restriction fragment length polymorphism (RFLP) patterns were analyzed. Of 102 PCR-positive samples, the PCR-RFLP patterns resulted in four different types, 32 samples being classified into type MN 1, 16 samples into type MN 2, 43 samples into type MN 3, and 11 samples into type MN 4. When the sequences of each RFLP type were compared to those reported in databases by using BLAST software, types MN 1, MN 2, MN 3, and MN 4 showed homologies of 97.3, 95.4, 96.7, and 99.5% with the 16S ribosomal DNA of
Helicobacter flexispira
taxon 3,
Helicobacter
sp. strains MIT 94-022 and MZ 640285, and
Helicobacter suis
, respectively. None of the 102 samples positive for the
Helicobacter
genus were positive with a primer set specific for
Helicobacter pylori
. Attempts to culture the organisms from selected stomachs in vitro were unsuccessful.
Molecular Analysis of
Neorickettsia risticii
in Adult Aquatic Insects in Pennsylvania, in Horses Infected by Ingestion of Insects, and Isolated in Cell Culture
Authors: Mott Jason , Muramatsu Yasukazu , Seaton Elizabeth , Martin Carol , Reed Stephen , Rikihisa Yasuko
Published: July 2002

Upon ingestion of adult aquatic insects, horses developed clinical signs of Potomac horse fever, and
Neorickettsia risticii
was isolated from the blood. 16S rRNA and 51-kDa antigen gene sequences from blood, isolates, and caddis flies fed to the horses were identical, proving oral transmission of
N. risticii
from caddis flies to horses.
Initial Concentration of
Staphylococcus epidermidis
in Simulated Pediatric Blood Cultures Correlates with Time to Positive Results with the Automated, Continuously Monitored BACTEC Blood Culture System
Authors: Haimi-Cohen Yishai , Vellozzi Ernestine M. , Rubin Lorry G.
Published: July 2002

The relationship of initial concentration of
Staphylococcus epidermidis
in blood cultures and time to positivity (TTP) in an automated, continuously monitored blood culture system was assessed. Blood and 1 to 1,000 CFU of
S. epidermidis
per ml in stationary or exponential phase were inoculated in BACTEC Pediatric Plus F bottles and incubated. The TTP was inversely proportional to the initial inoculated concentration. Blood culture bottles with initial bacterial densities of <10 CFU/ml had a TTP of >20 h (upper limit of 95% prediction interval, 20.7 h) and bottles with initial bacterial densities of ≥50 CFU/ml had a TTP of ≤15 h (lower limit of 95% prediction interval, 13.4 h).
Enterococcus gilvus
sp. nov. and
Enterococcus pallens
sp. nov. Isolated from Human Clinical Specimens
Authors: Tyrrell Gregory J. , Turnbull LeeAnn , Teixeira Lúcia M. , Lefebvre Johanne , Carvalho Maria da Glória S. , Facklam Richard R. , Lovgren Marguerite
Published: July 2002

Light yellow-pigmented (strain PQ1) and yellow-pigmented (strain PQ2), gram-positive, non-spore-forming, nonmotile bacteria consisting of pairs or chains of cocci were isolated from the bile of a patient with cholecystitis (PQ1) and the peritoneal dialysate of another patient with peritonitis (PQ2). Morphologically and biochemically, the organisms phenotypically belonged to the genus
Enterococcus
. Whole-cell protein (WCP) analysis and sequence analysis of a segment of the 16S rRNA gene suggested that they are new species within the genus
Enterococcus
. PQ1 and PQ2 displayed less than 70% identities to other enterococcal species by WCP analysis. Sequence analysis showed that PQ1 shared the highest level of sequence similarity with
Enterococcus raffinosus
and
E. malodoratus
(sequence similarities of 99.8% to these two species). Sequence analysis of PQ2 showed that it had the highest degrees of sequence identity with the group I enterococci
E. malodoratus
(98.7%),
E. raffinosus
(98.6%),
E. avium
(98.6%), and
E. pseudoavium
(98.6%). PQ1 and PQ2 can be differentiated from the other
Enterococcus
spp. in groups II, III, IV, and V by their phenotypic characteristics: PQ1 and PQ2 produce acid from mannitol and sorbose and do not hydrolyze arginine, placing them in group I. The yellow pigmentation differentiates these strains from the other group I enterococci. PQ1 and PQ2 can be differentiated from each other since PQ1 does not produce acid from arabinose, whereas PQ2 does. Also, PQ1 is
Enterococcus
Accuprobe assay positive and pyrrolidonyl-β-naphthylamide hydrolysis positive, whereas PQ2 is negative by these assays. The name
Enterococcus gilvus
sp. nov. is proposed for strain PQ1, and the name
Enterococcus pallens
sp. nov. is proposed for strain PQ2. Type strains have been deposited in culture collections as
E. gilvus
ATCC BAA-350 (CCUG 45553) and
E. pallens
ATCC BAA-351 (CCUG 45554).
Dynamics of Dissemination and Outer Surface Protein Expression of Different European
Borrelia burgdorferi
Sensu Lato Strains in Artificially Infected
Ixodes ricinus
Nymphs
Authors: Fingerle Volker , Rauser Sandra , Hammer Bettina , Kahl Olaf , Heimerl Christiane , Schulte-Spechtel Ulrike , Gern Lise , Wilske Bettina
Published: July 2002

