bluuebunny/crossref_metadata_2025 huggingface dataset:
Use of DNA and Peptide Nucleic Acid Molecular Beacons for Detection and Quantification of rRNA in Solution and in Whole Cells
Authors: Xi Chuanwu , Balberg Michal , Boppart Stephen A. , Raskin Lutgarde
Published: September 2003

DNA and peptide nucleic acid (PNA) molecular beacons were successfully used to detect rRNA in solution. In addition, PNA molecular beacon hybridizations were found to be useful for the quantification of rRNA: hybridization signals increased in a linear fashion with the 16S rRNA concentrations used in this experiment (between 0.39 and 25 nM) in the presence of 50 nM PNA MB. DNA and PNA molecular beacons were successfully used to detect whole cells in fluorescence in situ hybridization (FISH) experiments without a wash step. The FISH results with the PNA molecular beacons were superior to those with the DNA molecular beacons: the hybridization kinetics were much faster, the signal-to-noise ratio was much higher, and the specificity was much better for the PNA molecular beacons. Finally, it was demonstrated that the combination of the use of PNA molecular beacons in FISH and flow cytometry makes it possible to rapidly collect quantitative FISH data. Thus, PNA molecular beacons might provide a solution for limitations of traditional FISH methods, such as variable target site accessibility, poor sensitivity for target cells with low rRNA content, background fluorescence, and applications of FISH in microfluidic devices.
Unusual Water Flux in the Extracellular Polysaccharide of the Cyanobacterium
Nostoc commune
Authors: Shaw Eric , Hill Donna R. , Brittain Nicole , Wright Debbie J. , Täuber Uwe , Marand Hervé , Helm Richard F. , Potts Malcolm
Published: September 2003

The speed of water uptake by desiccated
Nostoc commune
was found to depend upon the duration of desiccation. The rehydration of desiccated colonies led to marked, time-dependent changes in structure and ultrastructure and fluctuations in the composition of the transcriptome. Physical evaporative water loss is an active process that was influenced by inhibitors of transcription and translation.
Factors Influencing the Chlorine Susceptibility of
Mycobacterium avium
,
Mycobacterium intracellulare
, and
Mycobacterium scrofulaceum
Authors: Falkinham Joseph O.
Published: September 2003

The susceptibility of representative strains of
Mycobacterium avium
,
Mycobacterium intracellulare
, and
Mycobacterium scrofulaceum
(the MAIS group) to chlorine was studied to identify factors related to culture conditions and growth phase that influenced susceptibility.
M. avium
and
M. intracellulare
strains were more resistant to chlorine than were strains of
M. scrofulaceum
. Transparent and unpigmented colony variants were more resistant to chlorine than were their isogenic opaque and pigmented variants (respectively). Depending on growth stage and growth rate, MAIS strains differed in their chlorine susceptibilities. Cells from strains of all three species growing in early log phase at the highest growth rates were more susceptible than cells in log and stationary phase. Rapidly growing cells were more susceptible to chlorine than slowly growing cells. The chlorine susceptibility of
M. avium
cells grown at 30°C was increased when cells were exposed to chlorine at 40°C compared to susceptibility after exposure at 30°C. Cells of
M. avium
grown in 6% oxygen were significantly more chlorine susceptible than cells grown in air. Chlorine-resistant MAIS strains were more hydrophobic and resistant to Tween 80,
para
-nitrobenzoate, hydroxylamine, and nitrite than were the chlorine-sensitive strains.
Prevalence and Characteristics of
eae
-Positive
Escherichia coli
from Healthy Cattle in Japan
Authors: Kobayashi Hideki , Miura Akihiko , Hayashi Hiroko , Ogawa Torata , Endô Takayuki , Hata Eiji , Eguchi Masashi , Yamamoto Kôsi
Published: September 2003

The prevalence of
eae
-positive
Escherichia coli
(
eae
EC) in Japan was examined using rectal stool samples taken from 35 calves less than 1 month old, 107 calves more than 1 to 3 months old, 88 heifers more than 3 to 6 months old, 214 heifers over 6 months old, and cows from 95 farms. Screening with
eae
PCR revealed the prevalence to be, with increasing age, 31.4, 8.4, 26.1, and 14.5%, respectively. Of 51 selected
eae
EC strains, more than 40% were serotyped as O26, O103, O111, O145, or O157, which are frequently detected as enterohemorrhagic
E
.
coli
types. Four strains were identified as recently reported intimin types η, ι, and κ.
Reevaluation of Production of Paralytic Shellfish Toxin by Bacteria Associated with Dinoflagellates of the Portuguese Coast
Authors: Martins Claudia A. , Alvito Paula , Tavares Maria João , Pereira Paulo , Doucette Gregory , Franca Susana
Published: September 2003

