bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of a Rapid, Quantitative Real-Time PCR Method for Enumeration of Pathogenic
Candida
Cells in Water
Authors: Brinkman Nichole E. , Haugland Richard A. , Wymer Larry J. , Byappanahalli Muruleedhara , Whitman Richard L. , Vesper Stephen J.
Published: March 2003

Quantitative PCR (QPCR) technology, incorporating fluorigenic 5′ nuclease (TaqMan) chemistry, was utilized for the specific detection and quantification of six pathogenic species of
Candida
(
C. albicans
,
C. tropicalis
,
C. krusei
,
C. parapsilosis
,
C. glabrata
and
C. lusitaniae
) in water. Known numbers of target cells were added to distilled and tap water samples, filtered, and disrupted directly on the membranes for recovery of DNA for QPCR analysis. The assay's sensitivities were between one and three cells per filter. The accuracy of the cell estimates was between 50 and 200% of their true value (95% confidence level). In similar tests with surface water samples, the presence of PCR inhibitory compounds necessitated further purification and/or dilution of the DNA extracts, with resultant reductions in sensitivity but generally not in quantitative accuracy. Analyses of a series of freshwater samples collected from a recreational beach showed positive correlations between the QPCR results and colony counts of the corresponding target species. Positive correlations were also seen between the cell quantities of the target
Candida
species detected in these analyses and colony counts of
Enterococcus
organisms. With a combined sample processing and analysis time of less than 4 h, this method shows great promise as a tool for rapidly assessing potential exposures to waterborne pathogenic
Candida
species from drinking and recreational waters and may have applications in the detection of fecal pollution.
Kinetics and Strain Specificity of Rhizosphere and Endophytic Colonization by Enteric Bacteria on Seedlings of
Medicago sativa
and
Medicago truncatula
Authors: Dong Yuemei , Iniguez A. Leonardo , Ahmer Brian M. M. , Triplett Eric W.
Published: March 2003

The presence of human-pathogenic, enteric bacteria on the surface and in the interior of raw produce is a significant health concern. Several aspects of the biology of the interaction between these bacteria and alfalfa (
Medicago sativa
) seedlings are addressed here. A collection of enteric bacteria associated with alfalfa sprout contaminations, along with
Escherichia coli
K-12,
Salmonella enterica
serotype Typhimurium strain ATCC 14028, and an endophyte of maize,
Klebsiella pneumoniae
342, were labeled with green fluorescent protein, and their abilities to colonize the rhizosphere and the interior of the plant were compared. These strains differed widely in their endophytic colonization abilities, with
K. pneumoniae
342 and
E. coli
K-12 being the best and worst colonizers, respectively. The abilities of the pathogens were between those of
K. pneumoniae
342 and
E. coli
K-12. All
Salmonella
bacteria colonized the interiors of the seedlings in high numbers with an inoculum of 10
2
CFU, although infection characteristics were different for each strain. For most strains, a strong correlation between endophytic colonization and rhizosphere colonization was observed. These results show significant strain specificity for plant entry by these strains. Significant colonization of lateral root cracks was observed, suggesting that this may be the site of entry into the plant for these bacteria. At low inoculum levels, a symbiosis mutant of
Medicago truncatula
,
dmi1
, was colonized in higher numbers on the rhizosphere and in the interior by a
Salmonella
endophyte than was the wild-type host. Endophytic entry of
M. truncatula
appears to occur by a mechanism independent of the symbiotic infections by
Sinorhizobium meliloti
or mycorrhizal fungi.
Enhanced Accumulation of Cd
2+
by a
Mesorhizobium
sp. Transformed with a Gene from
Arabidopsis thaliana
Coding for Phytochelatin Synthase
Authors: Sriprang Rutchadaporn , Hayashi Makoto , Ono Hisayo , Takagi Masahiro , Hirata Kazumasa , Murooka Yoshikatsu
Published: March 2003

We expressed the
Arabidopsis thaliana
gene for phytochelatin synthase (

PCS
At

) in
Mesorhizobium huakuii
subsp.
rengei
B3, a microsymbiont of
Astragalus sinicus
, a legume used as manure. The

PCS
At

gene was expressed under the control of the
nifH
promoter, which regulates the nodule-specific expression of the
nifH
gene. The expression of the

PCS
At

gene was demonstrated in free-living cells under low-oxygen conditions. Phytochelatin synthase (PCS) was expressed and catalyzed the synthesis of phytochelatins [(γ-Glu-Cys)

n

-Gly; PCs] in strain B3. A range of PCs, with values of
n
from 2 to 7, was synthesized by cells that expressed the

PCS
At

gene, whereas no PCs were found in control cells that harbored the empty plasmid. The presence of CdCl
2
activated PCS and induced the synthesis of substantial amounts of PCs. Cells that contained PCs accumulated 36 nmol of Cd
2+
/mg (dry weight) of cells. The expression of the

PCS
At

gene in
M. huakuii
subsp.
rengei
B3 increased the ability of cells to bind Cd
2+
approximately 9- to 19-fold. The PCS protein was detected by immunostaining bacteroids of mature nodules of
A. sinicus
containing the

PCS
At

gene. When recombinant
M. huakuii
subsp.
rengei
B3 established the symbiotic relationship with
A. sinicus
, the symbionts increased Cd
2+
accumulation in nodules 1.5-fold.
Strong Synergy between a Eukaryotic Antimicrobial Peptide and Bacteriocins from Lactic Acid Bacteria
Authors: Lüders Torben , Birkemo Gunn Alice , Fimland Gunnar , Nissen-Meyer Jon , Nes Ingolf F.
Published: March 2003

