bluuebunny/crossref_metadata_2025 huggingface dataset:
Flow Cytometric Assessment of Membrane Integrity of Ethanol-StressedOenococcus oeniCells
Authors: Graça da Silveira M. , Vitória San Romão M. , Loureiro-Dias Maria C. , Rombouts Frans M. , Abee Tjakko
Published: November 2002

The practical application of commercial malolactic starter cultures ofOenococcus oenisurviving direct inoculation in wine requires insight into the mechanisms involved in ethanol toxicity and tolerance in this organism. Exposure to ethanol resulted in an increase in the permeability of the cytoplasmic membrane, enhancing passive proton influx and concomitant loss of intracellular material (absorbing at 260 nm). Cells grown in the presence of 8% (vol/vol) ethanol revealed adaptation to ethanol stress, since these cells showed higher retention of compounds absorbing at 260 nm. Moreover, for concentrations higher than 10% (vol/vol), lower rates of passive proton influx were observed in these ethanol-adapted cells, especially at pH 3.5. The effect of ethanol onO. oenicells was studied as the ability to efficiently retain carboxyfluorescein (cF) as an indicator of membrane integrity and enzyme activity and the uptake of propidium iodide (PI) to assess membrane damage. Flow cytometric analysis of both ethanol-adapted and nonadapted cells with a mixture of the two fluorescent dyes, cF and PI, revealed three main subpopulations of cells: cF-stained intact cells; cF- and PI-stained permeable cells, and PI-stained damaged cells. The subpopulation ofO. oenicells that maintained their membrane integrity, i.e., cells stained only with cF, was three times larger in the population grown in the presence of ethanol, reflecting the protective effect of ethanol adaptation. This information is of major importance in studies of microbial fermentations in order to assign bulk activities measured by classical methods to the very active cells that are effectively responsible for the observations.
Impact of Protozoan Grazing on Bacterial Community Structure in Soil Microcosms
Authors: Rønn Regin , McCaig Allison E. , Griffiths Bryan S. , Prosser James I.
Published: November 2002

The influence of grazing by a mixed assemblage of soil protozoa (seven flagellates and one amoeba) on bacterial community structure was studied in soil microcosms amended with a particulate resource (sterile wheat roots) or a soluble resource (a solution of various organic compounds). Sterilized soil was reinoculated with mixed soil bacteria (obtained by filtering and dilution) or with bacteria and protozoa. Denaturing gradient gel electrophoresis (DGGE) of PCR amplifications of 16S rRNA gene fragments, as well as community level physiological profiling (Biolog plates), suggested that the mixed protozoan community had significant effects on the bacterial community structure. Excising and sequencing of bands from the DGGE gels indicated that high-G+C gram-positive bacteria closely related toArthrobacterspp. were favored by grazing, whereas the excised bands that decreased in intensity were related to gram-negative bacteria. The percentages of intensity found in bands related to high G+C gram positives increased from 4.5 and 12.6% in the ungrazed microcosms amended with roots and nutrient solution, respectively, to 19.3 and 32.9% in the grazed microcosms. Protozoa reduced the average bacterial cell size in microcosms amended with nutrient solution but not in the treatment amended with roots. Hence, size-selective feeding may explain some but not all of the changes in bacterial community structure. Five different protozoan isolates (Acanthamoebasp., two species ofCercomonas,Thaumatomonassp., andSpumellasp.) had different effects on the bacterial communities. This suggests that the composition of protozoan communities is important for the effect of protozoan grazing on bacterial communities.
Linking of Microorganisms to Phenanthrene Metabolism in Soil by Analysis of
13
C-Labeled Cell Lipids
Authors: Johnsen Anders R. , Winding Anne , Karlson Ulrich , Roslev Peter
Published: November 2002

Phenanthrene-metabolizing soil microbial communities were characterized by examining mineralization of [
14
C]phenanthrene, by most-probable-number (MPN) counting, by 16S-23S spacer DNA analysis of the numerically dominant, culturable phenanthrene-degrading isolates, and by examining incorporation of [
13
C]phenanthrene-derived carbon into sterols and polar lipid fatty acids (PLFAs). An unpolluted agricultural soil, a roadside soil diffusely polluted with polycyclic aromatic hydrocarbons (PAHs), and two highly PAH-polluted soils from industrial sites were analyzed. Microbial phenanthrene degraders were not detected by MPN counting in the agricultural soil and the roadside soil. In the industrial soils, phenanthrene degraders constituted 0.04 and 3.6% of the total number of CFU. 16S-23S spacer DNA analysis followed by partial 16S DNA sequencing of representative isolates from one of the industrial soils showed that one-half of the isolates belonged to the genus
Sphingomonas
and the other half were closely related to an unclassified beta-proteobacterium. The
13
C-PLFA profiles of the two industrial soils were relatively similar and resembled the profiles of phenanthrene-degrading
Sphingomonas
reference strains and unclassified beta-proteobacterium isolates but did not match the profiles of
Pseudomonas
,
Mycobacterium
, or
Nocardia
reference strains. The
13
C-PLFA profiles of phenanthrene degraders in the agricultural soil and the roadside soil were different from each other and different from the profiles of the highly polluted industrial soils. Only in the roadside soil were 10me/12me18:0 PLFAs enriched in
13
C, suggesting that actinomycetes metabolized phenanthrene in this soil. The
13
C-PLFA profiles of the unpolluted agricultural soil did not resemble the profiles of any of the reference strains. In all of the soils investigated, no excess
13
C was recovered in the 18:2ω6,9 PLFA, suggesting that fungi did not contribute significantly to assimilation of [
13
C]phenanthrene.
Molecular Analysis of the Nitrate-Reducing Community from Unplanted and Maize-Planted Soils
Authors: Philippot Laurent , Piutti Séverine , Martin-Laurent Fabrice , Hallet Stéphanie , Germon Jean Claude
Published: November 2002

