bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of Extracellular Products of
Pseudoalteromonas atlantica
on the Edible Crab
Cancer pagurus
Authors: Costa-Ramos Carolina , Rowley Andrew F.
Published: February 2004

Previous studies have shown that injection of extracellular products (ECP) of
Pseudoalteromononas atlantica
isolated from shell disease-infected edible crabs (
Cancer pagurus
) into healthy crabs causes rapid death. In this study we examined the nature of the active lethal factor(s) in ECP. Injection of ECP into crabs caused a rapid decline in the total number of circulating hemocytes (blood cells), and the crabs died within 60 to 90 min. The individuals that died showed eyestalk retraction, limb paralysis, and lack of antennal sensitivity, suggesting that the active factor(s) targeted the nervous system. Histopathological investigations showed that affected crabs had large aggregates of hemocytes in the gills, and there was destruction of the tubules in the hepatopancreas. The active factor in ECP was not sensitive to heat treatment (100°C for 30 min) and proteinase K digestion. As lipopolysaccharide (LPS) was a potential candidate for the lethal factor, it was purified from whole
P. atlantica
bacteria or ECP and subsequently injected into crabs. These crabs had all of the external symptoms observed previously with ECP, such as limb paralysis and eyestalk retraction, and they died within 90 min after challenge, although no significant decline in the number of circulating hemocytes was observed. Similarly, in vitro incubation of hemocytes with purified LPS (1 to 20 μg) from
P. atlantica
did not result in the clumping reaction observed with ECP but did result in a degranulation reaction and eventual cell lysis. Injection of crabs with
Escherichia coli
or
Pseudomonas aeruginosa
LPS (1 μg g of body weight
−1
) did not cause any of the characteristic symptoms observed following exposure to
P. atlantica
LPS. No mortality of crabs followed the injection of
E. coli
LPS, but
P. aeruginosa
LPS caused ca. 80% mortality at 2 h after injection. Overall, these results show that the main virulence factor of
P. atlantica
for edible crabs is LPS either alone or in combination with other heat-stable factors.
Stable Carbon Isotope Ratios of Lipid Biomarkers of Sulfate-Reducing Bacteria
Authors: Londry K. L. , Jahnke L. L. , Des Marais D. J.
Published: February 2004

We examined the potential use of natural-abundance stable carbon isotope ratios of lipids for determining substrate usage by sulfate-reducing bacteria (SRB). Four SRB were grown under autotrophic, mixotrophic, or heterotrophic growth conditions, and the δ
13
C values of their individual fatty acids (FA) were determined. The FA were usually
13
C depleted in relation to biomass, with Δδ
13
C
(FA − biomass)
of −4 to −17‰; the greatest depletion occurred during heterotrophic growth. The exception was
Desulfotomaculum acetoxidans
, for which substrate limitation resulted in biomass and FA becoming isotopically heavier than the acetate substrate. The δ
13
C values of FA in
Desulfotomaculum acetoxidans
varied with the position of the double bond in the monounsaturated C
16
and C
18
FA, with FA becoming progressively more
13
C depleted as the double bond approached the methyl end. Mixotrophic growth of
Desulfovibrio desulfuricans
resulted in little depletion of the i17:1 biomarker relative to biomass or acetate, whereas growth with lactate resulted in a higher proportion of i17:1 with a greater depletion in
13
C. The relative abundances of 10Me16:0 in
Desulfobacter hydrogenophilus
and
Desulfobacterium autotrophicum
were not affected by growth conditions, yet the Δδ
13
C
(FA − substrate)
values of 10Me16:0 were considerably greater during autotrophic growth. These experiments indicate that FA δ
13
C values can be useful for interpreting carbon utilization by SRB in natural environments.
Role of Calf-Adapted
Escherichia coli
in Maintenance of Antimicrobial Drug Resistance in Dairy Calves
Authors: Khachatryan Artashes R. , Hancock Dale D. , Besser Thomas E. , Call Douglas R.
Published: February 2004

The prevalence of antimicrobial drug-resistant bacteria is typically highest in younger animals, and prevalence is not necessarily related to recent use of antimicrobial drugs. In dairy cattle, we hypothesize that antimicrobial drug-resistant, neonate-adapted bacteria are responsible for the observed high frequencies of resistant
Escherichia coli
in calves. To explore this issue, we examined the age distribution of antimicrobial drug-resistant
E. coli
from Holstein cattle at a local dairy and conducted an experiment to determine if low doses of oxytetracycline affected the prevalence of antimicrobial drug-resistant
E. coli
. Isolates resistant to tetracycline (>4 μg/ml) were more prevalent in <3-month-old calves (79%) compared with lactating cows (14%). In an experimental trial where calves received diets supplemented with or without oxytetracycline, the prevalence of tetracycline-resistant
E. coli
was slightly higher for the latter group (
P
= 0.039), indicating that drug use was not required to maintain a high prevalence of resistant
E. coli
. The most common resistance pattern among calf
E. coli
isolates included resistance to streptomycin (>12 μg/ml), sulfadiazine (>512 μg/ml), and tetracycline (>4 μg/ml) (SSuT), and this resistance pattern was most prevalent during the period when calves were on milk diets. To determine if prevalence was a function of differential fitness, we orally inoculated animals with nalidixic acid-resistant strains of SSuT
E. coli
and susceptible
E. coli
. Shedding of SSuT
E. coli
was significantly greater than that of susceptible strains in neonatal calves (
P
< 0.001), whereas there was no difference in older animals (
P
= 0.5). These data support the hypothesis that active selection for traits linked to the SSuT phenotype are responsible for maintaining drug-resistant
E. coli
in this population of dairy calves.
Phylogenetic Composition of Arctic Ocean Archaeal Assemblages and Comparison with Antarctic Assemblages
Authors: Bano Nasreen , Ruffin Shomari , Ransom Briana , Hollibaugh James T.
Published: February 2004