The antimicrobial effect obtained upon combining the prokaryotic antimicrobial peptides (AMPs; more commonly referred to as bacteriocins) pediocin PA-1, sakacin P, and curvacin A (all produced by lactic acid bacteria [LAB]) with the eukaryotic AMP pleurocidin (from fish) has been investigated. The three LAB AMPs alone were active against gram-positive
Listeria ivanovii
bacteria at nanomolar concentrations, whereas they were inactive against gram-negative
Escherichia coli
bacteria. Pleurocidin alone was active against both of these types of bacteria at micromolar concentrations. Little if any synergy between the LAB AMPs and pleurocidin against the gram-positive
L. ivanovii
strain was obtained. In contrast, the LAB AMPs and pleurocidin acted highly synergistically against the gram-negative
E. coli
strain. Nanomolar concentrations of LAB AMPs increased the growth inhibitory potency of pleurocidin by about fourfold. When micromolar concentrations of LAB AMPs were combined with 2 μg of pleurocidin/ml, 100% growth inhibition was attained, whereas pleurocidin alone at a concentration of 2 μg/ml gave no growth inhibition. Most noteworthy, when high concentrations (128 μg/ml) of pleurocidin in the absence of LAB AMPs were used over a long period of incubation (1 week), some growth of
E. coli
was observed, whereas 16 μg of pleurocidin/ml completely abolished growth in the presence of 64 to 128 ng of LAB AMPs/ml over the same period of time. The results clearly demonstrate that combining eukaryotic and prokaryotic AMPs can greatly increase the specific activity and broaden the target-cell range of these peptides.
Soil Type Is the Primary Determinant of the Composition of the Total and Active Bacterial Communities in Arable Soils
Authors: Girvan Martina S. , Bullimore Juliet , Pretty Jules N. , Osborn A. Mark , Ball Andrew S.
Published: March 2003

Degradation of agricultural land and the resulting loss of soil biodiversity and productivity are of great concern. Land-use management practices can be used to ameliorate such degradation. The soil bacterial communities at three separate arable farms in eastern England, with different farm management practices, were investigated by using a polyphasic approach combining traditional soil analyses, physiological analysis, and nucleic acid profiling. Organic farming did not necessarily result in elevated organic matter levels; instead, a strong association with increased nitrate availability was apparent. Ordination of the physiological (BIOLOG) data separated the soil bacterial communities into two clusters, determined by soil type. Denaturing gradient gel electrophoresis and terminal restriction fragment length polymorphism analyses of 16S ribosomal DNA identified three bacterial communities largely on the basis of soil type but with discrimination for pea cropping. Five fields from geographically distinct soils, with different cropping regimens, produced highly similar profiles. The active communities (16S rRNA) were further discriminated by farm location and, to some degree, by land-use practices. The results of this investigation indicated that soil type was the key factor determining bacterial community composition in these arable soils. Leguminous crops on particular soil types had a positive effect upon organic matter levels and resulted in small changes in the active bacterial population. The active population was therefore more indicative of short-term management changes.
PCR Detection of Virulence Genes in
Yersinia enterocolitica
and
Yersinia pseudotuberculosis
and Investigation of Virulence Gene Distribution
Authors: Thoerner P. , Bin Kingombe C. I. , Bögli-Stuber K. , Bissig-Choisat B. , Wassenaar T. M. , Frey J. , Jemmi T.
Published: March 2003

PCR-based assays were developed for the detection of plasmid- and chromosome-borne virulence genes in
Yersinia enterocolitica
and
Yersinia pseudotuberculosis
, to investigate the distribution of these genes in isolates from various sources. The results of PCR genotyping, based on 5 virulence-associated genes of 140 strains of
Y. enterocolitica
, were compared to phenotypic tests, such as biotyping and serotyping, and to virulence plasmid-associated properties such as calcium-dependent growth at 37°C and Congo red uptake. The specificity of the PCR results was validated by hybridization. Genotyping data correlated well with biotype data, and most biotypes resulted in (nearly) homogeneous genotypes for the chromosomal virulence genes (
ystA
,
ystB
, and
ail
); however, plasmid-borne genes (
yadA
and
virF
) were detected with variable efficiency, due to heterogeneity within the bacterial population for the presence of the virulence plasmid. Of the virulence genes, only
ystB
was present in biotype 1A; however, within this biotype, pathogenic and apathogenic isolates could not be distinguished based on the detection of virulence genes. Forty
Y. pseudotuberculosis
isolates were tested by PCR for the presence of
inv
,
yadA
, and
lcrF
. All isolates were
inv
positive, and 88% of the isolates contained the virulence plasmid genes
yadA
and
lcrF
. In conclusion, this study shows that genotyping of
Yersinia
spp., based on both chromosome- and plasmid-borne virulence genes, is feasible and informative and can provide a rapid and reliable genotypic characterization of field isolates.
Inactivation of
gacS
Does Not Affect the Competitiveness of
Pseudomonas chlororaphis
in the
Arabidopsis thaliana
Rhizosphere
Authors: Schmidt-Eisenlohr Heike , Gast Astrid , Baron Christian
Published: March 2003