Microorganisms that use nitrate as an alternative terminal electron acceptor play an important role in the global nitrogen cycle. The diversity of the nitrate-reducing community in soil and the influence of the maize roots on the structure of this community were studied. The
narG
gene encoding the membrane bound nitrate reductase was selected as a functional marker for the nitrate-reducing community. The use of
narG
is of special interest because the phylogeny of the
narG
gene closely reflects the 16S ribosomal DNA phylogeny. Therefore, targeting the
narG
gene provided for the first time a unique insight into the taxonomic composition of the nitrate-reducing community in planted and unplanted soils. The PCR-amplified
narG
fragments were cloned and analyzed by restriction fragment length polymorphism (RFLP). In all, 60 RFLP types represented by two or more clones were identified in addition to the 58 RFLP types represented by only one clone. At least one clone belonging to each RFLP type was then sequenced. Several of the obtained sequences were not related to the
narG
genes from cultivated bacteria, suggesting the existence of unidentified nitrate-reducing bacteria in the studied soil. However, environmental sequences were also related to NarG from many bacterial divisions, i.e.,
Actinobacteria
and α, β, and γ
Proteobacteria
. The presence of the plant roots resulted in a shift in the structure of the nitrate-reducing community between the unplanted and planted soils. Sequencing of RFLP types dominant in the rhizosphere or present only in the rhizosphere revealed that they are related to NarG from the
Actinobacteria
in an astonishingly high proportion.
Novel Food-Grade Plasmid Vector Based on Melibiose Fermentation for the Genetic Engineering of
Lactococcus lactis
Authors: Boucher Isabelle , Parrot Marc , Gaudreau Hélène , Champagne Claude P. , Vadeboncoeur Christian , Moineau Sylvain
Published: November 2002

The α-galactosidase gene (
aga
) and a gene coding for a putative transcriptional regulator from the LacI/GalR family (
galR
) of
Lactococcus raffinolactis
ATCC 43920 were cloned and sequenced. When transferred into
Lactococcus lactis
and
Pediococcus acidilactici
strains,
aga
modified the sugar fermentation profile of the strains from melibiose negative (Mel
−
) to melibiose positive (Mel
+
). Analysis of
galA
mutants of
L. lactis
subsp.
cremoris
MG1363 indicated that the putative galactose permease GalA is also needed to obtain the Mel
+
phenotype. Consequently, GalA may also transport melibiose into this strain. We demonstrated that when
aga
was associated with the theta-type replicon of a natural
L. lactis
plasmid, it constituted the selectable marker of a cloning vector named pRAF800. Transcriptional analysis by reverse transcriptase PCR suggests that this vector is also suitable for gene expression. The α-galactosidase activity conferred by pRAF800 was monitored in an industrial strain grown in the presence of various carbon sources. The results indicated that the enzymatic activity was induced by galactose and melibiose, but not by glucose or lactose. The gene encoding the phage defense mechanism, AbiQ, was cloned into pRAF800, and the resulting clone (pRAF803) was transferred into an industrial
L. lactis
strain that became highly phage resistant. The measurements of various growth parameters indicated that cells were not affected by the presence of pRAF803. Moreover, the plasmid was highly stable in this strain even under starter production conditions. The
L. raffinolactis aga
gene represents the basis of a novel and convenient food-grade molecular tool for the genetic engineering of lactic acid bacteria.
Relatedness of Chromosomal and Plasmid DNAs of
Erwinia pyrifoliae
and
Erwinia amylovora
Authors: McGhee Gayle C. , Schnabel Elise L. , Maxson-Stein Kimberly , Jones Beatrix , Stromberg Verlyn K. , Lacy George H. , Jones Alan L.
Published: November 2002

The plant pathogen
Erwinia pyrifoliae
has been classified as a separate species from
Erwinia amylovora
based in part on differences in molecular properties. In this study, these and other molecular properties were examined for
E. pyrifoliae
and for additional strains of
E. amylovora
, including strains from brambles (
Rubus
spp.). The nucleotide composition of the internal transcribed spacer (ITS) region was determined for six of the seven 16S-23S rRNA operons detected in these species with a 16S rRNA gene probe. Each species contained four operons with a tRNA
Glu
gene and two with tRNA
Ile
and tRNA
Ala
genes, and analysis of the operons from five strains of
E. amylovora
indicated a high degree of ITS variability among them. One tRNA
Glu
-containing operon from
E. pyrifoliae
Ep1/96 was identical to one in
E. amylovora
Ea110, but three tRNA
Glu
operons and two tRNA
Ile
and tRNA
Ala
operons from
E. pyrifoliae
contained unique nucleotide changes. When
groEL
sequences were used for species-specific identification,
E. pyrifoliae
and
E. amylovora
were the closest phylogenetic relatives among a set of 12 bacterial species. The placement of
E. pyrifoliae
distinct from
E. amylovora
corroborated molecular hybridization data indicating low DNA-DNA similarity between them. Determination of the nucleotide sequence of plasmid pEP36 from
E. pyrifoliae
Ep1/96 revealed a number of presumptive genes that matched genes previously found in pEA29 from
E. amylovora
and similar organization for the genes and origins of replication. Also, pEP36 and pEA29 were incompatible with clones containing the reciprocal origin regions. Finally, the ColE1-like plasmid pEP2.6 from strain Ep1/96 contained sequences found in small plasmids in
E. amylovora
strains IL-5 and IH3-1.
Aerobic Metabolism of 4-Hydroxybenzoic Acid in Archaea via an Unusual Pathway Involving an Intramolecular Migration (NIH Shift)
Authors: Fairley D. J. , Boyd D. R. , Sharma N. D. , Allen C. C. R. , Morgan P. , Larkin M. J.
Published: November 2002

