bluuebunny/crossref_metadata_2025 huggingface dataset:
Incidence and Diversity of Microorganisms within the Walls of an Active Deep-Sea Sulfide Chimney
Authors: Schrenk Matthew O. , Kelley Deborah S. , Delaney John R. , Baross John A.
Published: June 2003

A large, intact sulfide chimney, designated Finn, was recovered from the Mothra Vent Field on the Juan de Fuca Ridge in 1998. Finn was venting 302°C fluids on the seafloor and contained complex mineralogical zones surrounding a large open central conduit. Examination of microorganisms within these zones, followed by community analysis with oligonucleotide probes, showed that there were variations in the abundance and diversity of eubacteria and archaea from the exterior to the interior of the chimney. The microbial abundance based upon epifluorescence microscopy and quantitative fatty acid analyses varied from >10
8
cells/g of sulfide 2 to 10 cm within the chimney wall to <10
5
cells/g in interior zones. Direct microscopic observation indicated that microorganisms were attached to mineral surfaces throughout the structure. Whole-cell hybridization results revealed that there was a transition from a mixed community of eubacteria and archaea near the cool exterior of the chimney to primarily archaea near the warm interior. Archaeal diversity was examined in three zones of Finn by cloning and sequencing of the 16S rRNA gene. The majority of sequences from the exterior of the chimney were related to marine group I of the
Crenarchaeota
and uncultured
Euryarchaeota
from benthic marine environments. In contrast, clone libraries from interior regions of the chimney contained sequences closely related to methanogens,
Thermococcales
, and
Archaeoglobales
, in addition to uncultured crenarchaeal phylotypes obtained from deep subsurface sites. These observations of microbial communities within an active hydrothermal chimney provide insight into the microbial ecology within such structures and may facilitate follow-up exploration into expanding the known upper temperature limits of life.
Development and Application of an Assay for Uranyl Complexation by Fungal Metabolites, Including Siderophores
Authors: Renshaw Joanna C. , Halliday Verity , Robson Geoffrey D. , Trinci Anthony P. J. , Wiebe Marilyn G. , Livens Francis R. , Collison David , Taylor Robin J.
Published: June 2003

An assay to detect UO
2
2+
complexation was developed based on the chrome azurol S (CAS) assay for siderophores (B. Schwyn and J. B. Neilands, Anal. Biochem. 160:47-56, 1987) and was used to investigate the ability of fungal metabolites to complex actinides. In this assay the discoloration of two dyed agars (one containing a CAS-Fe
3+
dye and the other containing a CAS-UO
2
2+
dye) caused by ligands was quantified. The assay was tested by using the siderophore desferrioxamine B (DFO), and the results showed that there was a regular, reproducible relationship between discoloration and the amount of siderophore added. The ratio of the discoloration on the CAS-UO
2
2+
agar to the discoloration on the CAS-Fe
3+
agar was independent of the amount of siderophore added. A total of 113 fungi and yeasts were isolated from three soil samples taken from the Peak District National Park. The fungi were screened for the production of UO
2
2+
chelators by using the CAS-based assay and were also tested specifically for hydroxamate siderophore production by using the hydroxamate siderophore auxotroph
Aureobacterium flavescens
JG-9. This organism is highly sensitive to the presence of hydroxamate siderophores. However, the CAS-based assay was found to be less sensitive than the
A. flavescens
JG-9 assay. No significant difference between the results for each site for the two tests was found. Three isolates were selected for further study and were identified as two
Pencillium
species and a
Mucor
species. Our results show that the new assay can be effectively used to screen fungi for the production of UO
2
2+
chelating ligands. We suggest that hydroxamate siderophores can be produced by mucoraceous fungi.
Combining Culture-Dependent and -Independent Methodologies for Estimation of Richness of Estuarine Bacterioplankton Consuming Riverine Dissolved Organic Matter
Authors: Kisand Veljo , Wikner Johan
Published: June 2003

Three different methods for analyzing natural microbial community diversity were combined to maximize an estimate of the richness of bacterioplankton catabolizing riverine dissolved organic matter (RDOM). We also evaluated the ability of culture-dependent quantitative DNA-DNA hybridization, a 16S rRNA gene clone library, and denaturing gradient gel electrophoresis (DGGE) to detect bacterial taxa in the same sample. Forty-two different cultivatable strains were isolated from rich and poor solid media. In addition, 50 unique clones were obtained by cloning of the bacterial 16S rDNA gene amplified by PCR from the community DNA into an
Escherichia coli
vector. Twenty-three unique bands were sequenced from 12 DGGE profiles, excluding a composite fuzzy band of the
Cytophaga-Flavobacterium
group. The different methods gave similar distributions of taxa at the genus level and higher. However, the match at the species level among the methods was poor, and only one species was identified by all three methods. Consequently, all three methods identified unique subsets of bacterial species, amounting to a total richness of 97 operational taxonomic units in the experimental system. The confidence in the results was, however, dependent on the current precision of the phylogenetic determination and definition of the species. Bacterial consumers of RDOM in the studied estuary were primarily both cultivatable and uncultivable taxa of the
Cytophaga-Flavobacterium
group, a concordant result among the methods applied. Culture-independent methods also suggested several not-yet-cultivated β-proteobacteria to be RDOM consumers.
Mapping of Genomic Regions (Quantitative Trait Loci) Controlling Production and Quality in Industrial Cultures of the Edible Basidiomycete
Pleurotus ostreatus
Authors: Larraya Luis M. , Alfonso Mikel , Pisabarro Antonio G. , Ramírez Lucía
Published: June 2003