Archaea
assemblages from the Arctic Ocean and Antarctic waters were compared by PCR-denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA genes amplified using the
Archaea
-specific primers 344f and 517r. Inspection of the DGGE fingerprints of 33 samples from the Arctic Ocean (from SCICEX submarine cruises in 1995, 1996, and 1997) and 7 Antarctic samples from Gerlache Strait and Dallman Bay revealed that the richness of
Archaea
assemblages was greater in samples from deep water than in those from the upper water column in both polar oceans. DGGE banding patterns suggested that most of the
Archaea
ribotypes were common to both the Arctic Ocean and the Antarctic Ocean. However, some of the
Euryarchaeota
ribotypes were unique to each system. Cluster analysis of DGGE fingerprints revealed no seasonal variation but supported depth-related differences in the composition of the Arctic Ocean
Archaea
assemblage. The phylogenetic composition of the
Archaea
assemblage was determined by cloning and then sequencing amplicons obtained from the
Archaea
-specific primers 21f and 958r. Sequences of 198 clones from nine samples covering three seasons and all depths grouped with marine group I
Crenarchaeota
(111 clones), marine group II
Euryarchaeota
(86 clones), and group IV
Euryarchaeota
(1 clone). A sequence obtained only from a DGGE band was similar to those of the marine group III
Euryarchaeota
.
Impact of Virioplankton on Archaeal and Bacterial Community Richness as Assessed in Seawater Batch Cultures
Authors: Winter Christian , Smit Arjan , Herndl Gerhard J. , Weinbauer Markus G.
Published: February 2004

During cruises in the tropical Atlantic Ocean (January to February 2000) and the southern North Sea (December 2000), experiments were conducted to monitor the impact of virioplankton on archaeal and bacterial community richness. Prokaryotic cells equivalent to 10 to 100% of the in situ abundance were inoculated into virus-free seawater, and viruses equivalent to 35 to 360% of the in situ abundance were added. Batch cultures with microwave-inactivated viruses and without viruses served as controls. The apparent richness of archaeal and bacterial communities was determined by terminal restriction fragment length polymorphism (T-RFLP) analysis of PCR-amplified 16S rRNA gene fragments. Although the estimated richness of the prokaryotic communities generally was greatly reduced within the first 24 h of incubation due to confinement, the effects of virus amendment were detected at the level of individual operational taxonomic units (OTUs) in the T-RFLP patterns of both groups,
Archaea
and
Bacteria
. One group of OTUs was detected in the control samples but was absent from the virus-treated samples. This negative response of OTUs to virus amendment probably was caused by viral lysis. Additionally, we found OTUs not responding to the amendments, and several OTUs exhibited variable responses to the addition of inactive or active viruses. Therefore, we conclude that individual members of pelagic archaeal and bacterial communities can be differently affected by the presence of virioplankton.
Effect of Environmental Factors on the Relationship between Concentrations of Coprostanol and Fecal Indicator Bacteria in Tropical (Mekong Delta) and Temperate (Tokyo) Freshwaters
Authors: Isobe Kei O. , Tarao Mitsunori , Chiem Nguyen H. , Minh Le Y. , Takada Hideshige
Published: February 2004

A reliable assessment of microbial indicators of fecal pollution (total coliform,
Escherichia coli
, and fecal streptococcus) is critical in tropical environments. Therefore, we investigated the relationship between concentrations of indicator bacteria and a chemical indicator, coprostanol (5β-cholestan-3β-ol), in tropical and temperate regions. Water samples were collected from the Mekong Delta, Vietnam, during wet and dry seasons, and from Tokyo, Japan, during summer, the aftermath of a typhoon, and winter. During the wet season in the Mekong Delta, higher bacterial densities were observed in rivers, probably due to the higher bacterial inputs from soil particles with runoff. In Tokyo, higher bacterial densities were usually observed during summer, followed by those in the typhoon aftermath and winter. A strong logarithmic correlation between the concentrations of
E. coli
and coprostanol was demonstrated in all surveys. Distinctive seasonal fluctuations were observed, as concentrations of coprostanol corresponding to 1,000 CFU of
E. coli
/100 ml were at their lowest during the wet season in the Mekong Delta and the typhoon aftermath in Tokyo (30 ng/liter), followed by the dry season in the Mekong Delta and the summer in Tokyo (100 ng/liter), and they were much higher during the winter in Tokyo (400 ng/liter). These results suggested that
E. coli
is a specific indicator of fecal contamination in both tropical and temperate regions but that the densities are affected by elevated water temperature and input from runoff of soil particles. The concurrent determination of
E. coli
and coprostanol concentrations could provide a possible approach to assessing the reliability of fecal pollution monitoring data.
Genotypic and Antibiotic Susceptibility Characteristics of a
Campylobacter coli
Population Isolated from Dairy Farmland in the United Kingdom
Authors: Leatherbarrow A. J. H. , Hart C. A. , Kemp R. , Williams N. J. , Ridley A. , Sharma M. , Diggle P. J. , Wright E. J. , Sutherst J. , French N. P.
Published: February 2004