A novel haloarchaeal strain,
Haloarcula
sp. strain D1, grew aerobically on 4-hydroxybenzoic acid (4HBA) as a sole carbon and energy source and is the first member of the domain
Archaea
reported to do so. Unusually, D1 metabolized 4HBA via gentisic acid rather than via protocatechuic acid, hydroquinone, or catechol. Gentisate was detected in 4HBA-grown cultures, and gentisate 1,2-dioxygenase activity was induced in 4HBA-grown cells. Stoichiometric accumulation of gentisate from 4HBA was demonstrated in 4HBA-grown cell suspensions containing 2,2′-dipyridyl (which strongly inhibits gentisate 1,2-dioxygenase). To establish whether initial 1-hydroxylation of 4HBA with concomitant 1,2-carboxyl group migration to yield gentisate occurred, 2,6-dideutero-4HBA was synthesized and used as a substrate. Deuterated gentisate was recovered from cell suspensions and identified as 3-deutero-gentisate, using gas chromatography-mass spectrometry and proton nuclear magnetic resonance spectroscopy. This structural isomer would be expected only if a 1,2-carboxyl group migration had taken place, and it provides compelling evidence that the 4HBA pathway in
Haloarcula
sp. strain D1 involves a hydroxylation-induced intramolecular migration. To our knowledge, this is the first report of a pathway which involves such a transformation (called an NIH shift) in the domain
Archaea
.
Conspicuous Veils Formed by Vibrioid Bacteria on Sulfidic Marine Sediment
Authors: Thar Roland , Kühl Michael
Published: November 2002

We describe the morphology and behavior of a hitherto unknown bacterial species that forms conspicuous veils (typical dimensions, 30 by 30 mm) on sulfidic marine sediment. The new bacteria were enriched on complex sulfidic medium within a benthic gradient chamber in oxygen-sulfide countergradients, but the bacteria have so far not been isolated in pure culture, and a detailed characterization of their metabolism is still lacking. The bacteria are colorless, gram-negative, and vibrioid-shaped (1.3- to 2.5- by 4- to 10-μm) cells that multiply by binary division and contain several spherical inclusions of poly-β-hydroxybutyric acid. The cells have bipolar polytrichous flagella and exhibit a unique swimming pattern, rotating and translating along their short axis. Free-swimming cells showed aerotaxis and aggregated at ca. 2 μM oxygen within opposing oxygen-sulfide gradients, where they were able to attach via a mucous stalk, forming a cohesive whitish veil at the oxic-anoxic interface. Bacteria attached to the veil kept rotating and adapted their stalk lengths dynamically to changing oxygen concentrations. The joint action of rotating bacteria on the veil induced a homogeneous water flow from the oxic water region toward the veil, whereby the oxygen uptake rate could be enhanced up to six times, as shown by model calculations. The veils showed a pronounced succession pattern. New veils were generated de novo within 24 h and had a homogeneous whitish translucent appearance. Bacterial competitors or eukaryotic predators were apparently kept away by the low oxygen concentration prevailing at the veil surface. Frequently, within 2 days the veil developed a honeycomb pattern of regularly spaced holes. After 4 days, most veils were colonized by grazing ciliates, leading to the fast disappearance of the new bacteria. Several-week-old veils finally developed into microbial mats consisting of green, purple, and colorless sulfur bacteria.
Genetic and Phenotypic Analysis of
Acinetobacter baumannii
Insertion Derivatives Generated with a Transposome System
Authors: Dorsey Caleb W. , Tomaras Andrew P. , Actis Luis A.
Published: November 2002

Acinetobacter baumannii
is a metabolically versatile pathogen that causes severe infections in compromised patients. However, little is known about the genes and factors involved in its basic physiology and virulence properties. Insertion mutagenesis was used to initiate the identification and characterization of some of these factors and genes in the prototype strain 19606. The utilization of the pLOFKm suicide delivery vector, which harbors a suicide mini-Tn
10
derivative, proved to be unsuccessful for this purpose. The EZ::TN 〈R6Kγ
ori
/KAN-2〉 Tnp transposome system available from Epicentre was then used in conjunction with electroporation to generate isogenic insertional derivatives of
A. baumannii
19606. Replica plating showed that 2% of the colonies that grew after electroporation on agar plates without antibiotics also grew in the presence of 40 μg of kanamycin per ml. DNA hybridization proved that all of the kanamycin-resistant derivatives contained the EZ::TN 〈R6Kγ
ori
/KAN-2〉 insertion element, which was mapped to different genomic locations. Replica plating on Simmons citrate agar and microtiter plate-plastic tube assays identified growth- and biofilm-defective derivatives, respectively. The location of the insertion in several of these derivatives was determined by self-ligation of
Nde
I- or
Eco
RI-digested genomic DNA and electroporation of
Escherichia coli
TransforMax EC100D (
pir
+
). Sequence analysis of the recovered plasmids showed that some of the
A. baumannii
19606 growth-defective derivatives contain insertions within genes encoding activities required for the generation of energy and cell wall components and for the biosynthesis of amino acids and purines. A gene encoding a protein similar to the GacS sensor kinase was interrupted in four derivatives, while another had an insertion in a gene coding for a hypothetical sensor kinase.
A. baumannii
19606 derivatives with defective attachment or biofilm phenotypes had insertions within genes that appear to be part of a chaperone-usher transport system described for other bacteria. DNA hybridization experiments showed that the presence of strain 19606 genes encoding regulatory and attachment or biofilm functions is widespread among other
A. baumannii
clinical isolates.
Field Studies Using a Recombinant Mycoinsecticide (
Metarhizium anisopliae
) Reveal that It Is Rhizosphere Competent
Authors: Hu Gang , St. Leger Raymond J.
Published: November 2002