Industrial production of the edible basidiomycete
Pleurotus ostreatus
(oyster mushroom) is based on a solid fermentation process in which a limited number of selected strains are used. Optimization of industrial mushroom production depends on improving the culture process and breeding new strains with higher yields and productivities. Traditionally, fungal breeding has been carried out by an empirical trial and error process. In this study, we used a different approach by mapping quantitative trait loci (QTLs) controlling culture production and quality within the framework of the genetic linkage map of
P. ostreatus
. Ten production traits and four quality traits were studied and mapped. The production QTLs identified explain nearly one-half of the production variation. More interestingly, a single QTL mapping to the highly polymorphic chromosome VII appears to be involved in control of all the productivity traits studied. Quality QTLs appear to be scattered across the genome and to have less effect on the variation of the corresponding traits. Moreover, some of the new hybrid strains constructed in the course of our experiments had production or quality values higher than those of the parents or other commercial strains. This approach opens the possibility of marker-assisted selection and breeding of new industrial strains of this fungus.
Characterization of the First Molluscicidal Lipopolysaccharide from
Moraxella osloensis
Authors: Tan Li , Grewal Parwinder S.
Published: June 2003

Moraxella osloensis
is a bacterium that is mutualistically associated with
Phasmarhabditis hermaphrodita,
a nematode that has potential for the biocontrol of mollusk pests, especially the slug
Deroceras reticulatum.
We discovered that purified
M. osloensis
lipopolysaccharide (LPS) possesses a lethal toxicity to
D. reticulatum
when administered by injection but no contact or oral toxicity to this slug. The toxicity of the LPS resides in the lipid A moiety.
M. osloensis
LPS was semiquantitated at 6 × 10
7
endotoxin units per mg. The LPS is a rough-type LPS with an estimated molecular weight of 5,300. Coinjection of galactosamine with the LPS increased the LPS's toxicity to the slug two- to four-fold. The galactosamine-induced sensitization of the slug to the LPS was reversed completely by uridine.
On the Origins of Cyanuric Acid Hydrolase: Purification, Substrates, and Prevalence of AtzD from
Pseudomonas
sp. Strain ADP
Authors: Fruchey Isaac , Shapir Nir , Sadowsky Michael J. , Wackett Lawrence P.
Published: June 2003

Cyanuric acid hydrolase (AtzD) from
Pseudomonas
sp. strain ADP was purified to homogeneity. Of 22 cyclic amides and triazine compounds tested, only cyanuric acid and
N
-methylisocyanuric acid were substrates. Other cyclic amidases were found not to hydrolyze cyanuric acid. Ten bacteria that use cyanuric acid as a sole nitrogen source for growth were found to contain either
atzD
or
trzD
, but not both genes.
Linkage of High Rates of Sulfate Reduction in Yellowstone Hot Springs to Unique Sequence Types in the Dissimilatory Sulfate Respiration Pathway
Authors: Fishbain Susan , Dillon Jesse G. , Gough Heidi L. , Stahl David A.
Published: June 2003

Diversity, habitat range, and activities of sulfate-reducing prokaryotes within hot springs in Yellowstone National Park were characterized using endogenous activity measurements, molecular characterization, and enrichment. Five major phylogenetic groups were identified using PCR amplification of the dissimilatory sulfite reductase genes (
dsrAB
) from springs demonstrating significant sulfate reduction rates, including a warm, acidic (pH 2.5) stream and several nearly neutral hot springs with temperatures reaching 89°C. Three of these sequence groups were unrelated to named lineages, suggesting that the diversity and habitat range of sulfate-reducing prokaryotes exceeds that now represented in culture.
Behavior of Psychrotrophic Lactic Acid Bacteria Isolated from Spoiling Cooked Meat Products
Authors: Hamasaki Yoshikatsu , Ayaki Mitsuko , Fuchu Hidetaka , Sugiyama Masaaki , Morita Hidetoshi
Published: June 2003

Three kinds of lactic acid bacteria were isolated from spoiling cooked meat products stored below 10°C. They were identified as
Leuconostoc mesenteroides
subsp.
mesenteroides
,
Lactococcus lactis
subsp.
lactis
, and
Leuconostoc citreum
. All three strains grew well in MRS broth at 10°C. In particular,
L. mesenteroides
subsp.
mesenteroides
and
L. citreum
grew even at 4°C, and their doubling times were 23.6 and 51.5 h, respectively. On the other hand, although the bacteria were initially below the detection limit (<10 CFU/g) in model cooked meat products, the bacterial counts increased to 10
8
CFU/g at 10°C after 7 to 12 days.
Microorganisms Associated with Uranium Bioremediation in a High-Salinity Subsurface Sediment
Authors: Nevin Kelly P. , Finneran Kevin T. , Lovley Derek R.
Published: June 2003

Although stimulation of dissimilatory metal reduction to promote the reductive precipitation of uranium has been shown to successfully remove uranium from some aquifer sediments, the organisms in the family
Geobacteraceae
that have been found to be associated with metal reduction in previous studies are not known to grow at the high salinities found in some uranium-contaminated groundwaters. Studies with a highly saline uranium-contaminated aquifer sediment demonstrated that the addition of acetate could stimulate the removal of U(VI) from the groundwater. This removal was associated with an enrichment in microorganisms most closely related to
Pseudomonas
and
Desulfosporosinus
species.
Direct Detection of
Vibrio cholerae
and
ctxA
in Peruvian Coastal Water and Plankton by PCR
Authors: Lipp Erin K. , Rivera Irma N. G. , Gil Ana I. , Espeland Eric M. , Choopun Nipa , Louis Valérie R. , Russek-Cohen Estelle , Huq Anwar , Colwell Rita R.
Published: June 2003