Campylobacter
infections are the most common cause of bacterial enteritis in humans, and nearly 8% of such infections are caused by
Campylobacter coli
. Most studies have concentrated on
Campylobacter jejuni
, frequently isolated from intensively farmed poultry and livestock production units, and few studies have examined the spread and relatedness of
Campylobacter
across a range of geographical and host boundaries. Systematic sampling of a 100-km
2
area of mixed farmland in northwest England yielded 88 isolates of
C. coli
from a range of sample types and locations, and water was heavily represented. Screening for antibiotic resistance revealed a very low prevalence of resistance, while genotyping performed by using three methods (
flaA
PCR restriction fragment length polymorphism [RFLP], pulsed-field gel electrophoresis [PFGE], and fluorescent amplified fragment length polymorphism [fAFLP]) provided insights into the genomic relatedness of isolates from different locations and hosts. Isolates were classified into 23
flaA
groups, 34 PFGE groups, and five major fAFLP clusters. PFGE banding analysis revealed a high level of variability and no clustering by sample type. fAFLP and
flaA
analyses successfully grouped the isolates by sample type. We report preliminary findings suggesting that there is a strain of
C. coli
which may have become adapted to survival or persistence in water and that there is a group of mainly water-derived isolates from which unusual
flaA
PCR fragments were recovered.
Genetic Diversity of
Cryptosporidium
spp. in Captive Reptiles
Authors: Xiao Lihua , Ryan Una M. , Graczyk Thaddeus K. , Limor Josef , Li Lixia , Kombert Mark , Junge Randy , Sulaiman Irshad M. , Zhou Ling , Arrowood Michael J. , Koudela Břetislav , Modrý David , Lal Altaf A.
Published: February 2004

The genetic diversity of
Cryptosporidium
in reptiles was analyzed by PCR-restriction fragment length polymorphism and sequence analysis of the small subunit rRNA gene. A total of 123 samples were analyzed, of which 48 snake samples, 24 lizard samples, and 3 tortoise samples were positive for
Cryptosporidium
. Nine different types of
Cryptosporidium
were found, including
Cryptosporidium serpentis
,
Cryptosporidium
desert monitor genotype,
Cryptosporidium muris
,
Cryptosporidium parvum
bovine and mouse genotypes, one
C. serpentis
-like parasite in a lizard, two new
Cryptosporidium
spp. in snakes, and one new
Cryptosporidium
sp. in tortoises.
C. serpentis
and the desert monitor genotype were the most common parasites and were found in both snakes and lizards, whereas the
C. muris
and
C. parvum
parasites detected were probably the result of ingestion of infected rodents. Sequence and biologic characterizations indicated that the desert monitor genotype was
Cryptosporidium saurophilum
. Two host-adapted
C. serpentis
genotypes were found in snakes and lizards.
Biodiversity of Exopolysaccharides Produced by
Streptococcus thermophilus
Strains Is Reflected in Their Production and Their Molecular and Functional Characteristics
Authors: Vaningelgem Frederik , Zamfir Medana , Mozzi Fernanda , Adriany Tom , Vancanneyt Marc , Swings Jean , De Vuyst Luc
Published: February 2004

Twenty-six lactic acid bacterium strains isolated from European dairy products were identified as
Streptococcus thermophilus
and characterized by bacterial growth and exopolysaccharide (EPS)-producing capacity in milk and enriched milk medium. In addition, the acidification rates of the different strains were compared with their milk clotting behaviors. The majority of the strains grew better when yeast extract and peptone were added to the milk medium, although the presence of interfering glucomannans was shown, making this medium unsuitable for EPS screening. EPS production was found to be strain dependent, with the majority of the strains producing between 20 and 100 mg of polymer dry mass per liter of fermented milk medium. Furthermore, no straightforward relationship between the apparent viscosity and EPS production could be detected in fermented milk medium. An analysis of the molecular masses of the isolated EPS by gel permeation chromatography revealed a large variety, ranging from 10 to >2,000 kDa. A distinction could be made between high-molecular-mass EPS (>1,000 kDa) and low-molecular-mass EPS (<1,000 kDa). Based on the molecular size of the EPS, three groups of EPS-producing strains were distinguished. Monomer analysis of the EPS by high-performance anion-exchange chromatography with amperometric detection was demonstrated to be a fast and simple method. All of the EPS from the
S. thermophilus
strains tested were classified into six groups according to their monomer compositions. Apart from galactose and glucose, other monomers, such as (
N
-acetyl)galactosamine, (
N
-acetyl)glucosamine, and rhamnose, were also found as repeating unit constituents. Three strains were found to produce EPS containing (
N
-acetyl)glucosamine, which to our knowledge was never found before in an EPS from
S. thermophilus
. Furthermore, within each group, differences in monomer ratios were observed, indicating possible novel EPS structures. Finally, large differences between the consistencies of EPS solutions from five different strains were assigned to differences in their molecular masses and structures.
Insecticidal
Bacillus thuringiensis
Silences
Erwinia carotovora
Virulence by a New Form of Microbial Antagonism, Signal Interference
Authors: Dong Yi-Hu , Zhang Xi-Fen , Xu Jin-Ling , Zhang Lian-Hui
Published: February 2004