Quorum-sensing-controlled processes are considered to be important for the competitiveness of microorganisms in the rhizosphere. They affect cell-cell communication, biofilm formation, and antibiotic production, and the GacS-GacA two-component system plays a role as a key regulator. In spite of the importance of this system for the regulation of various processes, strains with a Gac
−
phenotype are readily recovered from natural habitats. To analyze the influence of quorum sensing and the influence of the production of the antibiotic phenazine-1-carboxamide on rhizosphere colonization by
Pseudomonas chlororaphis
, a gnotobiotic system based on
Arabidopsis thaliana
seedlings in soil was investigated. Transposon insertion mutants of
P. chlororaphis
isolate SPR044 carrying insertions in different genes required for the production of
N
-acyl-homoserine lactones and phenazine-1-carboxamide were generated. Analysis of solitary rhizosphere colonization revealed that after prolonged growth, the population of the wild type was significantly larger than that of the homoserine lactone-negative
gacS
mutant and that of a phenazine-1-carboxamide-overproducing strain. In cocultivation experiments, however, the population size of the
gacS
mutant was similar to that of the wild type after extended growth in the rhizosphere. A detailed analysis of growth kinetics was performed to explain this phenomenon. After cells grown to the stationary phase were transferred to fresh medium, the
gacS
mutant had a reduced lag phase, and production of the stationary-phase-specific sigma factor RpoS was strongly reduced. This may provide a relative competitive advantage in cocultures with other bacteria, because it permits faster reinitiation of growth after a change to nutrient-rich conditions. In addition, delayed entry into the stationary phase may allow more efficient nutrient utilization. Thus, GacS-GacA-regulated processes are not absolutely required for efficient rhizosphere colonization in populations containing the wild type and Gac
−
mutants.
The Competitiveness of
Pseudomonas chlororaphis
Carrying pJP4 Is Reduced in the
Arabidopsis thaliana
Rhizosphere
Authors: Schmidt-Eisenlohr Heike , Baron Christian
Published: March 2003

The effect of the large catabolic IncP plasmid pJP4 on the competitiveness of
Pseudomonas chlororaphis
SPR044 and on its derivatives SPR244 (GacS deficient), SPR344 (phenazine-1-carboxamide overproducer), and SPR644 (phenazine-1-carboxamide deficient) in the
Arabidopsis thaliana
rhizosphere was assessed. Solitary rhizosphere colonization by the wild type, SPR244, and SPR644 was not affected by the plasmid. The size of the population of SPR344 carrying pJP4, however, was significantly reduced compared to the size of the population of the plasmid-free derivative. The abiotic stress caused by phenazine-1-carboxamide overproduction probably resulted in a selective disadvantage for cells carrying pJP4. Next, the effect of biotic stress caused by coinoculation of other bacteria was analyzed. Cells carrying pJP4 had a selective disadvantage compared to plasmid-free cells in the presence of the efficient colonizer
Pseudomonas fluorescens
WCS417r. This effect was not observed after coinoculation with a variety of other bacteria, and it was independent of quorum sensing and phenazine-1-carboxamide production. Thus, the presence of large catabolic plasmids imposes a detectable metabolic burden in the presence of biotic stress. Plasmid transfer in the
A. thaliana
rhizosphere from
P. chlororaphis
and its derivatives to
Ralstonia eutropha
was determined by using culture-dependent and culture-independent techniques. With the cultivation-independent technique we detected a significantly higher portion of exconjugants, but pJP4 transfer was independent of the quorum-sensing system and of phenazine-1-carboxamide production.
Evidence for the Existence of Psychrophilic Methanogenic Communities in Anoxic Sediments of Deep Lakes
Authors: Nozhevnikova Alla N. , Zepp Kornelia , Vazquez Francisco , Zehnder Alexander J. B. , Holliger Christof
Published: March 2003

In order to obtain evidence for the existence of psychrophilic methanogenic communities in sediments of deep lakes that are low-temperature environments (4 to 5°C), slurries were first incubated at temperatures between 4 and 60°C for several weeks, at which time they were amended, or not, with an additional substrate, such as cellulose, butyrate, propionate, acetate, or hydrogen, and further incubated at 6°C. Initial methane production rates were highest in slurries preincubated at temperatures between 4 and 15°C, with maximal rates in slurries kept at 6°C. Hydrogen-amended cultures were the only exceptions, with the highest methane production rates at 6°C after preincubation at 30°C.
Comparison of Ribotyping and Repetitive Extragenic Palindromic-PCR for Identification of Fecal
Escherichia coli
from Humans and Animals
Authors: Carson C. Andrew , Shear Brian L. , Ellersieck Mark R. , Schnell Jennifer D.
Published: March 2003

This report compares the performances of two popular genotypic methods used for tracking the sources of fecal pollution in water, ribotyping and repetitive extragenic palindromic-PCR (rep-PCR). The rep-PCR was more accurate, reproducible, and efficient in associating DNA fingerprints of fecal
Escherichia coli
with human and animal hosts of origin.
Antigenic Hepatitis A Virus Structures May Be Produced in
Escherichia coli
Authors: Sánchez Glòria , Caballero Santiago , Guix Susana , Bosch Albert , Pintó Rosa M.
Published: March 2003

The synthesis of 14S pentamers and 70S empty capsids of hepatitis A virus (HAV) has been accomplished by expressing the viral genome for periods of time longer than 4 h in
Escherichia coli
. HAV pentamers (14S) self-assembled into capsids (70S) in vitro. The antibodies induced by these structures recognized and neutralized HAV.
Comparison of Methods for DNA Isolation from Food Samples for Detection of Shiga Toxin-Producing
Escherichia coli
by Real-Time PCR
Authors: Heller Loree C. , Davis Carisa R. , Peak K. Kealy , Wingfield David , Cannons Andrew C. , Amuso Philip T. , Cattani Jacqueline
Published: March 2003

In this study, food samples were intentionally contaminated with
Escherichia coli
O157:H7, and then DNA was isolated by using four commercial kits. The isolated DNA samples were compared by using real-time PCR detection of the Shiga toxin genes. The four kits tested worked similarly.
Cr(VI) Reduction by Sulfidogenic and Nonsulfidogenic Microbial Consortia
Authors: Arias Y. Meriah , Tebo Bradley M.
Published: March 2003