Seawater and plankton samples were collected over a period of 17 months from November 1998 to March 2000 along the coast of Peru. Total DNA was extracted from water and from plankton grouped by size into two fractions (64 μm to 202 μm and >202 μm). All samples were assayed for
Vibrio cholerae
,
V. cholerae
O1,
V. cholerae
O139, and
ctxA
by PCR. Of 50 samples collected and tested, 33 (66.0%) were positive for
V. cholerae
in at least one of the three fractions. Of these, 62.5% (
n
= 32) contained
V. cholerae
O1;
ctxA
was detected in 25% (
n
= 20) of the
V. cholerae
O1-positive samples. None were positive for
V. cholerae
O139. Thus, PCR was successfully employed in detecting toxigenic
V. cholerae
directly in seawater and plankton samples and provides evidence for an environmental reservoir for this pathogen in Peruvian coastal waters.
Generation of Food-Grade Lactococcal Starters Which Produce the Lantibiotics Lacticin 3147 and Lacticin 481
Authors: O'Sullivan Lisa , Ryan Maire P. , Ross R. Paul , Hill Colin
Published: June 2003

Transconjugant lactococcal starters which produce both lantibiotics lacticin 3147 and lacticin 481 were generated via conjugation of large bacteriocin-encoding plasmids. A representative of one of the resultant strains proved more effective at killing
Lactobacillus fermentum
and inhibiting the growth of
Listeria monocytogenes
LO28H than either of the single bacteriocin-producing parental strains, demonstrating the potential of these transconjugants as protection cultures for food safety applications.
Nationwide Surveillance for
Staphylococcus aureus
with Reduced Susceptibility to Vancomycin in Korea
Authors: Kim Hong Bin , Park Wan Beom , Lee Ki Deok , Choi Young Joo , Park Sang Won , Oh Myoung-don , Kim Eui-Chong , Choe Kang Won
Published: June 2003

Methicillin-resistant
Staphylococcus aureus
(MRSA) accounts for more than 70% of
S. aureus
isolates from tertiary hospitals in Korea. Clinical isolates of
S. aureus
were collected from eight provincial, university-affiliated hospitals during the period from June 1999 to January 2001 for nationwide surveillance. All isolates were screened for reduced susceptibility to vancomycin by using brain heart infusion agar containing 4 μg of vancomycin per milliliter. Population analysis and the determination of the MIC of vancomycin were done for the isolates which grew on the screening agar plates. Of 682 total isolates, MRSA accounted for 64% (439 of 682). Of 27 (4%) isolates that grew on the screening agar plates, none showed the heteroresistance phenotype. No strains with reduced susceptibility to vancomycin were identified.
Longitudinal Study ofSalmonella entericaSerotype Typhimurium Infection in Three Danish Farrow-to-Finish Swine Herds
Authors: Kranker Søren , Alban Lis , Boes Jaap , Dahl Jan
Published: June 2003

A longitudinal study of the infection dynamics ofSalmonella entericawas carried out with three Danish farrow-to-finish swine herds. To account for variations inSalmonellashedding over time, litters from each herd were divided into two cohorts. Each cohort consisted of 30 pigs, for a total of 180 pigs. Pigs were individually monitored by monthly bacteriologic and serologic examinations from weaning to slaughter. At weaning, individual sows were examined bacteriologically and serologically. At slaughter, cecal contents, ileocecal lymph nodes, and carcass swab samples were obtained from 131 pigs. A total of 88 pigs were found to be sheddingSalmonellaon one or more occasions. Only theSalmonellaserotype Typhimurium was detected during the study period. At weaning, no sows or piglets were found to be shedding, but a serological reaction was detected in 11 sows. The prevalence in culture peaked in the nursery and subsequently declined to undetectable levels before slaughter. The seroprevalence peaked approximately 60 days after the peak prevalence in culture.Salmonellawas detected in individual fecal samples at least once in 53% of the pigs, and 62% of the pigs were seropositive more than once. Only 3.7% of all pigs were found to be culture positive on more than one occasion. Piglets from seroreacting sows had a significantly (P= 0.0339) lower probability of shedding in the nursery. Under the assumption that shedding lasted at least 1 or 2 weeks, the average shedding time was estimated to have been 18 or 26 days. An association between serology, on-farm bacteriology, andSalmonellaprevalence in culture at slaughter was shown. Marked differences in prevalence in sera and prevalence in culture between cohorts and within herds were observed. These differences emphasize the need for caution when using point estimates in on-farm interventions and surveillance in subclinically infected swine herds.
Prevalence of Enteroaggregative
Escherichia coli
among Children with and without Diarrhea in Switzerland
Authors: Pabst Werner L. , Altwegg Martin , Kind Christian , Mirjanic Slavko , Hardegger Daniel , Nadal David
Published: June 2003

In a prospective study between July 1999 and September 2000, stool specimens of children below the age of 16 years with (
n
= 187) and without (
n
= 137) diarrhea were tested for the presence of enterovirulent bacteria by standard culture methods and by PCR. Targets for the PCR were the plasmid pCVD432 for enteroaggregative
Escherichia coli
(EAEC), the verotoxin 1 and verotoxin 2 genes for enterohemorrhagic
E. coli
,
ipaH
for enteroinvasive
E. coli
(EIEC) and
Shigella
spp., genes coding for heat-stable and heat-labile toxins for enterotoxigenic
E. coli
(ETEC), and the
eaeA
gene for enteropathogenic
E. coli.
The following bacteria could be associated with diarrhea:
Salmonella enterica
(
P
= 0.001),
Campylobacter
spp. (
P
= 0.036), ETEC (
P
= 0.012), and EAEC (
P
= 0.006). The detection of EAEC, ETEC, and
S. enterica
was strongly associated with a history of recent travel outside of Switzerland. EAEC isolates were found in the specimens of 19 (10.2%) of 187 children with diarrhea and in those of 3 (2.2%) of 137 children without diarrhea (
P
= 0.006) and were the most frequently detected bacteria associated with diarrhea. Among the children below the age of 5 years, the specimens of 18 (11.9%) of 151 with diarrhea were positive for EAEC, while this agent was found in the specimens of 2 (2.2%) of 91 controls (
P
= 0.007). Enteropathogenic
E. coli
isolates were found in the specimens of 30 (16.4%) of the patients and in those of 15 (10.9%) of the controls, with similar frequencies in all age groups (
P
> 0.05). We conclude that EAEC bacteria are involved in a significant proportion of diarrhea cases among children. Children younger than 5 years of age are more often affected by EAEC than older children.
Diagnostic Potential of Parechovirus Capsid Proteins
Authors: Alho Annu , Marttila Jane , Ilonen Jorma , Hyypiä Timo
Published: June 2003