It is commonly known that bacteria may produce antibiotics to interfere with the normal biological functions of their competitors in order to gain competitive advantages. Here we report that
Bacillus thuringiensis
suppressed the quorum-sensing-dependent virulence of plant pathogen
Erwinia carotovora
through a new form of microbial antagonism, signal interference.
E. carotovora
produces and responds to acyl-homoserine lactone (AHL) quorum-sensing signals to regulate antibiotic production and expression of virulence genes, whereas
B. thuringiensis
strains possess AHL-lactonase, which is a potent AHL-degrading enzyme.
B. thuringiensis
did not seem to interfere with the normal growth of
E. carotovora
; rather, it abolished the accumulation of AHL signal when they were cocultured. In planta,
B. thuringiensis
significantly decreased the incidence of
E. carotovora
infection and symptom development of potato soft rot caused by the pathogen. The biocontrol efficiency is correlated with the ability of bacterial strains to produce AHL-lactonase. While all the seven AHL-lactonase-producing
B. thuringiensis
strains provided significant protection against
E. carotovora
infection,
Bacillus fusiformis
and
Escherichia coli
strains that do not process AHL-degradation enzyme showed little effect in biocontrol. Mutation of
aiiA
, the gene encoding AHL-lactonase in
B. thuringiensis
, resulted in a substantial decrease in biocontrol efficacy. These results suggest that signal interference mechanisms existing in natural ecosystems could be explored as a new version of antagonism for prevention of bacterial infections.
Genetic Selection Scheme for Isolation of Signal Transduction Pathway Mutants
Authors: Anandan Shivanthi , Uram Jennifer
Published: February 2004

Genetic characterization of a signal transduction pathway requires the isolation of mutations in the pathway. Characterization of these mutated genes and their loci enumerates the components of the pathway and leads to an understanding of the role of each gene locus in the pathway under study. We have designed and developed a strategy based on resistance to the chemical flucytosine for the identification of mutations in a given pathway. In this study, the
Escherichia coli codA
gene, which encodes the enzyme cytosine deaminase, was fused to the light-intensity-regulated gene promoter
psbDII
. Cytosine deaminase converts 5′-fluorocytosine to the toxic product 5-fluorouracil. Wild-type cells containing an intact signal transduction pathway that regulates the
psbDII
promoter will die in the presence of this chemical. Cells that carry mutations in the pathway that inactivate the
psbDII
promoter will not express the
codA
gene and, consequently, will live on 5′-fluorocytosine, allowing the isolation and subsequent characterization of mutations in this signaling pathway. Utilizing this selection method, we have successfully isolated and characterized mutations in the
psbDII
pathway. This selection scheme can be used with a tissue-specific or phase-specific promoter fused to the
codA
gene to direct the timing of expression of
codA
to obtain mutants defective in temporal or cell-specific expression of a particular pathway. This scheme also allows the isolation of mutants even when a clearly identifiable phenotype is not available. The selection scheme presented here extends the molecular tools available for the genetic dissection of signal transduction pathways.
Impact of a Disruption of a Pathway Delivering Copper to Mitochondria onPodospora anserinaMetabolism and Life Span
Authors: Stumpferl Stefan W. , Stephan Oliver , Osiewacz Heinz D.
Published: February 2004

A global depletion of cellular copper as the result of a deficiency in high-affinity copper uptake was previously shown to affect the phenotype and life span of the filamentous fungusPodospora anserina. We report here the construction of a strain in which the delivery of copper to complex IV of the mitochondrial respiratory chain is affected. This strain, PaCox17::ble, is aPaCox17-null mutant that does not synthesize the molecular chaperone targeting copper to cytochromecoxidase subunit II. PaCox17::ble is characterized by a decreased growth rate, a reduction in aerial hyphae formation, reduced female fertility, and a dramatic increase in life span. The mutant respires via a cyanide-resistant alternative pathway, displays superoxide dismutase (SOD) activity profiles significantly differing from those of the wild-type strain and is characterized by a stabilization of the mitochondrial DNA. Collectively, the presented data define individual components of a molecular network effective in life span modulation and copper as an element with a dual effect. As a cofactor of complex IV of the respiratory chain, it is indirectly involved in the generation of reactive oxygen species (ROS) and thereby plays a life span-limiting role. In contrast, Cu/Zn SOD as a ROS-scavenging enzyme lowers molecular damage and thus positively affects life span. Such considerations explain the reported differences in life span of independent mutants and spread more light on the delicate tuning of the molecular network influencing biological ageing.
SmD1 Is Required for Spliced Leader RNA Biogenesis
Authors: Zeiner Gusti M. , Foldynová Silvie , Sturm Nancy R. , Lukeš Julius , Campbell David A.
Published: February 2004

The Sm-binding site of the kinetoplastid spliced leader RNA has been implicated in accurate spliced leader RNA maturation and
trans
-splicing competence. In
Trypanosoma brucei
, RNA interference-mediated knockdown of SmD1 caused defects in spliced leader RNA maturation, displaying aberrant 3′-end formation, partial formation of cap 4, and overaccumulation in the cytoplasm; U
28
pseudouridylation was unaffected.
Core Promoter Structure in the Oomycete
Phytophthora infestans
Authors: McLeod Adele , Smart Christine D. , Fry William E.
Published: February 2004

We have investigated the core promoter structure of the oomycete
Phytophthora infestans
. The transcriptional start sites (TSS) of three previously characterized
P. infestans
genes,
Piexo1
,
Piexo3
, and
Piendo1
, were determined by primer extension analyses. The TSS regions were homologous to a previously identified 16-nucleotide (nt) core sequence that overlaps the TSS in most oomycete genes. The core promoter regions of
Piexo1
and
Piendo1
were investigated by using a transient protoplast expression assay and the reporter gene β-glucuronidase. Mutational analyses of the promoters of
Piexo1
and
Piendo1
showed that there is a putative core promoter element encompassing the TSS (−2 to + 5) that has high sequence and functional homology to a known core promoter element present in other eukaryotes, the initiator element (Inr). Downstream and flanking the Inr is a highly conserved oomycete promoter region (+7 to + 15), hereafter referred to as FPR (flanking promoter region), which is also important for promoter function. The importance of the 19-nt core promoter region (Inr and FPR) in
Piexo1
and
Piendo1
was further investigated through electrophoretic mobility shift assays (EMSA). The EMSA studies showed that (i) both core promoters were able to specifically bind a protein or protein complex in a
P. infestans
whole-cell protein extract and (ii) the same mutations that reduced binding of the EMSA complex also reduced β-glucuronidase (GUS) levels in transient expression assays. The consistency of results obtained using two different assays (GUS transient assays [in vivo] and EMSA studies [in vitro]) supports a convergence of inference about the relative importance of specific nucleotides within the 19-nt core promoter region.
Analysis of Invasive
Haemophilus influenzae
Infections after Extensive Vaccination against
H. influenzae
Type b
Authors: Campos José , Hernando Margarita , Román Federico , Pérez-Vázquez María , Aracil Belén , Oteo Jesús , Lázaro Edurne , de Abajo Francisco ,   the Group of Invasive Haemophilus Infections of the Autonomous Community of Madrid, Spain
Published: February 2004