Unfed
Ixodes ricinus
nymphs were infected with eight different strains and clones of
Borrelia afzelii
and
B. garinii
by capillary feeding. Except one
B. afzelii
clone, all expressed OspC in culture. Tick midguts and salivary glands were investigated at different time intervals for the presence of borreliae and for OspA and OspC phenotypes by immunofluorescence with simultaneous staining of OspA and OspC with monoclonal antibodies. Both species were transmittable to
I. ricinus
. All OspC-expressing strains and clones were able to disseminate into the salivary glands. In contrast, the OspC-negative
B. afzelii
clone was not detectable in the salivary glands, an indication that OspC plays an important role in dissemination. OspA-positive borreliae prevailed in the midgut. OspC positives were more frequent in the salivary glands than in the midgut. Notably, simultaneously OspA- and OspC-negative borreliae were detected in both organs. Kinetics of dissemination varied with the strains. The OspC-positive
B. afzelii
clone and all
B. garinii
OspA type 4 strains were detectable in the salivary glands right after feeding, while one
B. garinii
OspA type 6 strain invaded the salivary glands with a delay of 24 h. These findings support the hypothesis that OspA is abundantly expressed in unfed ticks while upregulation of OspC is also a prerequisite for dissemination in the vector for the Eurasian species
B. afzelii
and
B. garinii
. However, we found strain-specific dynamics of Osp expression and strain-specific kinetics of systemic infection in the vector tick and it appears that additional factors are involved in the initiation and regulation of the dissemination process.
Evaluation of the Abbott LCx Quantitative Assay for Measurement of Human Immunodeficiency Virus RNA in Plasma
Authors: de Mendoza Carmen , Alcamí José , Sainz María , Folgueira Dolores , Soriano Vincent
Published: July 2002

Plasma human immunodeficiency virus RNA in 491 clinical specimens was measured by LCx. There was a strong correlation with the results provided by other methods (
r
2
values of 0.93 with Cobas-Monitor version 1.5 and of 0.95 with Quantiplex version 3.0). However, values were uniformly higher with LCx than with Quantiplex when non-B subtypes were tested.
Evaluation of the Directigen FluA+B Test for Rapid Diagnosis of Influenza Virus Type A and B Infections
Authors: Chan K. H. , Maldeis N. , Pope W. , Yup A. , Ozinskas A. , Gill J. , Seto W. H. , Shortridge K. F. , Peiris J. S. M.
Published: July 2002

Directigen FluA+B (BD Diagnostic Systems, Sparks, Md.), a new rapid test for the detection of influenza virus types A and B, was evaluated with nasopharyngeal aspirate specimens collected from 250 patients in comparison with culture and direct fluorescent antigen (DFA) detection tests. The patients studied were predominantly children, 80% being ≤6 years old. Specimens negative by culture but positive by the Directigen FluA+B or DFA tests were analyzed by reverse transcription-PCR to resolve the discrepant results. The resolved sensitivity, specificity, and positive and negative predictive values of the Directigen FluA+B test for influenza virus type A were 96%, 99.6%, 96%, and 99.6%, respectively, and for influenza virus type B they were 87.5%, 96.8%, 80%, and 98%, respectively. Storage of nasopharyngeal aspirates in virus transport medium at 2 to 8°C for 48 h had little adverse effect on the detection of influenza virus type A, but diagnosis of influenza virus type B is best carried out with fresh specimens. The test detected a range of human and animal influenza virus A subtypes, including the H5N1 and H9N2 viruses that recently caused human disease in Hong Kong.
Epidemiological Dynamics of Hepatitis C Virus among 747 German Individuals: New Subtypes on the Advance
Authors: Schröter Matthias , Zöllner Bernhard , Schäfer Peter , Reimer Andreas , Müller Matthias , Laufs Rainer , Feucht Heinz-Hubert
Published: July 2002