In time course experiments, bacterial community compositions were compared between a sulfidogenic and two nonsulfidogenic Cr(VI)-reducing consortia enriched from metal-contaminated sediments. The consortia were subjected to 0 and 0.85 mM or 1.35 mM Cr(VI), and Cr(VI) reduction, growth, and denaturing gradient gel electrophoresis profiles of PCR products of small-subunit (16S) ribosomal genes were compared. Results showed that although Cr(VI) was completely reduced by the three consortia, Cr(VI) inhibited cell growth, with sulfate-reducing bacteria being particularly sensitive to Cr(VI) toxicity relative to other bacteria in the consortia.
A Pediocin-Producing
Lactobacillus plantarum
Strain Inhibits
Listeria monocytogenes
in a Multispecies Cheese Surface Microbial Ripening Consortium
Authors: Loessner Melanie , Guenther Susanne , Steffan Sandra , Scherer Siegfried
Published: March 2003

The growth of
Listeria monocytogenes
WSLC 1364, originating from a cheese-borne outbreak, was examined in the presence and in the absence of a pediocin AcH-producing
Lactobacillus plantarum
strain on red smear cheese. Nearly complete inhibition was observed at 10
2
CFU of
L. monocytogenes
per ml of salt brine solution, while contamination with
Listeria
mutants resistant to pediocin resulted in high cell counts of the pathogen on the cheese surface. The inhibition was due to pediocin AcH added together with the
L. plantarum
culture to the brine solution but not to bacteriocin production in situ on cheese. Pediocin resistance developed in vitro at different but high frequencies in all 12
L. monocytogenes
strains investigated, and a resistant mutant remained stable in a microbial surface ripening consortium over a 4-month production process in the absence of selection pressure. In conclusion, the addition of a
L. plantarum
culture is a potent measure for combating
Listeria
in a contaminated production line, but because of the potential development of resistance, it should not be used continuously over a long time in a production line.
An Improved Enrichment Broth for Isolation of
Escherichia coli
O157, with Specific Reference to Starved Cells, from Radish Sprouts
Authors: Sata Shin , Fujisawa Tomohiko , Osawa Ro , Iguchi Atsushi , Yamai Shiro , Shimada Toshio
Published: March 2003

An enrichment broth was developed for the efficient isolation of
Escherichia coli
O157 from radish sprouts. The broth was buffered peptone water containing 0.5% sodium thioglycolate (STG-BPW), which was designed to allow growth of
E. coli
O157 in starved and unstarved states. However, this medium suppressed the growth of non-carbohydrate-fermenting obligate aerobes whose colonial appearance on sorbitol MacConkey agar containing cefixime and tellurite (CT-SMAC) resembled that of
E. coli
O157. Both starved and unstarved cells of
E. coli
O157 experimentally inoculated into radish sprouts were successfully recovered with STG-BPW enrichment in all cases, most of which showed marked disappearance of
E. coli
O157-like colonies on CT-SMAC.
Heterologous Expression of the
Saccharomyces cerevisiae PGU1
Gene in
Schizosaccharomyces pombe
Yields an Enzyme with More Desirable Properties for the Food Industry
Authors: Sieiro C. , Poza M. , Vilanova M. , Villa T. G.
Published: March 2003

The
Saccharomyces cerevisiae PGU1
gene was successfully expressed in
Schizosaccharomyces pombe
. The optimum pH and temperature for the recombinant enzyme were 5 and 40°C, respectively, these being around 0.5 U higher and 5°C lower than those shown by the native enzyme. The

K
m

value was about fourfold higher than that of the
S. cerevisiae
enzyme. The recombinant endopolygalacturonase was more efficient in reducing the viscosity of polygalacturonic acid and was also more stable at different pHs and temperatures than the native enzyme.
Anaerobic Mineralization of Quaternary Carbon Atoms: Isolation of Denitrifying Bacteria on Pivalic Acid (2,2-Dimethylpropionic Acid)
Authors: Probian Christina , Wülfing Annika , Harder Jens
Published: March 2003

The degradability of pivalic acid was established by the isolation of several facultative denitrifying strains belonging to
Zoogloea resiniphila
, to
Thauera
and
Herbaspirillum
, and to
Comamonadaceae
, related to [
Aquaspirillum
] and
Acidovorax
, and of a nitrate-reducing bacterium affiliated with
Moraxella osloensis
. Pivalic acid was completely mineralized to carbon dioxide. The catabolic pathways may involve an oxidation to dimethylmalonate or a carbon skeleton rearrangement, a putative 2,2-dimethylpropionyl coenzyme A mutase.
Biodegradation of the Nitramine Explosive CL-20
Authors: Trott Sandra , Nishino Shirley F. , Hawari Jalal , Spain Jim C.
Published: March 2003

The cyclic nitramine explosive CL-20 (2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane) was examined in soil microcosms to determine whether it is biodegradable. CL-20 was incubated with a variety of soils. The explosive disappeared in all microcosms except the controls in which microbial activity had been inhibited. CL-20 was degraded most rapidly in garden soil. After 2 days of incubation, about 80% of the initial CL-20 had disappeared. A CL-20-degrading bacterial strain,
Agrobacterium
sp. strain JS71, was isolated from enrichment cultures containing garden soil as an inoculum, succinate as a carbon source, and CL-20 as a nitrogen source. Growth experiments revealed that strain JS71 used 3 mol of nitrogen per mol of CL-20.
Invasive
Streptococcus iniae
Infections Outside North America
Authors: Lau Susanna K. P. , Woo Patrick C. Y. , Tse Herman , Leung Kit-Wah , Wong Samson S. Y. , Yuen Kwok-Yung
Published: March 2003