To study humoral and cellular immunity against human parechovirus type 1 (HPEV1), the viral capsid proteins VP0, VP1, and VP3 were expressed and purified as glutathione
S
-transferase (GST)-tagged recombinant proteins. The fusion proteins were used to raise antisera in rabbits. VP0 and VP1 antisera specifically detected HPEV1-infected cells in culture by immunoperoxidase staining and immunofluorescence. Furthermore, antisera against the VP0 and VP1 proteins had neutralizing effects against HPEV1 infection. When the HPEV1 antibody titers of 20 adults and 55 children were determined by a microneutralization test, the prevalence of HPEV1 antibodies in the adult population was 96%, while 50% of children were seropositive. Selected sera were used to evaluate HPEV1 fusion proteins as antigens in an enzyme immunoassay. The VP3 capsid protein appeared to be suitable for the purpose, with specificity of 100% and sensitivity of 96% compared to the neutralization test. Furthermore, T-cell responses to the purified HPEV1 and HPEV1 capsid fusion proteins were studied in 20 adults. Sixty percent of the subjects had T-cell proliferation responses to purified HPEV1, and 90% of the subjects also had positive T-cell responses to at least one of the GST capsid proteins.
Studies of Epidemiology and Seroprevalence of Bovine Noroviruses in Germany
Authors: Deng Y. , Batten C. A. , Liu B. L. , Lambden P. R. , Elschner M. , Günther H. , Otto P. , Schnürch P. , Eichhorn W. , Herbst W. , Clarke I. N.
Published: June 2003

Jena virus (JV) is a bovine enteric calicivirus that causes diarrhea in calves. The virus is approximately 30 nm in diameter and has a surface morphology similar to the human Norwalk virus. The genome sequence of JV was recently described, and the virus has been assigned to the genus
Norovirus
of the family
Caliciviridae
. In the present study, the JV capsid gene encoded by open reading frame 2 was cloned into the baculovirus transfer vector pFastBac 1, and this was used to transform
Escherichia coli
to generate a recombinant bacmid. Transfection of insect cells with the recombinant baculovirus DNA resulted in expression of the JV capsid protein. The recombinant JV capsid protein undergoes self-assembly into virus-like particles (VLPs) similar to JV virions in size and appearance. JV VLPs were released into the cell culture supernatant, concentrated, and then purified by CsCl equilibrium gradient centrifugation. Purified JV VLPs were used to hyperimmunize laboratory animals. An antigen capture enzyme-linked immunosorbent assay (ELISA) was developed and characterized initially with clinical specimens containing defined human noroviruses and bovine diarrheal samples from calves experimentally infected with JV; the ELISA was specific only for JV. The ELISA was used to screen 381 diarrheal samples collected from dairy herds in Thuringia, Hesse, and Bavaria, Germany, from 1999 to 2002; 34 of these samples (8.9%) were positive for JV infection. The unexpectedly high prevalence of JV was confirmed in a seroepidemiological study using 824 serum or plasma samples screened using an anti-JV ELISA, which showed that 99.1% of cattle from Thuringia have antibodies to JV.
Molecular Epidemiology of
Vibrio cholerae
O139 in China: Polymorphism of Ribotypes and CTX Elements
Authors: Qu Mei , Xu Jing , Ding Yanpeng , Wang Ruibai , Liu Peng , Kan Biao , Qi Guoming , Liu Yanqing , Gao Shouyi
Published: June 2003

Vibrio cholerae
O139, the second etiological serogroup of cholera, triggered the first outbreak of O139 cholera in China in 1993. To analyze the clone polymorphism of O139 isolates in China, 117 strains of
V. cholerae
O139, isolated from different areas in China between 1993 and 1999, were selected to characterize the phylogenetic relationships by molecular techniques. Analysis of restriction fragment length polymorphism in the conserved 16S rRNA gene revealed seven different ribotypes within the 117 strains. Among these strains, there were eight that lacked the cholera toxin gene (
ctxAB
),
zot
, and the repetitive sequence (RS); these eight strains belonged to three individual ribotypes. Our results suggested that
V. cholerae
O139 strains in China had clone diversity in phylogeny. The results of our hybridization patterns for CTX genetic elements (
ctxAB
,
zot
, and RS) showed that CTXΦ genomes in most
V. cholerae
O139 strains had two or more copies and had extensive restriction patterns even for the strains which belong to the same ribotype. For 22 (20.1%) strains, the copies of
ctxAB
were different from those of
zot
, suggesting that a
ctxAB
-negative CTXΦ genome may exist in O139 strains. This
ctxAB
-negative CTXΦ genome may coexist with the intact CTXΦ genome in a strain. In addition, the dendrogram for I-
Ceu
I-generated pulsed-field gel electrophoresis patterns showed that
V. cholerae
serogroup O139 has a closer relationship with one strain of serogroup O22 than with the strains of serogroup O1. The results of this study showed the clonal diversity and the distribution of O139 strains in China, suggesting multiple origins of the O139 cholera epidemic or sporadic events.
Genetic Variability among Group A and B Respiratory Syncytial Virus Isolates from a Large Referral Hospital in New Delhi, India
Authors: Rajala Maitreyi S. , Sullender Wayne M. , Prasad A. K. , Dar Lalit , Broor Shobha
Published: June 2003