Little clinical and microbiological information is available about invasive
Haemophilus influenzae
infection after widespread vaccination against
H. influenzae
type b (Hib). We conducted an active community surveillance study on invasive
H. influenzae
during a 2-year period in a community of more than 5 million people after vaccination against Hib in children was introduced. The median incidence was 16.3 cases/100,000 persons per year in children less than 1-year-old and 4.41 cases/100,000 persons in children less than <5 years old. The highest incidence in adults was observed in patients greater than 70 years old. Clinical diagnoses included bacteremia, pneumonia, and meningitis. Of the
H. influenzae
-infected patients, 74.3% had underlying predisposing conditions, including impaired immunity and respiratory diseases. A total of 73.6% of the isolates were nontypeable and 16.5, 6.6, and 3.3% were types b, f, and e, respectively. Infections due to capsulated strains b, e, and f were evenly distributed between children and adults. Ampicillin and cotrimoxazole resistance occurred at frequencies of 24.2 and 48.4%, respectively. Antibiotic resistance was more prevalent in capsulated than in noncapsulated
H. influenzae
. Invasive isolates were highly resistant to antibiotics that were used infrequently in the community. Nontypeable
H. influenzae
were genetically much more heterogeneous than capsulated strains. Capsule-deficient mutants (b
−
) were not detected. Plasmid carriage was linked to antibiotic resistance and capsulated strains. Over the study period, the incidence of invasive
H. influenzae
infections, either encapsulated or not, did not increase. In the post-Hib vaccination era, most invasive infections were due to noncapsulated strains and occurred in the extreme ages of life in patients with predisposing conditions.
Use of Insertion Sequence Element IS
1126
in a Genotyping and Transmission Study of
Porphyromonas gingivalis
Authors: Park Ok-Jin , Min Kyung-Man , Choe Son-Jin , Choi Bong-Kyu , Kim Kack-Kyun
Published: February 2004

Porphyromonas gingivalis
is strongly associated with periodontal diseases and is regarded as one of the risk factors for periodontitis. Insertion sequence element IS
1126
-based PCR was used to investigate the genetic heterogeneity of
P. gingivalis
from periodontitis patients and to examine the frequency of the parent-child and spouse-spouse transmission. Two sets of IS
1126
-specific primers were used for the PCR. The inward primer set (PI1 and PI2), which amplifies the IS
1126
fragment of approximately 690 bp, was used to identify
P. gingivalis
. The outward primer set (PI1RC and PI2RC), which is reverse complementary to PI1 and PI2, respectively, and amplifies the gene fragments between the adjacent IS
1126
elements was used to characterize the genotypes of the
P. gingivalis
strains. PCR of
P. gingivalis
with PI1RC and PI2RC resulted in the production of two to seven amplicons, which showed a unique electrophoretic pattern in each strain (4 laboratory strains and 37 clinical isolates cultured from 12 patients with aggressive periodontitis). The usefulness of the method for transmission study was confirmed by detecting identical genotypes between the isolates and the plaque samples from which the isolates were cultured and between the plaque samples from different tooth sites in the same patient. Thirty probands with periodontal diseases and their thirty immediate family members were included in the transmission study. In 11 of 14 parent-child pairs (78.6%),
P. gingivalis
revealed an identical or similar band pattern, whereas 5 of 16 spouse pairs (31.25%) had this similarity. These results show that IS
1126
-based PCR for genotyping
P. gingivalis
has a highly discriminating potential with reproducible data and is a simple and reliable method for a transmission study.
Characterization of Cell-to-Cell Signaling-Deficient
Pseudomonas aeruginosa
Strains Colonizing Intubated Patients
Authors: Dénervaud Valérie , TuQuoc Patrick , Blanc Dominique , Favre-Bonté Sabine , Krishnapillai Viji , Reimmann Cornelia , Haas Dieter , van Delden Christian
Published: February 2004

Cell-to-cell signaling involving
N
-acyl-homoserine lactone compounds termed autoinducers (AIs) is instrumental to virulence factor production and biofilm development by
Pseudomonas aeruginosa
. In order to determine the importance of cell-to-cell signaling during the colonization of mechanically ventilated patients, we collected 442
P. aeruginosa
pulmonary isolates from 13 patients. Phenotypic characterization showed that 81% of these isolates produced the AI-dependent virulence factors elastase, protease, and rhamnolipids. We identified nine genotypically distinct
P. aeruginosa
strains. Six of these strains produced AIs [
N
-butanoyl-homoserine lactone or
N
-(3-oxo-dodecanoyl)-homoserine lactone] and extracellular virulence factors (elastase, total exoprotease, rhamnolipid, hydrogen cyanide, or pyocyanin) in vitro. Three of the nine strains were defective in the production of both AIs and extracellular virulence factors. Two of these strains had mutational defects in both the
lasR
and
rhlR
genes, which encode the
N
-acyl-homoserine lactone-dependent transcriptional regulators LasR and RhlR, respectively. The third of these AI-deficient strains was only mutated in the
lasR
gene. Our observations suggest that most, but not all, strains colonizing intubated patients are able to produce virulence factors and that mutations affecting the cell-to-cell signaling circuit are preferentially located in the transcriptional regulator genes.
Experience with the MicroSeq D2 Large-Subunit Ribosomal DNA Sequencing Kit for Identification of Filamentous Fungi Encountered in the Clinical Laboratory
Authors: Hall Leslie , Wohlfiel Sherri , Roberts Glenn D.
Published: February 2004