In the summer of 2000, we released genetically altered insect-pathogenic fungi onto a plot of cabbages at a field site on the Upper Marlboro Research Station, Md. The transformed derivatives of
Metarhizium anisopliae
ARSEF 1080, designated GPMa and GMa, carried the
Aequorea victoria
green fluorescent protein (
gfp
) gene alone (GMa) or with additional protease genes (
Pr1
) (GPMa). The study (i) confirmed the utility of
gfp
for monitoring pathogen strains in field populations over time, (ii) demonstrated little dissemination of transgenic strains and produced no evidence of transmission by nontarget insects, (iii) found that recombinant fungi were genetically stable over 1 year under field conditions, and (iv) determined that deployment of the transgenic strains did not depress the culturable indigenous fungal microflora. The major point of the study was to monitor the fate (survivorship) of transformants under field conditions. In nonrhizosphere soil, the amount of GMa decreased from 10
5
propagules/g at depths of 0 to 2 cm to 10
3
propagules/g after several months. However, the densities of GMa remained at 10
5
propagules/g in the inner rhizosphere, demonstrating that rhizospheric soils are a potential reservoir for
M. anisopliae
. These results place a sharp focus on the biology of the soil/root interphase as a site where plants, insects, and pathogens interact to determine fungal biocontrol efficacy, cycling, and survival. However, the rhizospheric effect was less marked for GPMa, and overall it showed reduced persistence in soils than did GMa.
Bacterial Group II Introns in a Deep-Sea Hydrothermal Vent Environment
Authors: Podar Mircea , Mullineaux Lauren , Huang Hon-Ren , Perlman Philip S. , Sogin Mitchell L.
Published: November 2002

Group II introns are catalytic RNAs and mobile retrotransposable elements known to be present in the genomes of some nonmarine bacteria and eukaryotic organelles. Here we report the discovery of group II introns in a bacterial mat sample collected from a deep-sea hydrothermal vent near 9°N on the East Pacific Rise. One of the introns was shown to self-splice in vitro. This is the first example of marine bacterial introns from molecular population structure studies of microorganisms that live in the proximity of hydrothermal vents. These types of mobile genetic elements may prove useful in improving our understanding of bacterial genome evolution and may serve as valuable markers in comparative studies of bacterial communities.
Xylanase and Acetyl Xylan Esterase Activities of XynA, a Key Subunit of the
Clostridium cellulovorans
Cellulosome for Xylan Degradation
Authors: Kosugi Akihiko , Murashima Koichiro , Doi Roy H.
Published: November 2002

The
Clostridium cellulovorans xynA
gene encodes the cellulosomal endo-1,4-β-xylanase XynA, which consists of a family 11 glycoside hydrolase catalytic domain (CD), a dockerin domain, and a NodB domain. The recombinant acetyl xylan esterase (rNodB) encoded by the NodB domain exhibited broad substrate specificity and released acetate not only from acetylated xylan but also from other acetylated substrates. rNodB acted synergistically with the xylanase CD of XynA for hydrolysis of acetylated xylan. Immunological analyses revealed that XynA corresponds to a major xylanase in the cellulosomal fraction. These results indicate that XynA is a key enzymatic subunit for xylan degradation in
C. cellulovorans
.
Existence of a True Phosphofructokinase in
Bacillus sphaericus
: Cloning and Sequencing of the
pfk
Gene
Authors: Alice Alejandro F. , Pérez-Martínez Gaspar , Sánchez-Rivas Carmen
Published: November 2002

Some strains of
Bacillus sphaericus
are entomopathogenic to mosquito larvae, which transmit diseases, such as filariasis and malaria, affecting millions of people worldwide. This species is unable to use hexoses and pentoses as unique carbon sources, which was proposed to be due to the lack of glycolytic enzymes, such as 6-phosphofructokinase (PFK). In this study, PFK activity was detected and the
pfk
gene was cloned and sequenced. Furthermore, this gene was shown to be present in strains belonging to all the homology groups of this heterogeneous species, in which PFK activity was also detected. A careful sequence analysis revealed the conservation of different catalytic and regulatory residues, as well as the enzyme's phylogenetic affiliation with the family of allosteric ATP-PFK enzymes.
Quantitative Determination of Bile Salt Hydrolase Activity in Bacteria Isolated from the Small Intestine of Chickens
Authors: Knarreborg Ane , Engberg Ricarda M. , Jensen Søren K. , Jensen Bent B.
Published: November 2002

A quantitative assay based on high-performance liquid chromatography analysis of bile salts and bacterial protein determination was established for investigating bile salt hydrolase (BSH) activity in bacteria isolated from the small intestine of chickens. Bacteria were isolated using various media and were subsequently grouped according to cell morphology, fermentation profile, and 16S ribosomal DNA sequence. Representative isolates from each bacterial group were assayed for BSH activity. The isolates differed in BSH activity with respect to the state of growth and preculturing with and without taurochenodeoxycholate. The highest levels of BSH activity were found with
Enterococcus faecium
and
Clostridium perfringens
.
Incorporation of DNA and Protein Precursors into Macromolecules by Bacteria at −15
o
C
Authors: Christner Brent C.
Published: November 2002

DNA and protein precursors were incorporated into trichloroacetic acid-precipitated material by bacterial cell suspensions during incubation for 50 to 100 days at −15
o
C. Incorporation did not occur at −70
o
C and was inhibited by antibiotics. The results demonstrate that bacteria can perform macromolecular synthesis under conditions that mimic entrapment in glacial ice.
Virulence Properties and Serotypes of Shiga Toxin-Producing
Escherichia coli
from Healthy Australian Cattle
Authors: Hornitzky Michael A. , Vanselow Barbara A. , Walker Keith , Bettelheim Karl A. , Corney Bruce , Gill Paul , Bailey Graham , Djordjevic Steven P.
Published: November 2002