Respiratory syncytial virus (RSV) is an important childhood pathogen of acute lower respiratory infections in developed and developing countries. The molecular epidemiology of RSV in India is largely unknown. The present study was undertaken to standardize and evaluate reverse transcription-PCR (RT-PCR) for the rapid and simultaneous detection of RSV groups A and B in clinical samples and to study intragroup genetic variability. RT-PCR was evaluated by comparing the results of seminested RT-PCR with centrifugation-enhanced cultures on 200 nasopharyngeal aspirates from children with acute lower respiratory infections. RSV was isolated in 34 nasopharyngeal aspirates by centrifugation-enhanced cultures and identified in 45 samples by RT-PCR. In 15 samples RSV was identified by seminested RT-PCR alone and in four by centrifugation-enhanced cultures alone. Of the 45 samples positive for RSV by nested PCR, 15 belonged to group A, 29 to group B, and one sample suggested a mixed infection. Group B RSV predominated in both years of the 2-year study. Genetic variability within RSV groups was studied by restriction fragment analysis of 35 PCR products. Among both group A and group B RSV, two different composite patterns were observed. Thus, RSV was found to be a major pathogen of acute lower respiratory tract infections in India, as it was detected in 24.5% of children by RT-PCR. RT-PCR provides a sensitive method for detection and typing of RSV group A and B viruses in clinical samples as well as a means to study intragroup variations. However, a higher sensitivity of detection of RSV in clinical samples can be obtained by its combination with additional techniques, such as virus cultivation.
Changing Patterns of Rotavirus Genotypes in Ghana: Emergence of Human Rotavirus G9 as a Major Cause of Diarrhea in Children
Authors: Armah George E. , Steele Andrew Duncan , Binka Fred N. , Esona Mathew D. , Asmah Richard Harry , Anto Francis , Brown David , Green Jon , Cutts Felicity , Hall Andy
Published: June 2003

Genotyping of human rotaviruses was performed on 312 rotavirus-positive samples collected from 2,205 young children with diarrhea in the Upper East District of Ghana, a rural community. Of the 271 (86.9%) rotavirus strains that could be VP7 (G) or VP4 (P) characterized, 73 (26.9%) were of G9 specificity. The predominant G9 genotype was G9P[8], which constituted 79.5% of all G9 strains detected, followed by G9P[6] (12.3%), G9P[10] (2.7%), and G9P[4] (1.3%). G9 strains with mixed P types constituted 2.7% of all G9 strains found in the study. All the G9P[8] strains had a long RNA electrophoretic pattern with VP6 subgroup II specificity. Four G9 isolates, GH1319, GH1416, GH3550, and GH3574, which were selected based on the abundance of stool material and were representative of the three electropherotypes observed, were cloned and sequenced. The Ghanaian isolates shared more than 98% sequence nucleotide homology with other G9 strains from the United States (US1205), Malawi (MW69), Brazil (R160), Japan (95H115), and Nigeria (Bulumkutu). However, they showed only 95% nucleotide homology with the Thai G9 strain Mc345. Phylogenetic analysis of the nucleic acid sequence revealed the existence of at least three clusters, with Ghanaian strains forming one cluster, Nigerian and Brazilian strains forming a second cluster, and U.S., Malawian, and Japanese strains forming a third.
Rapid Diagnosis of Extrapulmonary Tuberculosis by PCR: Impact of Sample Preparation and DNA Extraction
Authors: Honoré-Bouakline S. , Vincensini J. P. , Giacuzzo V. , Lagrange P. H. , Herrmann J. L.
Published: June 2003

In cases of suspected extrapulmonary tuberculosis, rapid and accurate laboratory diagnosis is of prime importance, since traditional techniques of detecting acid-fast bacilli have limitations. The major difficulty with mycobacteria is achieving optimal cell lysis. Buffers used in commercial kits do not allow this complete lysis in a number of clinical specimens. A comparison of two sample preparation methods, pretreatment with proteinase K (PK-Roche) and complete DNA purification (cetyltrimethylammonium bromide [CTAB]-Roche), was conducted on 144 extrapulmonary specimens collected from 120 patients to evaluate the impact on the Cobas-Amplicor method. Thirty patients were diagnosed with tuberculosis, with 15 patients culture positive for
Mycobacterium tuberculosis
. Amplification and detection of the amplicons were impaired by a high number of inhibitory specimens (39 to 52%). CTAB-Roche allowed the detection of more culture-positive specimens by PCR than PK-Roche. Comparison with the final diagnoses of tuberculosis confirmed that CTAB-Roche produced the best sensitivity (53.8%) compared to culture (43.3%), PK-Roche (16%), and smear (13%). However, the specificity of the PCR assay with CTAB-Roche-extracted material was always lower (78.8%) than those with culture (100%) and PK-Roche (96.5%). False-positive specimens were lung biopsy material, lymph node biopsy material and aspirate, or bone marrow aspirate, mainly from immunocompromised patients. Despite the efficiency of complete DNA extraction for the rapid diagnosis by PCR of extrapulmonary tuberculosis, the false-positive results challenge our understanding of PCR results.
Molecular Characterization of Ampicillin-Resistant
Enterococcus faecium
Isolates from Hospitalized Patients in Norway
Authors: Jureen Roland , Top Janetta , Mohn Stein Christian , Harthug Stig , Langeland Nina , Willems Rob J. L.
Published: June 2003