Streptococcus iniae
, a fish pathogen causing infections in aquaculture farms worldwide, has only been reported to cause human infections in North America. In this article, we report the first two cases of invasive
S. iniae
infections in two Chinese patients outside North America. While the first patient presented with bacteremic cellulitis, which is the most common presentation in previous cases, the second patient represents the first recognized case of
S. iniae
osteomyelitis. Both
S. iniae
strains isolated from the two patients were either misidentified or unidentified by three commercial systems and were only identified by 16S rRNA gene sequencing. Since no currently available commercial system for bacterial identification includes
S. iniae
in its database, 16S rRNA gene sequencing is the most practical and reliable method to identify the bacterium at the moment. In contrast to the distinct genetic profile described previously in clinical isolates from Canada, the present two isolates and a clinical isolate from a Canadian patient were found to be genetically unrelated, as demonstrated by pulsed-field gel electrophoresis. Morphologically, colonies of both isolates were also larger, more beta-hemolytic and mucoid, which differ from the usual morphotype described for
S. iniae
. Owing to their habit of cooking and eating fresh fish, the Asian population is strongly associated with
S. iniae
infections. As a result of the difficulty in making microbiological diagnosis in patients with cellulitis and the problem of identification in most clinical microbiology laboratories, the prevalence of
S. iniae
infections, especially in the Asian population, may have been under-estimated.
Conventional Methods versus 16S Ribosomal DNA Sequencing for Identification of Nontuberculous Mycobacteria: Cost Analysis
Authors: Cook Victoria J. , Turenne Christine Y. , Wolfe Joyce , Pauls Ryan , Kabani Amin
Published: March 2003

The clinical profile of nontuberculous mycobacteria (NTM) has been raised by the human immunodeficiency virus and AIDS pandemic. Different laboratory techniques, often molecular based, are available to facilitate the rapid and accurate identification of NTM. The expense of these advanced techniques has been questioned. At the National Reference Center for Mycobacteriology and the Health Sciences Center, University of Manitoba, in Winnipeg, Canada, we performed a direct cost analysis of laboratory techniques for commercial DNA probe-negative (Gen-Probe, Inc., San Diego, Calif.), difficult-to-identify NTM. We compared the costs associated with conventional phenotypic methodology (biochemical testing, pigment production, growth, and colony characteristics) and genotypic methodology (16S ribosomal DNA [rDNA] sequence-based identification). We revealed a higher cost per sample with conventional methods, and this cost varied with organism characteristics: $80.93 for slowly growing, biochemically active NTM; $173.23 for slowly growing, biochemically inert NTM; and $129.40 for rapidly growing NTM. The cost per sample using 16S rDNA sequencing was $47.91 irrespective of organism characteristics, less than one-third of the expense associated with phenotypic identification of biochemically inert, slow growers. Starting with a pure culture, the turnaround time to species identification is 1 to 2 days for 16S rDNA sequencing compared to 2 to 6 weeks for biochemical testing. The accuracy of results comparing both methodologies is briefly discussed. 16S rDNA sequencing provides a cost-effective alternative in the identification of clinically relevant forms of probe-negative NTM. This concept is not only useful in mycobacteriology but also is highly applicable in other areas of clinical microbiology.
p24 Antigen Detection Assay Modified with a Booster Step for Diagnosis and Monitoring of Human Immunodeficiency Virus Type 1 Infection
Authors: Sutthent Ruengpung , Gaudart Narintorn , Chokpaibulkit Kulkanya , Tanliang Nattaya , Kanoksinsombath Chinda , Chaisilwatana Pongsakdi
Published: March 2003

We modified a p24 antigen enzyme-linked immunosorbent assay as a method for diagnosis and monitoring of human immunodeficiency virus type 1 (HIV-1) subtype E infection. This modified assay is based on the use of preheated immune complex dissociation combined with a booster step using a regular Vironostika HIV-1 p24 antigen assay (bioMerieux) to decrease the lower limit of p24 antigen detection from 10 pg/ml (lower limit achievable when using a regular p24 antigen assay) to 0.5 pg/ml (100 virions/ml) by the new method. The correlation between the values obtained by the HIV-1 RNA (Amplicor HIV-1 Monitor) assay and the p24 antigen assay modified with a booster step antigen assay in 160 frozen plasma samples with known viral load and 80 blind fresh plasma samples by Spearman rank were 0.671 (R2= 0.450;P< 0.01) and 0.782 (R2= 0.612;P< 0.01). During antiretroviral treatment, the change of p24 antigen level at ≥0.5 log correlated well with the level of HIV-1 in plasma. In order to improve the early diagnosis of HIV-1 infection in 121 infants born to HIV-1-infected mothers, a heat-denatured plasma p24 antigen assay modified with a booster step was compared with DNA-PCR and HIV RNA (nucleic acid sequence-based amplification) assays. The sensitivity of the antigen test modified with a booster step was similar to that of the HIV-1 RNA (NASBA QL) assay and better than that of the DNA-PCR assay (100 versus 61.90%) for subjects 1 to 2 months old. The overall results from this study might renew interest in p24 antigen detection as an easily affordable alternative method for diagnosis of HIV-1 infection and monitoring of disease progression in developing countries.
Epidemiology of Tetracycline Resistance Determinants in
Shigella
spp. and Enteroinvasive
Escherichia coli
: Characterization and Dissemination of
tet
(A)-
1
Authors: Hartman Antoinette B. , Essiet Idongesit I. , Isenbarger Daniel W. , Lindler Luther E.
Published: March 2003