Described herein is our experience with the MicroSeq D2 large-subunit rDNA sequencing kit for the identification of filamentous fungi encountered in the mycology laboratory at the Mayo Clinic. A total of 234 filamentous fungi recovered from clinical specimens were used in the evaluation. All were identified by using phenotypic characteristics as observed macroscopically and microscopically on any medium or a combination of media, which included Sabouraud's dextrose, inhibitory mold, cornmeal, Czapek-Dox, potato dextrose, and V8 juice agars; all isolates were sequenced using the MicroSeq D2 large-subunit rDNA sequencing kit. Of the of 234 isolates, 158 were correctly identified to the appropriate genus or genus and species by using nucleic acid sequencing. Sequences for 70 (29.9%) of the isolates (27 genera) were not included in the MicroSeq library. Of the 80 dematiaceous and 154 hyaline fungi sequenced, 65 and 51.2%, respectively, gave results concordant with those determined by phenotypic identification. Nucleic acid sequencing using the MicroSeq D2 large-subunit rDNA sequencing kit offers promise of being an accurate identification system; however, the associated library needs to include more of the clinically important genera and species.
Evaluation of the RealArt Malaria LC Real-Time PCR Assay for Malaria Diagnosis
Authors: Farcas Gabriella A. , Zhong Kathleen J. Y. , Mazzulli Tony , Kain Kevin C.
Published: February 2004

PCR-based methods have advantages over traditional microscopic methods for the diagnosis of malaria, especially in cases of low parasitemia and mixed infections. However, current PCR-based assays are often labor-intensive and not readily quantifiable and have the potential for contamination due to a requirement for postamplification sample handling. Real-time PCR can address these limitations. This study evaluated the performance characteristics of a commercial malaria real-time PCR assay (RealArt Malaria LC Assay; Artus GmbH, Hamburg, Germany) on the LightCycler platform for the detection of malaria parasites in 259 febrile returned travelers. Compared to nested PCR as the reference standard, the real-time assay had a sensitivity of 99.5%, specificity of 100%, positive predictive value of 100%, and negative predictive value of 99.6% for the detection of malaria. Our results indicate that the RealArt assay is a rapid (<45 min), sensitive, and specific method for the detection of malaria in returned travelers.
Association of Presence of
Aspergillus
Antibodies with Hemoptysis in Patients with Old Tuberculosis or Bronchiectasis but No Radiologically Visible Mycetoma
Authors: Chu Chung-ming , Woo Patrick C. Y. , Chong Ken T. K. , Leung Wah-shing , Chan Veronica L. , Yuen Kwok-yung
Published: February 2004

Old tuberculosis and bronchiectasis are the two most important causes of chronic structural changes of lungs in our locality. In the absence of radiologically visible mycetoma, the cause of hemoptysis in these two groups of patients is largely unknown. A 17-month prospective study was carried out to compare the prevalence of
Aspergillus fumigatus
and
Aspergillus flavus
antibodies in hemoptysis patients with old tuberculosis or bronchiectasis but no radiologically visible mycetoma (cases,
n
= 38), hemoptysis patients with other diagnosis (control group 1,
n
= 29), and patients with old tuberculosis or bronchiectasis but no hemoptysis (control group 2,
n
= 47) by a recently developed sensitive and specific
A. fumigatus
and
A. flavus
antibody assay. There were a significantly larger number of patients with antibody against
A. fumigatus
or
A. flavus
among the cases than among the patients in control groups 1 and 2 (
P
< 0.05 in both comparisons). Molds were not recovered from any of the patients. Among the 10 cases with
Aspergillus
antibody, eight and two had antibody against
A. flavus
and
A. fumigatus
, respectively. We conclude that there was an association between the presence of
Aspergillus
antibodies and hemoptysis in patients with old tuberculosis or bronchiectasis, suggesting that these patients probably had occult infections caused by the corresponding fungi. Development of serological tests against other
Aspergillus
species as well as other causes of mycetoma will probably increase the detection of occult mold infections in patients with existing parenchymal lung diseases, and treatment of fungal microinvasion may help to alleviate hemoptysis in these patients with bronchiectasis or old tuberculosis who have
Aspergillus
antibodies.
Identification of Subgenus C Adenoviruses by Fiber-Based Multiplex PCR
Authors: Adhikary Arun Kumar , Inada Toshiki , Banik Urmila , Numaga Jiro , Okabe Nobuhiko
Published: February 2004