The genetic relationship of 81 ampicillin-resistant and 21 ampicillin-susceptible
Enterococcus faecium
isolates from clinical infections and rectal screening in hospitalized patients in Norway was studied by pulsed-field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP). PFGE showed 55 different banding patterns, and 65 of the isolates could be grouped into one large group. With AFLP, 46 patterns were discerned, and 74 isolates clustered in one group. In general, the isolates had a higher degree of similarity than with PFGE. The
purK
gene, which is one of the targets of the
E. faecium
multilocus sequence typing scheme, was sequenced. Eleven different
purK
alleles could be discerned, with the majority of isolates (
n
= 80) harboring allele 1. With only two exceptions, all strains carrying
purK-1
clustered in the same PFGE and AFLP groups, indicating a good correlation between PFGE type, AFLP type, and
purK
allele. Genetic polymorphism of a 571-bp PCR fragment of the C-terminal domain of the penicillin-binding protein 5 gene (
pbp5
) was determined, and sequence differences were associated with the level of ampicillin resistance. This study indicates that the majority of ampicillin-resistant
E. faecium
strains in Norway belong to a distinct genetic lineage of closely related genotypes. Rectal and clinical isolates were generally indistinguishable, and differences in clonal distribution and allele polymorphism were found mainly between ampicillin-resistant and -susceptible isolates.
Emergence of Rifampin-Resistant
Rhodococcus equi
with Several Types of Mutations in the
rpoB
Gene among AIDS Patients in Northern Thailand
Authors: Asoh Norichika , Watanabe Hiroshi , Fines-Guyon Marguerite , Watanabe Kiwao , Oishi Kazunori , Kositsakulchai Weerayut , Sanchai Tippaya , Kunsuikmengrai Khemrassamee , Kahintapong Sumpun , Khantawa Banyong , Tharavichitkul Prasit , Sirisanthana Thira , Nagatake Tsuyoshi
Published: June 2003

The antimicrobial susceptibilities of 30
Rhodococcus equi
isolates obtained from 30 patients between 1993 and 2001 in northern Thailand were investigated. The MICs showed a tendency toward resistance to various antibiotics but sensitivity to imipenem, minocycline, vancomycin, and teicoplanin (MICs, ≤0.5 μg/ml) and relative sensitivity to meropenem, clarithromycin, and ciprofloxacin (MICs, ≤2 μg/ml). Of the 30 isolates, 26 were susceptible (MICs, ≤1 μg/ml), 1 showed low-level resistance (MIC, 8 μg/ml), and 3 showed high-level resistance (MICs, ≥64 μg/ml) to rifampin. PCR amplification and DNA sequencing of the
rpoB
gene and molecular typing by pulsed-field gel electrophoresis (PFGE) were performed for eight
R. equi
isolates from eight AIDS patients with pneumonia or lung abscess caused by
R. equi
between 1998 and 2001, including one low- and three high-level rifampin-resistant isolates. As a result, two high-level rifampin-resistant strains with PFGE pattern A had a Ser531Trp (
Escherichia coli
numbering) mutation, and one high-level rifampin-resistant strain with PFGE pattern B had a His526Tyr mutation, whereas one low-level rifampin-resistant strain with PFGE pattern C had a Ser509Pro mutation. Four rifampin-susceptible strains with PFGE patterns D and E showed an absence of mutation in the
rpoB
region. Our results indicate the presence of several types of rifampin-resistant
R. equi
strains among AIDS patients in northern Thailand.
Distribution of the Secondary Type III Secretion System Locus Found in Enterohemorrhagic
Escherichia coli
O157:H7 Isolates among Shiga Toxin-Producing
E. coli
Strains
Authors: Makino Sou-Ichi , Tobe Toru , Asakura Hiroshi , Watarai Masahisa , Ikeda Testuya , Takeshi Koichi , Sasakawa Chihiro
Published: June 2003

The ability of the complete genome sequence of enterohemorrhagic
Escherichia coli
O157 led to the identification of a 17-kb chromosomal region which contained a type III secretion system gene cluster at min 64.5. This locus contains open reading frames whose amino acid sequences show high degrees of similarity with those of proteins that make up the type III secretion apparatus, which is encoded by the
inv-spa-prg
locus on a
Salmonella
SPI-1 pathogenicity island. This locus was designated ETT2 (
E. coli
type III secretion 2) and consisted of the
epr
,
epa
, and
eiv
genes. ETT2 was found in enteropathogenic
E. coli
strains and also in some non-O157 Shiga toxin-producing
E. coli
(STEC) strains, but most of them contained a truncated portion of ETT2. Most O157 isolates had a complete collection of toxin-encoding genes
eae
and
hlyA
and the ETT2 locus, while most O26 strains had toxin-encoding genes
eae
and
hlyA
genes but an incomplete ETT2 locus. Thus, an intact copy of ETT2 might mark a pathogenic distinction for particular STEC strains. Therefore, the presence of the ETT2 locus can be used for identification of truly pathogenic STEC strains and for molecular fingerprinting of the epidemic strains in humans and animals.
The Genus
Aeromonas
: Biochemical Characteristics, Atypical Reactions, and Phenotypic Identification Schemes
Authors: Abbott Sharon L. , Cheung Wendy K. W. , Janda J. Michael
Published: June 2003