To make a comprehensive study of tetracycline resistance determinant distribution in the genus
Shigella
, a collection of 577 clinical isolates of
Shigella
spp. and enteroinvasive
Escherichia coli
(EIEC) from a variety of geographical locations was screened to identify tetracycline-resistant strains. The 459 tetracycline-resistant isolates identified were then screened by PCR analysis to determine the distribution in these strains of tetracycline efflux resistance determinants belonging to classes A to E, G, and H that have been identified in gram-negative bacteria. Only classes A to D were represented in these strains. Although Tet B was the predominant determinant in all geographical locations, there were geographical and species differences in the distribution of resistance determinants. An allele of
tet
(A), designated
tet
(A)-
1
, was identified and sequenced, and the 8.6-kb plasmid containing determinant Tet A-1, designated pSSTA-1, was found to have homologies to portions of a
Salmonella enterica
cryptic plasmid and the broad-host-range resistance plasmid RSF1010. This allele and pSSTA-1 were used as epidemiological markers to monitor clonal and horizontal transmission of determinant Tet A-1. An analysis of serotype, distribution of tetracycline resistance determinants, and resistance profiles indicated that both clonal spread and horizontal transfer had contributed to the spread of specific tetracycline resistance determinants in these populations and demonstrated the use of these parameters as an epidemiological tool to follow the transmission of determinants and strains.
Serology and Genetics of the Flagellar Antigen ofEscherichia coliO157:H7a,7c
Authors: Ratiner Yuli A. , Salmenlinna Saara , Eklund Marjut , Keskimäki Markku , Siitonen Anja
Published: March 2003

AmongEscherichia colistrains of the O55:H7 serovar, which is considered the ancestor of Shiga toxin-producingE. coli(STEC) O157:H7, two subtypes, H7a,7b and H7a,7c (briefly, H7a,b and H7a,c, respectively), of the H7 flagellar antigen have been described previously [J. Wright and R. Villanueva, J. Hyg. (Camb.) 51:39-48, 1953; Y. A. Ratiner and V. A. Sinelnikova, Zh. Microbiol. Epidemiol. Immunobiol. 3:111-116, 1969). We have now studied 13 STEC O157:H7 strains and 1 O55:H7 strain that were epidemiologically unrelated, that originated from six countries on two continents, and that had different profiles when analyzed by multilocus enzyme electrophoresis, pulsed-field gel electrophoresis, and PCR forstxandeae. They were all found to possess the H7a,c flagellar antigen. Serum cross-absorption assays confirmed that their H antigens were indistinguishable from each other and from that ofE. coliO55:H7a,c but differed from the standard H7a,b antigen ofE. coliH test strain U5/41. It was shown by phage-mediated transduction that the flagellin genes for these two H-antigen subserotypes were alleles of theE. coli fliClocus. On the basis of the serological data obtained in this study and the molecular characteristics ofE. coli fliCH7alleles recently published, it is inferred that H7a,c and H7a,b are the main serological subtypes of the group ofE. coliH7 flagellins.
Epitope-Blocking Enzyme-Linked Immunosorbent Assays for the Detection of Serum Antibodies to West Nile Virus in Multiple Avian Species
Authors: Blitvich Bradley J. , Marlenee Nicole L. , Hall Roy A. , Calisher Charles H. , Bowen Richard A. , Roehrig John T. , Komar Nicholas , Langevin Stanley A. , Beaty Barry J.
Published: March 2003

We report the development of epitope-blocking enzyme-linked immunosorbent assays (ELISAs) for the rapid detection of serum antibodies to West Nile virus (WNV) in taxonomically diverse North American avian species. A panel of flavivirus-specific monoclonal antibodies (MAbs) was tested in blocking assays with serum samples from WNV-infected chickens and crows. Selected MAbs were further tested against serum samples from birds that represented 16 species and 10 families. Serum samples were collected from birds infected with WNV or Saint Louis encephalitis virus (SLEV) and from noninfected control birds. Serum samples from SLEV-infected birds were included in these experiments because WNV and SLEV are closely related antigenically, are maintained in similar transmission cycles, and have overlapping geographic distributions. The ELISA that utilized MAb 3.1112G potentially discriminated between WNV and SLEV infections, as all serum samples from WNV-infected birds and none from SLEV-infected birds were positive in this assay. Assays with MAbs 2B2 and 6B6C-1 readily detected serum antibodies in all birds infected with WNV and SLEV, respectively, and in most birds infected with the other virus. Two other MAbs partially discriminated between infections with these two viruses. Serum samples from most WNV-infected birds but no SLEV-infected birds were positive with MAb 3.67G, while almost all serum samples from SLEV-infected birds but few from WNV-infected birds were positive with MAb 6B5A-5. The blocking assays reported here provide a rapid, reliable, and inexpensive diagnostic and surveillance technique to monitor WNV activity in multiple avian species.
Detection and Characterization of the Hemolysin Genes in
Aeromonas hydrophila
and
Aeromonas sobria
by Multiplex PCR
Authors: Wang Gehua , Clark Clifford G. , Liu Chenyi , Pucknell Chad , Munro Cindy K. , Kruk Tamara M. A. C. , Caldeira Richard , Woodward David L. , Rodgers Frank G.
Published: March 2003