The virulence properties and serotypes of complex Shiga toxin-producing
Escherichia coli
(cSTEC) were determined in two studies of healthy cattle in eastern Australia. In the first, a snapshot study, 84 cSTEC isolates were recovered from 37 of 1,692 (2.2%) fecal samples collected from slaughter-age cattle from 72 commercial properties. The second, a longitudinal study of three feedlots and five pasture beef properties, resulted in the recovery of 118 cSTEC isolates from 104 animals. Of the 70 serotypes identified, 38 had not previously been reported.
Identification of a Replicon from pTXL1, a Small Cryptic Plasmid from
Leuconostoc mesenteroides
subsp.
mesenteroides
Y110, and Development of a Food-Grade Vector
Authors: Biet Franck , Cenatiempo Yves , Fremaux Christophe
Published: November 2002

A 2,665-bp cryptic plasmid, pTXL1, isolated from
Leuconostoc mesenteroides
subsp.
mesenteroides
Y110 was identified. This plasmid harbors a replicon localized on a 1,300-bp fragment. Two observations suggested that pTXL1 does not belong to rolling-circle replication (RCR)-type plasmids and most likely replicates via a theta mechanism. These hypotheses are supported by the observation that no detectable single-stranded intermediate was found for the replicon and that, unlike in RCR-type plasmids, the pTXL1 replicon sequence lacks an open reading frame encoding a replicase. The small-sized pTXL1 plasmid is stable and, according to its origin, can be considered in the “generally recognized as safe” category. Its ability to replicate in several lactic acid bacteria was exploited to develop a vector producing mesentericin Y105, a class II anti-
Listeria
bacteriocin. With this new vector, a recombinant industrial
Leuconostoc cremoris
strain able to produce mesentericin Y105 was constructed.
Vancomycin-Resistant Enterococci in Humans and Imported Chickens in Japan
Authors: Ozawa Yoshiyuki , Tanimoto Koichi , Nomura Takahiro , Yoshinaga Masao , Arakawa Yoshichika , Ike Yasuyoshi
Published: November 2002

The phenotypes and genotypes of 22 VanA-type vancomycin-resistant enterococci that had been isolated in Japan were examined. The VanA resistance determinant was plasmid mediated in each of the 22 strains. Of the 22 strains, 8 were isolated from different patients and 11 and 3 were obtained from different samples of chickens imported from Thailand and France, respectively. Three of the strains that were isolated from patients and the 11 strains isolated from the Thai chickens showed high-level vancomycin resistance (MICs, 512 to 1,024 μg/ml) and low-level teicoplanin resistance (MICs, 0.5 to 4 μg/ml). Each of these strains had three amino acid substitutions in the N-terminal region of the deduced VanS sequence. L50 was converted to V, E54 was converted to Q, and Q69 was converted to H compared to the
vanS
gene sequence of Tn
1546
.
Disruption of
narH
,
narJ
, and
moaE
Inhibits Heterotrophic Nitrification in
Pseudomonas
Strain M19
Authors: Nemergut D. R. , Schmidt S. K.
Published: November 2002

Interruptions in three nitrate reductase-related genes,
narH
,
narJ,
and
moaE
, inhibited heterotrophic nitrification in
Pseudomonas
strain M19. No nitrate was detected in the medium, and nitrification proceeded in the presence of a nitrate reductase inhibitor. Heterotrophic nitrification was greatly stimulated by the addition of nitrate.
Enumeration of
Legionella
CFU by Colony Hybridization Using Specific DNA Probes
Authors: Satoh Wakako , Nakata Mayumi , Yamamoto Hiroyuki , Ezaki Takayuki , Hiramatsu Keiichi
Published: November 2002

Oligonucleotide probes and colony hybridization (CH) were applied to enumerate organisms of the genus
Legionella
in cooling tower water. The CH counts indicated almost the same results as CFU counts in cultivated samples derived from the water. It was concluded that it is possible to substitute the CH procedure for the conventional one.
Real-Time PCR for Detection and Differentiation ofEntamoeba histolyticaandEntamoeba disparin Fecal Samples
Authors: Blessmann Joerg , Buss Heidrun , Nu Phuong A. Ton , Dinh Binh T. , Ngo Quynh T. Viet , Van An Le , Alla Mohamed D. Abd , Jackson Terry F. H. G. , Ravdin Jonathan I. , Tannich Egbert
Published: November 2002

A closed-tube, real-time PCR assay was developed for sensitive and specific detection and differentiation of the two closely related intestinal protozoan parasitesEntamoeba histolyticaandEntamoeba dispardirectly from human feces. The assay is performed with the LightCycler system using fluorescence-labeled detection probes and primers amplifying a 310-bp fragment from the high-copy-number, ribosomal DNA-containing ameba episome. The assay was able to detect as little as 0.1 parasite per g of feces. The two pairs of primers used were specific for the respective ameba species, and results were not influenced by the presence of otherEntamoebaspecies even when present in exceeding amounts. PCR was evaluated using several hundred stool samples from areas of amebiasis endemicity in Vietnam and South Africa, and results were compared with those of microscopy and ameba culture. PCR was found to be significantly more sensitive than microscopy or culture, as all samples positive by microscopy and 22 out of 25 (88%) samples positive by culture were also positive by PCR, but PCR revealed a considerable number of additionalE. histolytica-orE. dispar-positive samples. Compared to culture and subsequent ameba differentiation by isoenzyme analysis, PCR was 100% specific for each of the twoEntamoebaspecies. Interestingly, the comparison with PCR revealed that culture, in particular, underestimatesE. histolyticainfections. Given the high sensitivity and specificity of the developed PCR assay, the inability of microscopy to distinguish between the two ameba species, and the time it takes to culture and subsequently differentiate entamoebae by isoenzyme analysis, this assay is more suitable than microscopy or culture to correctly diagnose intestinalE. histolyticaorE. disparinfection.
Detection of Human Respiratory Syncytial Virus in Respiratory Samples by LightCycler Reverse Transcriptase PCR
Authors: Whiley David M. , Syrmis Melanie W. , Mackay Ian M. , Sloots Theo P.
Published: November 2002