A total of 193 strains representing 14 different
Aeromonas
genomospecies were evaluated for 63 phenotypic properties to create useful tables for the reference identification of mesophilic aeromonads. Only 9 of 62 biochemical tests (14%) yielded uniform results, and the fermentation of certain carbohydrates was found to be linked to specific species. A number of unusual or aberrant properties for the genus
Aeromonas
were also detected in the collection of 428 strains (193 in the phenotypic study, 235 in a retrospective review). These tests included susceptibility to the vibriostatic agent, fermentation of
m-
inositol and
d
-xylose, hydrolysis of urea, and the lack of cytochrome oxidase activity. Fermentation of melibiose was linked to raffinose fermentation in all
Aeromonas
species except
A. jandaei
. Keys are provided for clinical laboratories choosing to identify aeromonads to species level based upon initial Møeller decarboxylase and dihydrolase reactions. In addition, several new tests were identified that help to separate members of the
A. caviae
complex (
A. caviae, A. media,
and
A. eucreonophila
).
Devices for Rapid Diagnosis of Malaria: Evaluation of Prototype Assays That Detect
Plasmodium falciparum
Histidine-Rich Protein 2 and a
Plasmodium vivax
-Specific Antigen
Authors: Forney J. Russ , Wongsrichanalai Chansuda , Magill Alan J. , Craig Leslie G. , Sirichaisinthop Jeeraphat , Bautista Christian T. , Miller R. Scott , Ockenhouse Christian F. , Kester Kent E. , Aronson Naomi E. , Andersen Ellen M. , Quino-Ascurra Higinio A. , Vidal Carlos , Moran Kimberly A. , Murray Clinton K. , DeWitt Caroline Cady , Heppner D. Gray , Kain Kevin C. , Ballou W. Ripley , Gasser Robert A.
Published: June 2003

The
Para
Sight F test was developed as a pioneer industry effort in the large-scale, process-controlled production of a device for the rapid diagnosis of malaria. This device performed well in field settings but was limited to the detection of a single malaria species,
Plasmodium falciparum.
The
Para
Sight F+V assay advanced upon the
Para
Sight F test format by incorporating a monoclonal antibody directed against a proprietary
Plasmodium vivax
-specific antigen, in addition to the antibody directed against
P. falciparum
histidine-rich protein 2, which was used in the
Para
Sight F assay. The modified assay was developed to add the capability to detect
P. falciparum
and
P. vivax
in a single-test-strip format. The present study evaluated three distinct
Para
Sight F+V prototypes with samples from symptomatic patients in regions of Thailand and Peru where malaria is endemic. Over a 2-year enrollment period (1998 and 1999), a total of 4,894 patients consented to participation in the study. Compared with the results for duplicate microscopic examinations of Giemsa-stained blood smears as the reference diagnostic standard, each successive prototype showed substantial improvement in performance. The final
Para
Sight F+V prototype, evaluated in 1999, had an overall sensitivity for detection of asexual
P. falciparum
parasites of 98%. The sensitivity of the device was 100% for
P. falciparum
densities of >500 parasites/μl, with a sensitivity of 83% for parasite densities of ≤500/μl. The specificity for the exclusion of
P. falciparum
was 93%. For
P. vivax
, the overall sensitivity was 87% for the final 1999 prototype. The sensitivities calculated for different levels of
P. vivax
parasitemia were 99% for parasite densities of >5,000/μl, 92% for parasite densities of 1,001 to 5,000/μl, 94% for parasite densities of 501 to 1,000/μl, and 55% for parasite densities of 1 to 500/μl. The specificity for the exclusion of
P. vivax
was 87%. The areas under the receiver operating characteristic curves for the diagnostic performance of the assay for the detection of
P. falciparum
and
P. vivax
were 0.8907 and 0.8522, respectively. These findings indicate that assays for rapid diagnosis have the potential to enhance diagnostic capabilities in those instances in which skilled microscopy is not readily available.
Precise Characterization of Norovirus (Norwalk-Like Virus)-Specific Monoclonal Antibodies with Broad Reactivity
Authors: Yoda Tomoko , Suzuki Yasuhiko , Terano Yoshitake , Yamazaki Kenji , Sakon Naomi , Kuzuguchi Tsuyoshi , Oda Hajime , Tsukamoto Teizo
Published: June 2003

We have been characterizing monoclonal antibodies against Norovirus (Norwalk-like virus). In the course of our study, two monoclonal antibodies generated against Norovirus genogroup II capsid protein were found to react not only to genogroup II but also to genogroup I recombinant capsid proteins. In addition, we showed that these two monoclonal antibodies reacted to a 40-amino-acid-fragment located close to the N-terminal region of genogroup II Norovirus. Similar reactivity was observed with the equivalent region of genogroup I Norovirus. In this study, we confirmed that the epitopes of the two monoclonal antibodies existed within an 11-amino-acid peptide. To obtain an idea of the reactive ranges of the two monoclonal antibodies toward different strains of Norovirus, their reactivities were investigated using 16 types of peptide constructed according to the data in GenBank and 8 recombinant capsid proteins (7 whole capsid proteins and 1 short [80-amino-acid] protein fragment). A characteristic broad reactivity of the two monoclonal antibodies is clearly shown by the results of this study. Thus, these monoclonal antibodies could be useful tools for detecting a broad range of Norovirus strains.
The World Health Organization's External Quality Assurance System Proficiency Testing Program Has Improved the Accuracy of Antimicrobial Susceptibility Testing and Reporting among Participating Laboratories Using NCCLS Methods
Authors: Chaitram Jasmine M. , Jevitt Laura A. , Lary Sara , Tenover Fred C.
Published: June 2003