A multiplex PCR assay was designed to amplify the
Aeromonas hydrophila
and
A. veronii
bv. sobria hemolysin and aerolysin genes. The assay was evaluated by using 121 clinical isolates and 7 reference strains of
Aeromonas
spp., and these were divided into five genotypes on the basis of the results of the multiplex PCR. The five genotypes were characterized as type 1 for those carrying the
ahh1
gene only (36% of isolates), type 2 for those carrying the
asa1
gene only (8.5% of isolates), type 3 for those carrying both the
ahh1
and the
asa1
genes (4% of isolates), type 4 for those carrying the
ahh1
gene and the A. hydrophila aerA (aerolysin) gene (37.5% of isolates), and type 5 for those in which no hemolysin genes were detected (14% of isolates). The most common single hemolysin gene carried among all the
Aeromonas
isolates examined was
ahh1
, with 99 of 128 (77%) of isolates testing positive for this gene either alone or in combination with other hemolysin genes. Phenotypic expression of toxins was evaluated in a Vero cell culture cytotoxicity assay. These results indicated that there is a statistically significant correlation between the cytotoxin titers and the hemolysin genotype. Isolates belonging to genotype 4 (carrying both the
ahh1
gene and the aerolysin and hemolysin
aerA
genes) expressed higher cytotoxin titers than isolates of the other genotypes (
P
< 0.001). These isolates were more cytotoxic in cell culture and may have greater clinical significance.
Use of Listeriolysin O and Internalin A in a Seroepidemiological Study of Listeriosis in Swiss Dairy Cows
Authors: Boerlin Patrick , Boerlin-Petzold Franziska , Jemmi Thomas
Published: March 2003

Recombinant listeriolysin O and internalin A were used as antigens in an enzyme-linked immunosorbent assay (ELISA) for the specific detection of anti-Listeria monocytogenesantibodies in cattle. The results showed sensitivities and specificities of 82 and 92%, respectively, for the listeriolysin O ELISA, and 100 and 90%, respectively, for the internalin A ELISA, respectively. The test may be useful for the confirmation of listeria-related abortions and mastitis but does not seem to be indicated for use in the diagnosis of listeria-related encephalitis in cattle. A representative sample of 1,652 serum samples from the healthy dairy cattle population in Switzerland was tested by both ELISAs. The results showed that 11% of the healthy dairy cows in Switzerland simultaneously presented antibodies toward listeriolysin O and internalin A, and 48% of the farms had one or several animals simultaneously positive by assays with both antigens. Multivariable analysis at the farm level confirmed that feeding of silage represents a significant risk factor for a positive listeria serology. Detailed analysis identified corn silage but not grass silage as the major factor in this association. Cattle breed and hygiene on the farm were also identified as significant factors associated with the serological status of farms. In conclusion, the results of the study show that internalin A is a promising new antigen for use in listeria serology and that specific anti-L. monocytogenesantibodies are found in a significant proportion of healthy dairy cows in Switzerland.
Comparison of Broth Microdilution, E Test, and Agar Dilution Methods for Antibiotic Susceptibility Testing of
Campylobacter jejuni
and
Campylobacter coli
Authors: Luber Petra , Bartelt Edda , Genschow Elke , Wagner Jutta , Hahn Helmut
Published: March 2003

A standardized broth microdilution method was compared to the E test and an agar dilution method for the antimicrobial susceptibility testing of
Campylobacter jejuni
and
C. coli
isolates. A group of 47 human clinical isolates, 37 isolates from retail poultry, and 29 isolates from living turkeys (total, 113 isolates) was included in the study. These encompassed 92
C. jejuni
and 21
C. coli
strains. The MICs of six antimicrobial agents were determined by the broth microdilution and E test methods, and the strains of human origin were additionally tested by the agar dilution method. In general, broth microdilution MICs agreed within 1 log
2
MIC increment with 90.0% of E test results and 78.7% of agar dilution test results. The agar dilution method gave much lower gentamicin MICs than the broth microdilution method, but the data were significantly (
P
< 0.01) correlated and there was 100% agreement in the sensitivities and specificities in the comparison of the tests. The broth microdilution method had the highest sensitivity for analysis of the susceptibilities of
Campylobacter
to nalidixic acid and trimethoprim-sulfamethoxazole. The MICs of ciprofloxacin and erythromycin complied numerically by all three methods. The classification of the results and the correlation of the data demonstrated a high degree of agreement. All methods were equally suitable for the testing of the sensitivity of
Campylobacter
to tetracycline. Thus, the broth microdilution method appears to be an easy and reliable method for determination of the MICs of antibiotics for
C. jejuni
and
C. coli
, and it may offer an interesting alternative to MIC determination by the agar dilution technique or the E test.
Detection and Identification of
Bartonella
Species Pathogenic for Humans by PCR Amplification Targeting the Riboflavin Synthase Gene (
ribC
)
Authors: Johnson G. , Ayers M. , McClure S. C. C. , Richardson S. E. , Tellier R.
Published: March 2003

Several
Bartonella
species have now been implicated as human pathogens. The recovery of these fastidious organisms in the clinical microbiology laboratory remains difficult, and current methods are still relatively insensitive. Thus, the bartonellae are good candidates for detection by PCR. We have developed a PCR assay which uses a single primer pair targeting the riboflavin synthase gene (
ribC
) and detected six
Bartonella
species that have been implicated in human disease,
B. henselae
,
B. quintana
,
B. bacilliformis
,
B. clarridgeiae
,
B. elizabethae
, and
B. vinsonii
subsp.
berkhoffii.
Species identification is achieved simply by restriction enzyme digestion of the amplicon. This PCR assay appears to be specific for the
Bartonella
genus because it failed to amplify DNA from several other bacterial species.
Quantitative Microbiologic Models for Preterm Delivery
Authors: Onderdonk Andrew B. , Lee Mei-Ling , Lieberman Ellice , Delaney Mary L. , Tuomala Ruth E.
Published: March 2003