Laboratory diagnosis of human respiratory syncytial virus (hRSV) infections has traditionally been performed by virus isolation in cell culture and the direct fluorescent-antibody assay (DFA). Reverse transcriptase PCR (RT-PCR) is now recognized as a sensitive and specific alternative for detection of hRSV in respiratory samples. Using the LightCycler instrument, we developed a rapid RT-PCR assay for the detection of hRSV (the LC-RT-PCR) with a pair of hybridization probes that target the hRSV L gene. In the present study, 190 nasopharyngeal aspirate samples from patients with clinically recognized respiratory tract infections were examined for hRSV. The results were then compared to the results obtained with a testing algorithm that combined DFA and a culture-augmented DFA (CA-DFA) assay developed in our laboratory. hRSV was detected in 77 (41%) specimens by LC-RT-PCR and in 75 (39%) specimens by the combination of DFA and CA-DFA. All specimens that were positive by the DFA and CA-DFA testing algorithm were positive by the LC-RT-PCR. The presence of hRSV RNA in the two additional LC-RT-PCR-positive specimens was confirmed by a conventional RT-PCR method that targets the hRSV N gene. The sensitivity of LC-RT-PCR was 50 PFU/ml; and this, together with its high specificity and rapid turnaround time, makes the LC-RT-PCR suitable for the detection of hRSV in clinical specimens.
Laboratory Diagnosis and Genetic Analysis of an Echovirus 30-Associated Outbreak of Aseptic Meningitis in Taiwan in 2001
Authors: Wang Jen-Ren , Tsai Huey-Pin , Huang Sen-Wen , Kuo Pin-Hwa , Kiang David , Liu Ching-Chuan
Published: November 2002

A large outbreak of aseptic meningitis occurred from April to November 2001 in Taiwan. Of the 1,130 enterovirus-infected patients, echovirus 30 (E30) infection was diagnosed in 188 (16.6%), with the patients having various clinical manifestations including aseptic meningitis (73.9%), young infant fever (6.9%), respiratory symptoms or herpangina (13.3%), or others (5.9%). The majority of the E30-infected patients were between 3 and 10 years old. Of the 264 E30 strains identified, 94.3, 71, and 67.4% were isolated from RD, MRC-5, and A549 cells, respectively. Primary isolation of E30 required mean times of 3.7 days for RD cells and 4.1 days for MRC-5 and A549 cells. Among all E30-positive patients, virus was most frequently isolated from throat swab specimens (85.2%) and, to a lesser extent, stool (76.4%) or cerebrospinal fluid (70.1%) specimens. The virus isolates were initially identified as echovirus 4 (E4) on the basis of immunofluorescence staining with anti-E4 and anti-E30 (Bastianni prototype) monoclonal antibodies. However, upon performance of the neutralization test, E30-specific reverse transcription-PCR, and sequencing of the VP1 gene, the results identified these isolates as E30, not E4, indicating that the reagent used to type E30, which is produced with the Bastianni strain as the immunogen, is inadequate for the identification of recent E30 isolates in Taiwan. Phylogenetic analyses of the VP1 genes of these isolates showed that their sequences differed from those of E30 isolates from the GenBank database by 9.1 to 25.2%, suggesting that this outbreak was caused by a new variant strain of E30 introduced into Taiwan in 2000 that resulted in the widespread aseptic meningitis epidemic in 2001.
Multiple Genetic Typing of
Salmonella enterica
Serotype Typhimurium Isolates of Different Phage Types (DT104, U302, DT204b, and DT49) from Animals and Humans in England, Wales, and Northern Ireland
Authors: Liebana Ernesto , Garcia-Migura Lourdes , Clouting Carol , Clifton-Hadley Felicity A. , Lindsay Elisabeth , Threlfall E. John , McDowell Stanley W. J. , Davies Robert H.
Published: November 2002

Salmonella enterica
serotype Typhimurium is a common cause of salmonellosis among humans and animals in England, Wales, and Northern Ireland. Phage types DT104 and U302 were the most prevalent types in both livestock and humans in 2001. In addition,
Salmonella
serotype Typhimurium DT204b was responsible for a recent international outbreak involving England. A total of 119 isolates from humans (
n
= 28) and animals or their environment (
n
= 91), belonging to DT104 (
n
= 66), U302 (
n
= 33), DT204b (
n
= 12), and DT49 (
n
= 8), were fingerprinted by a combination of well-established genetic methods (pulsed-field gel electrophoresis [PFGE],
Pst
I/
Sph
I [PS] ribotyping, and plasmid profiling). The different techniques identified different degrees of polymorphism (from greatest to least, plasmid profiling [40 types], PS ribotyping [34 types], and PFGE [23 types]). It seems clear that a prevalent genomic clone, as well as a variety of less frequent clones, is present for each of the phage types. In most cases, the prevalent clones appeared within isolates from several animal species and from several geographical locations. We did not find clear evidence of a higher degree of diversity for any of the animal species included, or of any link between isolates from particular animal species and humans. The data presented show the inaccuracy of drawing epidemiological conclusions based on a single fingerprinting method. Strains that share one of the markers do not necessarily belong to the same clone, and a multiple typing approach is required to enable enough discrimination to track strains for epidemiological investigations.
Two Different Families ofhopQAlleles inHelicobacter pylori
Authors: Cao Ping , Cover Timothy L.
Published: November 2002