A total of 150 laboratories in 33 countries that followed the NCCLS testing procedures participated in the World Health Organization's External Quality Assurance System for Antimicrobial Susceptibility Testing (EQAS-AST) from January 1998 through March 2001. Laboratories tested seven bacterial isolates for antimicrobial resistance and reported the results to the Centers for Disease Control and Prevention (CDC) in Atlanta, Ga. The results were compared to the results generated at the CDC with the NCCLS broth microdilution and disk diffusion reference methods. Although there were few testing errors with
Salmonella enterica
subsp.
enterica
serovar Enteritidis, drugs that are not appropriate for therapy of
Salmonella
infections were tested and reported by 136 (91%) of 150 laboratories. In addition, 29 (20%) of 150 laboratories used the
Staphylococcus aureus
breakpoints to report oxacillin results for
Staphylococcus saprophyticus
. For a
vanB
-containing
Enterococcus faecalis
strain, 124 (83%) of 150 laboratories correctly reported vancomycin results that were ±1 doubling dilution from the reference MIC or ±3 mm from the reference disk diffusion result. Of the laboratories that tested
Streptococcus agalactiae
by disk diffusion, 17% reported nonsusceptible results for penicillin in error. While 110 laboratories (73%) tested the
S
.
pneumoniae
challenge isolate against a fluoroquinolone, 83% tested it against ciprofloxacin, for which there are no NCCLS interpretive criteria. Ten of 12 laboratories testing levofloxacin and 4 of 4 laboratories testing ofloxacin by an MIC method correctly reported resistant results for the isolate. Feedback letters sent to participating laboratories highlighted areas of susceptibility testing in individual laboratories that needed improvement. The positive impact of the feedback letters and the overall effectiveness of the EQAS program were documented in repeat testing challenges with pneumococci and staphylococci. The 31 and 19% increases in the numbers of laboratories using appropriate testing methods for pneumococci and staphylococci, respectively, in 2000 versus 1998 indicate that laboratory performance is improving.
Serotyping
Streptococcus pneumoniae
by Multiplex PCR
Authors: Brito D. A. , Ramirez M. , de Lencastre H.
Published: June 2003

The capsule is a major virulence factor of pneumococci, and it was shown that some capsular variants are associated with antimicrobial resistance and certain types of disease. Moreover, pneumococcal capsular typing has received renewed interest since the availability of conjugate vaccines, which include serotypes frequently associated with pediatric disease. Our aim was to develop a simple, reliable, and economical method for detecting epidemiologically important serotypes present in the proposed 11-valent conjugate vaccine. We designed primers based on the sequences available for the capsular types 1, 3, 4, 6B, 14, 18C, 19F, 19A, and 23F and combined them into seven multiplex PCRs. The method involves streamlined DNA template preparation and agarose gel electrophoresis to analyze the amplification products. A total of 446 pneumococci selected from among isolates colonizing the nasopharynx of children attending day care centers in Lisbon, Portugal, were typed both by conventional immunological techniques and by multiplex PCR. Capsular types identified by the PCR method invariably produced results concordant with the conventional serotyping technique. Even when the method presented does not fully type an isolate, the PCR data can guide the experimenter when using immunological serotyping. Multiplex PCR for the analysis of pneumococci provides an accurate, expeditious, and cost-effective way of reducing the number of strains that have to be serotyped by conventional immunological techniques.
Survey of
emm
Gene Sequences from Pharyngeal
Streptococcus pyogenes
Isolates Collected in Spain and Their Relationship with Erythromycin Susceptibility
Authors: Albertí Sebastián , García-Rey César , Domínguez María A. , Aguilar Lorenzo , Cercenado Emilia , Gobernado Miguel , García-Perea Adela
Published: June 2003

We conducted a nationwide survey of the variable 5′
emm
(M protein gene) sequences from 614 pharyngeal
Streptococcus pyogenes
isolates susceptible (299 isolates) and resistant (315 isolates) to erythromycin that were isolated in Spain from 1996 to 1999. Almost 98% of these isolates had
emm
sequences in agreement with previously recorded M antigen association. We only identified a new 5′
emm
sequence in 17 isolates. Nine different
emm
types accounted for 85% of the
S. pyogenes
isolates susceptible to erythromycin. By contrast, only 3
emm
types accounted for 70% of the erythromycin-resistant isolates. Further characterization of these isolates by ribotyping and pulsed-field gel electrophoresis indicated that high frequency of erythromycin resistance in Spain is due to few clones.
Controlled Clinical Comparison of BacT/ALERT Standard Aerobic Medium with BACTEC Standard Aerobic Medium for Culturing Blood
Authors: Mirrett Stanley , Reller L. Barth , Petti Cathy A. , Woods Christopher W. , Vazirani Bindu , Sivadas Rekha , Weinstein Melvin P.
Published: June 2003

Standard aerobic media are widely used for culturing blood with the BacT/ALERT (BioMérieux, Inc., Durham, N.C.) (BM) and BACTEC 9240 (BD Diagnostic Systems, Sparks, Md.) (BD) automated continuously monitoring instrument systems. Although similarly composed of soybean-casein digest broths, the formulations of the standard aerobic media available for these instruments differ from each other in supplements and in sodium polyanetholesulfonate concentration. Therefore, we compared the standard aerobic media available for these systems at two university hospitals. Blood samples from adult patients with suspected bloodstream infection were inoculated at the bedside into nonvented BM and BD standard aerobic blood culture bottles and incubated in their respective instruments. The laboratories received 6,743 pairs of bottles that were each filled with 8 to 12 ml of blood. A total of 523 isolates representing true infections were recovered from 257 patients; of these isolates, 348 were recovered from both the BD and the BM bottles, 108 were recovered from the BM bottles only, and 67 were recovered from the BD bottles only (
P
< 0.005). More staphylococci (
P
< 0.05), especially coagulase-negative staphylococci (
P
< 0.05), and yeasts (
P
< 0.01) were recovered from BM bottles than from BD bottles. Of 291 unimicrobial episodes of bloodstream infection, 220 were detected with both bottles, 41 were detected with the BM bottles only, and 30 were detected with the BD bottles only (difference not significant). Among 335 cultures that were positive in both bottles within the first 72 h of incubation, the median times to detection were 14 h for BM bottles and 13 h for BD bottles. Rates for false-positive results were 0.5% for BM bottles and 0.1% for BD bottles. One BM bottle and seven BD bottles yielded false-negative results. We conclude that the BM medium provides improved recovery of microorganisms, especially staphylococci and yeasts, compared with that provided by the BD medium.