Subgenus C human adenoviruses, which include serotypes 1, 2, 5, and 6, are often associated with respiratory illness, ocular infections, gastroenteritis, and systemic infection among immunocompromised patients. To address the problems associated with the conventional typing methods, we developed a fiber-based multiplex PCR assay for simple and specific identification of adenovirus type 1, 2, 5, and 6 field isolates. To design type-specific primers, adenovirus type 1 and 6 fiber genes were sequenced. The assay correctly identified prototype strains of adenovirus serotypes 1, 2, 5, 6, as well as 21 previously typed adenovirus field isolates. Mixing two different prototype DNAs produced two amplicons of different lengths, thus clearly distinguishing the prototypes. The results correlated 100% with serological tests and 95% with the previously described PCR-restriction fragment length polymorphism method. The detection of dual infection is an added benefit of the assay. No nonspecific amplification was detected with other adenovirus serotypes or with nonadenoviral DNA. Our fiber-based multiplex PCR assay will provide a convenient tool for type-specific identification of subgenus C adenovirus isolates in various clinical situations and in epidemiological investigations and is a better alternative than the hexon-based assay.
Use of TaqMan 5′ Nuclease Real-Time PCR for Quantitative Detection of
Mycoplasma genitalium
DNA in Males with and without Urethritis Who Were Attendees at a Sexually Transmitted Disease Clinic
Authors: Jensen Jørgen Skov , Björnelius Eva , Dohn Birthe , Lidbrink Peter
Published: February 2004

Mycoplasma genitalium
is a cause of nongonococcal urethritis, particularly in patients not infected with
Chlamydia trachomatis
. A quantitative 5′ nuclease assay (TaqMan PCR) was developed and validated. The assay detected a fragment of the MgPa adhesin gene by use of a TaqMan MGB (minor groove binder) probe and included an internal processing control to detect PCR inhibition. Urethral swab specimens and first-void urine samples from
M. genitalium
-positive men were examined, and the
M. genitalium
DNA load was correlated to symptoms and signs. The assay consistently detected <5 genome copies without cross-reactions with other mycoplasmas. Urine and urethral swab specimens from men with urethritis had higher
M. genitalium
DNA loads than specimens from men without urethritis. However, a very broad overlap of DNA loads between patients with and without urethritis was observed. Urethral swab specimens from patients with urethral discharge had a significantly higher DNA load than specimens from patients without discharge. This correlation was not found in first-void urine specimens.
High Genetic Diversity among
Stenotrophomonas maltophilia
Strains Despite Their Originating at a Single Hospital
Authors: Valdezate Sylvia , Vindel Ana , Martín-Dávila Pilar , Del Saz Begoña Sánchez , Baquero Fernando , Cantón Rafael
Published: February 2004

The levels of genetic relatedness of 139
Stenotrophomonas maltophilia
strains recovered from 105 hospitalized non-cystic fibrosis patients (51% from medical wards, 35% from intensive care units, and 14% from surgical wards) and 7 environmental sources in the same hospital setting during a 4-year period were typed by the pulsed-field gel electrophoresis (PFGE) technique. A total of 99 well-defined distinct
Xba
I PFGE patterns were identified (Simpson's discrimination index, 0.996). The dendrogram showed a Dice similarity coefficient ranging from 28 to 80%. Two major clusters (I and II), three minor clusters (III, IV, and V), and two independent branches were observed when using a 36% Dice coefficient, indicating a high diversity of genetic relatedness. It is of note that 84% of strains were grouped within two major clonal lineages. No special cluster gathering was found among strains belonging to the same sample type specimen, patients' infection or colonization status, and ward of precedence. Despite this fact, three different clones (A, B, and C) recovered from respiratory samples from six, three, and two patients, respectively, and two clones, D and E, in two bacteremic patients each, were identified. Isolation of an
S. maltophilia
strain belonging to the clone A profile in a bronchoscope demonstrated a common source from this clone. This study revealed a high genetic diversity of
S. maltophilia
isolates despite their origin from a single hospital, which may be related to the wide environmental distribution of this pathogen. However, few clones could be transmitted among different patients, yielding outbreak situations.
Genotyping of
Mycobacterium leprae
on the Basis of the Polymorphism of TTC Repeats for Analysis of Leprosy Transmission
Authors: Matsuoka Masanori , Zhang Liangfen , Budiawan Teky , Saeki Keisuke , Izumi Shinzo
Published: February 2004

The polymorphism of TTC repeats in
Mycobacterium leprae
was examined using the bacilli obtained from residents in villages at North Maluku where
M. leprae
infections are highly endemic (as well as from patients at North Sulawesi of Indonesia) to elucidate the possible mode of leprosy transmission. TTC genotypes are stable for several generations of passages in nude mice footpads and, hence, are feasible for the genotyping of isolates and epidemiological analysis of leprosy transmission. It was found that bacilli with different TTC genotypes were distributed among residents at the same dwelling in villages in which leprosy is endemic and that some household contacts harbored bacilli with a different genotype from that harbored by the patient. Investigations of a father-and-son pair of patients indicated that infections of bacilli with 10 and 18 copies, respectively, had occurred. Genotypes of TTC repeats were found to differ between a son under treatment and two brothers. These results reveal the possibility that in addition to exposure via the presence of a leprosy patient with a multibacillary infection who was living with family members, there might have been some infectious sources to which the residents had been commonly exposed outside the dwellings. A limited discriminative capacity of the TTC polymorphism in the epidemiological analysis implies the need of searching other useful polymorphic loci for detailed subdivision of clinical isolates.
Hepatitis C Virus (HCV) Genotypes in the Caribbean Island of Martinique: Evidence for a Large Radiation of HCV-2 and for a Recent Introduction from Europe of HCV-4
Authors: Martial Jenny , Morice Yoann , Abel Sylvie , Cabié André , Rat Christelle , Lombard Frédéric , Edouard André , Pierre-Louis Serge , Garsaud Philippe , Béra Odile , Chout Roger , Gordien Emmanuel , Deny Paul , Césaire Raymond
Published: February 2004