Helicobacter pylorigenomes contain about 30 differenthopgenes, which encode outer membrane proteins. In this study, we analyzed genetic diversity in theH. pylori hopQ(omp27) locus, which corresponds to HP1177 in the genome ofH. pylorireference strain 26695.hopQand its flanking genes were PCR amplified from multipleH. pyloristrains, and the nucleotide sequences were determined. This analysis revealed the existence of two different families ofhopQalleles. Type IhopQalleles are present in the genomes of two fully sequencedH. pyloristrains, whereas the existence of type IIhopQalleles has not previously been recognized. Type I and type IIhopQalleles are 75 to 80% identical in nucleotide sequences and encode predicted outer membrane proteins that are 68 to 72% identical in amino acid sequences. PCR-based methods were developed to enable rapid differentiation between type I and type IIhopQalleles. Type IhopQalleles were found significantly more commonly incag+/type s1-vacAstrains from patients with peptic ulcer disease than incag-negative/s2-vacAstrains from patients without ulcer disease (P< 0.001). Determination ofhopQallelic types provides a new method for classification ofH. pyloristrains. Further studies in multiple populations of patients are indicated to evaluate the usefulness of this approach for distinguishing potentially ulcerogenicH. pyloristrains from less virulent strains.
Testing Genotypic and Phenotypic Resistance in Human Immunodeficiency Virus Type 1 Isolates of Clade B and Other Clades from Children Failing Antiretroviral Therapy
Authors: Brindeiro Patrícia A. , Brindeiro Rodrigo M. , Mortensen Cláudio , Hertogs Kurt , De Vroey Veronique , Rubini Norma P. M. , Sion Fernando S. , De Sá Carlos A. M. , Machado Deisy M. , Succi Regina C. M. , Tanuri Amilcar
Published: November 2002

The emergence of resistance to antiretroviral drugs is a major obstacle to the successful treatment of human immunodeficiency virus type 1 (HIV-1)-infected patients. In this work, we correlate clinical and virological trends such as viral load (VL) and CD4 counts to genotypic and phenotypic antiretroviral (ARV) resistance profiles of HIV-1 isolates from the B and non-B subtypes found in vertically infected children failing ARV therapy. Plasma samples were collected from 52 vertically HIV-1-infected children failing different ARV therapies. Samples underwent HIV-1
pol
sequencing and phenotyping and were clustered into subtypes by phylogenetic analysis. Clinical data from each patient were analyzed together with the resistance (genotypic and phenotypic) data obtained. Thirty-five samples were from subtype B, 10 samples were non-B (subtypes A, C, and F), and 7 were mosaic samples. There was no significant difference concerning treatment data between B and non-B clades. Prevalence of known drug resistance mutations revealed slightly significant differences among B and non-B subtypes: L10I, 21 and 64%, K20R, 13 and 43%, M36I, 34 and 100%, L63P, 76 and 36%, A71V/T, 24 and 0%, and V77I, 32 and 0%, respectively, in the protease (0.0001 ≤
P
≤ 0.0886), and D67N, 38 and 8%, K70R, 33 and 0%, R211K, 49 and 85%, and K219Q/E, 31 and 0%, respectively, in the reverse transcriptase (0.0256 ≤
P
≤ 0.0704). Significant differences were found only in secondary resistance mutations and did not reflect significant phenotypic variation between clade B and non-B.
Helicobacter cetorumsp. nov., a Urease-PositiveHelicobacterSpecies Isolated from Dolphins and Whales
Authors: Harper C. G. , Feng Y. , Xu S. , Taylor N. S. , Kinsel M. , Dewhirst F. E. , Paster B. J. , Greenwell M. , Levine G. , Rogers A. , Fox J. G.
Published: November 2002

A novel helicobacter with the proposed nameHelicobacter cetorum, sp. nov. (type strain MIT 99-5656; GenBank accession numberAF 292378), was cultured from the main stomach of two wild, stranded Atlantic white-sided dolphins (Lagenorhynchus acutus) and from the feces of three captive cetaceans (a Pacific white-sided dolphin [Lagenorhynchus obliquidens]; an Atlantic bottlenose dolphin [Tursiops truncatus]; and a beluga whale [Delphinapterus leucas]). The infected captive cetaceans were either subclinical, or clinical signs included intermittent regurgitation, inappetance, weight loss, and lethargy. Ulcers were observed in the esophagus and forestomach during endoscopic examination in two of the three captive animals. In the third animal, esophageal linear erosions were visualized endoscopically, and histopathological evaluation of the main stomach revealed multifocal lymphoplasmacytic gastritis with silver-stained spiral-shaped bacteria.Helicobacter cetorumis a fusiform gram-negative bacterium with a single bipolar flagellum. The isolates grow under microaerobic conditions at 37 and 42°C but not at 25°C.H. cetorumis urease, catalase, and oxidase positive, and it is sensitive to cephalothin. The isolates from the wild, stranded dolphins were sensitive to nalidixic acid, whereas the isolates from the collection animals were resistant. By 16S rRNA sequencing it was determined thatH. cetorumrepresented a distinct taxon that clusters most closely withH. pylori.Further studies are necessary to determine the role ofH. cetorumin the development of gastric ulcers and gastritis of cetaceans. This is the first description and formal naming of a novelHelicobacterspecies from a marine mammal.
Determining the Genetic Structure of the Natural Population of
Staphylococcus aureus
: a Comparison of Multilocus Sequence Typing with Pulsed-Field Gel Electrophoresis, Randomly Amplified Polymorphic DNA Analysis, and Phage Typing
Authors: Grundmann Hajo , Hori Satoshi , Enright Mark C. , Webster Carol , Tami Adriana , Feil Edward J. , Pitt Tyrone
Published: November 2002