This study demonstrates the dynamics in the epidemiology of hepatitis C virus subtypes. Subtypes 3a and 4a have become increasingly prevalent in patients where an infection within recent years can be assumed. Evidence is presented that the subtypes observed among younger patients can spread rapidly and lead to significant changes in the subtype distribution.
Development of a Sensitive and Specific Enzyme-Linked Immunosorbent Assay for Detecting and Quantifying CMY-2 and SHV β-Lactamases
Authors: Hujer Andrea M. , Page Malcolm G. P. , Helfand Marion S. , Yeiser Bethany , Bonomo Robert A.
Published: July 2002

Polyclonal rabbit antibodies against SHV-1 and CMY-2 β-lactamases were produced and characterized, and enzyme-linked immunosorbent assays (ELISAs) were developed. Immunoblots revealed that the anti-SHV-1 antibody recognized SHV-1 but did not recognize TEM-1, K-1, OXA-1, or any AmpC β-lactamase tested. The anti-CMY-2 antibody detected
Escherichia coli
CMY-2,
Enterobacter cloacae
P99,
Klebsiella pneumoniae
ACT-1, and the AmpC β-lactamases of
Enterobacter aerogenes
,
Morganella morganii
, and
Citrobacter freundii
. No cross-reactivity of the anti-CMY-2 antibody was seen against laboratory strains of
E. coli
possessing TEM-1, SHV-1, K-1, or OXA-1 β-lactamases. Operating conditions for performing ELISAs were optimized. Both anti-CMY-2 and anti-SHV-1 antibodies detected picogram quantities of purified protein in ELISAs. The reactivity of the anti-CMY-2 antibody was tested against a number of AmpC β-lactamases by assaying known quantities of purified enzymes in ELISAs (AmpC β-lactamases of
M. morganii
,
C. freundii
,
E. coli
, and
E. cloacae
). As the homology to CMY-2 β-lactamase decreased, the minimum level needed for detection increased (e.g., 94% homology recognized at 1 ng/ml and 71% homology recognized at 10 ng/ml). The ELISAs were used to assay unknown clinical isolates for AmpC and SHV β-lactamases, and the results were confirmed with PCR amplification of
bla
AmpC
and
bla
SHV
genes. Overall, we found that our ELISAs were at least 95% sensitive and specific for detecting SHV and AmpC β-lactamases. The ELISA format can facilitate the identification of AmpC and SHV β-lactamases and can be used to quantify relative amounts of β-lactamase enzymes in clinical and laboratory isolates.
Detection of Smallpox Virus DNA by LightCycler PCR
Authors: Espy Mark J. , Cockerill Franklin R. , Meyer Richard F. , Bowen Michael D. , Poland Gregory A. , Hadfield Ted L. , Smith Thomas F.
Published: July 2002

A 300-bp plasmid fragment of the hemagglutinin gene was used as target DNA to develop a rapid real-time LightCycler (Roche Applied Science, Indianapolis, Ind.) PCR assay for laboratory detection of smallpox virus. PCR primers and probes were designed specifically for detection of smallpox virus DNA, but all viruses of the genus
Orthopoxvirus
tested could be detected by use of the hemagglutinin gene target sequence. Base pair mismatches in the 204-bp amplicon allowed discrimination of cowpox virus (melting temperature [

T
m

], 56.40°C), monkeypox virus (

T
m

, 56.24°C), and vaccinia virus (

T
m

, 56.72°C), including the Dryvax vaccine strain, from smallpox virus (

T
m

, 62.45°C) by melting curve analysis. The analytical sensitivity was 5 to 10 copies of target DNA per sample. The assay was specific for members of the genus
Orthopoxvirus
; the DNAs of herpes simplex virus and varicella-zoster virus were not detected by the smallpox virus LightCycler PCR.