Molecular epidemiological studies of hepatitis C virus (HCV) in the Caribbean may help to specify the origin and spread of HCV infection. Indeed, the Caribbean population is intermixed from European and African origins and geographically close to the American continent. We characterized HCV genotypes in the Caribbean island of Martinique. HCV genotypes were analyzed by sequencing or reverse hybridization in the 5′ noncoding region (5′NC) in 250 HCV-monoinfected and 85 HCV-human immunodeficiency virus (HIV)-coinfected patients. In addition, sequencing in the nonstructural 5B (NS5B) gene was required to determine the subtype or to perform phylogenetic analysis in selected samples. Genotypes 1 to 6 were found, respectively, in 84.4, 6.8, 5.2, 2.8, 0.4, and 0.4% of 250 HCV-monoinfected patients and in 71.7, 7.1, 15.3, 5.9, 0, and 0% of 85 HCV-HIV-coinfected patients. HCV-1b was found in 66.4% of the HCV-monoinfected patients and was associated with blood transfusion, whereas HCV-1a was detected in 41.2% of the HCV-HIV-coinfected patients and was associated with intravenous drug use (IVDU). The HCV-3 strains belonged to subtype 3a and were linked to IVDU. Phylogenetic analyses were focused on HCV-2 and HCV-4, which are common in Africa. Two opposite patterns were evidenced.NS5Bsequences from 19 HCV-2 isolates were affiliated with many different subtypes described either in Europe or in West Africa, suggesting an ancient radiation. In contrast, seven of the nine HCV-4NS5Bsequences ranged within HCV-4a and HCV-4d clusters spreading in continental France by the IVDU route. Epidemiological data demonstrate the recent introduction of HCV-4a and -4d subtypes into the Caribbean.
spa
Typing Method for Discriminating among
Staphylococcus aureus
Isolates: Implications for Use of a Single Marker To Detect Genetic Micro- and Macrovariation
Authors: Koreen Larry , Ramaswamy Srinivas V. , Graviss Edward A. , Naidich Steven , Musser James M. , Kreiswirth Barry N.
Published: February 2004

Strain typing of microbial pathogens has two major aims: (i) to index genetic microvariation for use in outbreak investigations and (ii) to index genetic macrovariation for use in phylogenetic and population-based analyses. Until now, there has been no clear indication that one genetic marker can efficiently be used for both purposes. Previously, we had shown that DNA sequence analysis of the protein A gene variable repeat region (
spa
typing) provides a rapid and accurate method to discriminate
Staphylococcus aureus
outbreak isolates from those deemed epidemiologically unrelated. Here, using the hypothesis that the genetic macrovariation within a low-level recombinogenic species would accurately be characterized by a single-locus marker, we tested whether
spa
typing could congruently index the extensive genetic variation detected by a whole-genome DNA microarray in a collection of 36 isolates, which was recovered from 10 countries on four continents over a period of four decades, that is representative of the breadth of diversity within
S. aureus.
Using
spa
and
coa
typing, pulsed-field gel electrophoresis (PFGE), and microarray and multilocus enzyme electrophoresis (MLEE) data in molecular epidemiologic and evolutionary analyses, we determined that
S. aureus
likely has a primarily clonal population structure and that
spa
typing can singly index genetic variation with 88% direct concordance with the microarray and can correctly assign isolates to phylogenetic lineages.
spa
typing performed better than MLEE, PFGE, and
coa
typing in discriminatory power and in the degree of agreement with the microarray at various phylogenetic depths. This study showed that genetic analysis of the repeat region of protein A comprehensively characterizes both micro- and macrovariation in the primarily clonal population structure of
S. aureus
.
Comparison of LCx with Other Current Viral Load Assays for Detecting and Quantifying Human Immunodeficiency Virus Type 1 RNA in Patients Infected with the Circulating Recombinant Form A/G (CRF02)
Authors: Amendola Alessandra , Bordi Licia , Angeletti Claudio , Girardi Enrico , Ippolito Giuseppe , Capobianchi Maria R.
Published: February 2004

LCx was compared to other assays in measuring human immunodeficiency virus type 1 (HIV-1) CRF02 viremia. LCx showed significant but low correlation with the other methods. Values of <2.60 log
10
cp/ml were observed in 29.6% of specimens with LCx and in only 14.8% with bDNA and PCR, suggesting suboptimal performance of LCx with CRF02.
Aneruptive Fever Associated with Antibodies to
Rickettsia helvetica
in Europe and Thailand
Authors: Fournier Pierre-Edouard , Allombert Caroline , Supputamongkol Yupin , Caruso Giuseppe , Brouqui Philippe , Raoult Didier
Published: February 2004

We report that eight patients from France, Italy, and Thailand had serological evidence of
Rickettsia helvetica
infection. The infection presented as a mild disease in the warm season and was associated with fever, headache, and myalgia but not with a cutaneous rash.
R. helvetica
should be suspected in patients with unexplained fever, especially following a bite from an
Ixodes
sp. tick.
Community-Acquired Methicillin-Resistant
Staphylococcus aureus
Isolated in Switzerland Contains the Panton-Valentine Leukocidin or Exfoliative Toxin Genes
Authors: Liassine Nadia , Auckenthaler Raymond , Descombes Marie-Christine , Bes Michèle , Vandenesch François , Etienne Jerome
Published: February 2004

Among 10 strains of community-acquired methicillin-resistant
Staphylococcus aureus
(CA-MRSA) isolated in 2002 from patients with skin infections, seven harbored the Panton-Valentine leukocidin gene, two harbored the exfoliative toxin A gene, and one harbored neither of these genes. CA-MRSA isolates producing a variety of exotoxins are currently spreading in the Swiss community.