Preterm delivery (PTD) is the leading cause of infant morbidity and mortality in the United States. An epidemiological association between PTD and various bacteria that are part of the vaginal microflora has been reported. No single bacterial species has been identified as being causally associated with PTD, suggesting a multifactorial etiology. Quantitative microbiologic cultures have been used previously to define normal vaginal microflora in a predictive model. These techniques have been applied to vaginal swab cultures from pregnant women in an effort to develop predictive microbiologic models for PTD. Logistic regression analysis with microbiologic information was performed for various risk groups, and the probability of a PTD was calculated for each subject. Four predictive models were generated by using the quantitative microbiologic data. The area under the curve (AUC) for the receiver operating curves ranged from 0.74 to 0.94, with confidence intervals (CI) ranging from 0.62 to 1. The model for the previous PTD risk group with the highest percentage of PTDs had an AUC of 0.91 (CI, 0.79 to 1). It may be possible to predict PTD by using microbiologic risk factors measured once the gestation period has reached the 20-week time point.
International Proficiency Study of a Consensus L1 PCR Assay for the Detection and Typing of Human Papillomavirus DNA: Evaluation of Accuracy and Intralaboratory and Interlaboratory Agreement
Authors: Kornegay Janet R. , Roger Michel , Davies Philip O. , Shepard Amanda P. , Guerrero Nayana A. , Lloveras Belen , Evans Darren , Coutlée François
Published: March 2003

The PGMY L1 consensus primer pair combined with the line blot assay allows the detection of 27 genital human papillomavirus (HPV) genotypes. We conducted an intralaboratory and interlaboratory agreement study to assess the accuracy and reproducibility of PCR for HPV DNA detection and typing using the PGMY primers and typing amplicons with the line blot (PGMY-LB) assay. A test panel of 109 samples consisting of 29 HPV-negative (10 buffer controls and 19 genital samples) and 80 HPV-positive samples (60 genital samples and 20 controls with small or large amounts of HPV DNA plasmids) were tested blindly in triplicate by three laboratories. Intralaboratory agreement ranged from 86 to 98% for HPV DNA detection. PGMY-LB assay results for samples with a low copy number of HPV DNA were less reproducible. The rate of intralaboratory agreement excluding negative results for HPV typing ranged from 78 to 96%. Interlaboratory reliability for HPV DNA positivity and HPV typing was very good, with levels of agreement of >95% and kappa values of >0.87. Again, low-copy-number samples were more prone to generating discrepant results. The accuracy varied from 91 to 100% for HPV DNA positivity and from 90 to 100% for HPV typing. HPV testing can thus be accomplished reliably with PCR by using a standardized written protocol and quality-controlled reagents. The use of validated HPV DNA detection and typing assays demonstrating excellent interlaboratory agreement will allow investigators to better compare results between epidemiological studies.
Evaluation of Etest Method for Determining Fluconazole and Voriconazole MICs for 279 Clinical Isolates of
Candida
Species Infrequently Isolated from Blood
Authors: Maxwell M. J. , Messer S. A. , Hollis R. J. , Boyken L. , Tendolkar S. , Diekema D. J. , Pfaller M. A.
Published: March 2003

The performance of Etest in fluconazole and voriconazole testing of 279 isolates of uncommon
Candida
spp. was assessed in comparison with the National Committee for Clinical Laboratory Standards (NCCLS)-approved standard broth microdilution (BMD) method. The NCCLS method employed RPMI 1640 broth medium, and MICs were read after incubation for 48 h at 35°C. Etest MICs were determined with RPMI agar containing 2% glucose and were read after incubation for 48 h at 35°C. The isolates include
Candida krusei
,
C. lusitaniae
,
C. guilliermondii
,
C. kefyr
,
C. rugosa
,
C. lipolytica
,
C. pelliculosa
,
C. dubliniensis
,
C. famata
,
C. zeylanoides
,
C. inconspicua
, and
C. norvegensis
. Overall agreement between Etest and BMD MICs was 96% for fluconazole and 95% for voriconazole. Where a discrepancy was observed between Etest and the reference method, the Etest tended to give lower values with both fluconazole and voriconazole. The Etest method using RPMI agar appears to be a useful method for determining fluconazole and voriconazole susceptibilities of uncommon species of
Candida
.
Hepatitis C Virus Subtyping by a Core-Envelope 1-Based Reverse Transcriptase PCR Assay with Sequencing and Its Use in Determining Subtype Distribution among Danish Patients
Authors: Corbet Sylvie , Bukh Jens , Heinsen Anja , Fomsgaard Anders
Published: March 2003

A reverse transcriptase PCR (RT-PCR) assay using conserved primers deduced from the core-envelope 1 (C-E1) region of the hepatitis C virus (HCV) genome was developed for subtyping purposes. The sensitivity and specificity of this assay tested against two HCV reference panels containing genotype 1 through 5 subtypes were similar to those of an RT-PCR assay from the 5′-untranslated region (5′-UTR). The sensitivity of the RT-PCR typing assay in the more variable C-E1 region was, however, lower than that of the RT-PCR in the highly conserved 5′-UTR when testing multiple clinical samples. Thus, 71 (88%) of 81 consecutive samples from hospitalized Danish patients positive for HCV antibodies and RNA (5′-UTR) were positive also in the C-E1 RT-PCR assay. Phylogenetic analysis of the E1 sequences obtained by direct sequencing of HCV from two reference panels and 71 Danish patients allowed us to readily distinguish the subtypes. In contrast, phylogenetic analysis of their corresponding 5′-UTR sequences was able to predict only major genotypes. Three different genotypes and four subtypes were identified among Danish samples: 1a (43%), 1b (11%), 2b (6%), and 3a (39%). An isolate from a Somalian refugee was identified as a new HCV type related to Somalian isolates described as subtype 3h. The most common genotype in Denmark is genotype 1 (53%), which is the most difficult to treat. However, Denmark had the highest prevalence in Europe of subtype 3a, which responds more favorably to treatment. The described C-E1 RT-PCR with sequencing is suggested as an easy routine assay for definitive genotyping and subtyping of HCV.