We used a sample of
Staphylococcus aureus
strains that are carried by humans and that are representative of the natural population of
S. aureus
strains in order to assess the value of multilocus sequence typing (MLST), pulsed-field gel electrophoresis, randomly amplified polymorphic DNA analysis, and phage typing as epidemiological tools. Only MLST was able to define clonal complexes unambiguously. All DNA-based typing approaches achieved a high degree of agreement, implying phylogenetic concordance, but predicted epidemiological associations with variable accuracy.
Stability of Variable-Number Tandem Repeats of Mycobacterial Interspersed Repetitive Units from 12 Loci in Serial Isolates ofMycobacterium tuberculosis
Authors: Savine Evgueni , Warren Robin M. , van der Spuy Gian D. , Beyers Nulda , van Helden Paul D. , Locht Camille , Supply Philip
Published: November 2002

Variable number tandem repeats (VNTRs) of elements named mycobacterial interspersed repetitive units (MIRUs) have previously been identified in 12 minisatellite loci of theMycobacterium tuberculosisgenome. These markers allow reliable high-throughput genotyping ofM. tuberculosisand represent a portable approach to global molecular epidemiology ofM. tuberculosis. To assess their temporal stability, we genotyped 123 serial isolates, separated by up to 6 years and belonging to a variety of distinct IS6110restriction fragment length polymorphism (RFLP) families, from 56 patients who had positive sputum cultures. All 12 MIRU VNTR loci were completely identical within the groups of serial isolates in 55 out of 56 groups (98.2%), although 11 pairs of isolates from the same patients with conserved MIRU VNTRs displayed slightly different IS6110RFLP profiles. In a single case, serial isolates with an unchanged IS6110RFLP profile showed a change in 1 out of 12 MIRU VNTR loci. These results indicate that MIRU VNTRs are stable over time and therefore are suitable for reliable follow-up of patients chronically infected with tuberculosis over long periods. Moreover, they support MIRU VNTR genotyping as a powerful first-line method followed by subtyping by IS6110RFLP to define ongoing transmission clusters.
Comparison of a Repetitive Extragenic Palindromic Sequence-Based PCR Method and Clinical and Microbiological Methods for Determining Strain Sources in Cases of Nosocomial
Acinetobacter baumannii
Bacteremia
Authors: Martín-Lozano David , Cisneros José Miguel , Becerril Berta , Cuberos Lucila , Prados Trinidad , Ortíz-Leyba Carlos , Cañas Elías , Pachón Jerónimo
Published: November 2002

Using a repetitive extragenic palindromic PCR (REP-PCR), we genotypically characterized strains causing nosocomial
Acinetobacter baumannii
infections and analyzed the source of bacteremia in 67 patients from an institution in which infections by this bacterium were endemic. Six different genotypes were found, including 21, 27, 3, 9, 3, and 4 strains. The probable source of bacteremia, according to clinical and/or microbiological criteria, was known in 42 patients (63%): respiratory tract (
n
= 19), surgical sites (
n
= 12), intravascular catheters (
n
= 5), burns (
n
= 3), and urinary tract (
n
= 3). The definite source of bacteremia, according to REP-PCR, could be established in 30 (71%) out of the 42 patients with strains from blood and other sites; in these cases clinical and microbiological criteria for the source of bacteremia were thus confirmed. In the remaining 12 patients (29%) the probable source was refuted by the REP-PCR method. The definite sources of bacteremia according to genotype were as follows: respiratory tract in 13 patients (31%), surgical sites in 8 (19%), intravascular catheters in 4 (9%), burns in 3 (7%), and urinary tract in 2 (5%). A comparison of strains from blood cultures and other sites with regard to their REP-PCR and antimicrobial resistance profiles was also made. Taking the REP-PCR as the “gold standard,” the positive predictive value of antibiotype was 77% and the negative predictive value was 42%. In summary, the utility of the diagnosis of the source of nosocomial
A. baumannii
bacteremia using clinical and/or microbiological criteria, including antibiotyping, is limited, as demonstrated by REP-PCR.
Importance of Sample Preparation for Molecular Diagnosis of Lyme Borreliosis from Urine
Authors: Bergmann A. R. , Schmidt B. L. , Derler A.-M. , Aberer E.
Published: November 2002

Urine PCR has been used for the diagnosis of
Borrelia burgdorferi
infection in recent years but has been abandoned because of its low sensitivity and the irreproducibility of the results. Our study aimed to analyze technical details related to sample preparation and detection methods. Crucial for a successful urine PCR were (i) avoidance of the first morning urine sample; (ii) centrifugation at 36,000 ×
g
; and (iii) the extraction method, with only DNAzol of the seven different extraction methods used yielding positive results with patient urine specimens. Furthermore, storage of frozen urine samples at −80°C reduced the sensitivity of a positive urine PCR result obtained with samples from 72 untreated erythema migrans (EM) patients from 85% in the first 3 months to <30% after more than 3 months. Bands were detected at 276 bp on ethidium bromide-stained agarose gels after amplification by a nested PCR. The specificity of bands for 32 of 33 samples was proven by hybridization with a GEN-ETI-K-DEIA kit and for a 10 further positive amplicons by sequencing. By using all of these steps to optimize the urine PCR technique,
B. burgdorferi
infection could be diagnosed by using urine samples from EM patients with a sensitivity (85%) substantially better than that of serological methods (50%). This improved method could be of future importance as an additional laboratory technique for the diagnosis of unclear, unrecognized borrelia infections and diseases possibly related to Lyme borreliosis.