Paralytic shellfish toxins (PSTs) are potent neurotoxins produced by certain dinoflagellate and cyanobacterial species. The autonomous production of PSTs by bacteria remains controversial. In this study, PST production by two bacterial strains, isolated previously from toxic dinoflagellates, was evaluated using biological and analytical methods. Analyses were performed under conditions determined previously to be optimal for toxin production and detection. Our data are inconsistent with autonomous bacterial PST production under these conditions, thereby challenging previous findings for the same strains.
Phenotypic Changes Resulting from Distinct Point Mutations in the
Azospirillum brasilense glnA
Gene, Encoding Glutamine Synthetase
Authors: Van Dommelen Anne , Keijers Veerle , Wollebrants An , Vanderleyden Jozef
Published: September 2003

Sequencing the
glnA
genes of two chemically induced
Azospirillum brasilense
glutamine synthetase mutants revealed an Arg→Cys mutation, corresponding to the glutamate binding site, in one mutant and an Asp→Asn mutation, corresponding to the ammonium binding site, in the second mutant. The phenotypic changes in these mutants are discussed in relation to their genotypes.
Diversion of Electron Flow from Methanogenesis to Crystalline Fe(III) Oxide Reduction in Carbon-Limited Cultures of Wetland Sediment Microorganisms
Authors: Roden Eric E.
Published: September 2003

Electron flow in acetate-limited cultures of wetland sediment microorganisms was diverted from methane production to Fe(III) reduction in the presence of crystalline Fe(III) oxides at surface area loadings equivalent to that of amorphous Fe(III) oxide. The results indicate that inferences regarding the ability of microbial Fe(III) oxide reduction to compete with other terminal electron-accepting processes in anoxic soils and sediments should be based on estimates of bulk microbially available surface site abundance rather than assumed thermodynamic properties of the dominant oxide phase(s) in the soil or sediment.
Survival of F-Specific RNA Coliphage, Feline Calicivirus, andEscherichia coliin Water: a Comparative Study
Authors: Allwood Paul B. , Malik Yashpal S. , Hedberg Craig W. , Goyal Sagar M.
Published: September 2003

The relationship between the survival of enteric viral pathogens and their indicators (coliform bacteria and coliphages) is not well understood. We compared the survival rates of feline calicivirus (FCV),Escherichia coli, and a male-specific RNA coliphage MS2 at 4, 25, and 37°C for up to 28 days in dechlorinated water. The survival rates ofE. coliand FCV, a surrogate of noroviruses (NV), had a high degree of correlation at 4 and 25°C, while MS2 phage survived significantly longer (P< 0.05) at these two temperatures. At 37°C, the survival rates for all three organisms were highly correlated. Decimal reduction values indicating the number of days needed for 90% reduction in titer (D values) decreased for all three organisms as storage temperatures increased. FCV had the shortest D value among all three organisms at all temperatures investigated. These findings indicate that F-specific RNA phages may be useful indicators of NV in the environment.
Role for Mannose-Sensitive Hemagglutinin in Promoting Interactions between
Vibrio cholerae
El Tor and Mussel Hemolymph
Authors: Zampini Massimiliano , Canesi Laura , Betti Michele , Ciacci Caterina , Tarsi Renato , Gallo Gabriella , Pruzzo Carla
Published: September 2003

The role of mannose-sensitive hemagglutinin (MSHA) in
Vibrio cholerae
O1 El Tor interactions with hemolymph of the mussel
Mytilus galloprovincialis
was studied. Bacterial adherence to and association with hemocytes were evaluated at 4 and 18°C, respectively. In hemolymph serum, the wild-type strain N16961 adhered to and associated with hemocytes about twofold more efficiently than its mutant lacking MSHA. In artificial seawater (ASW), no significant differences between the two strains were observed. N16961 was also more sensitive to hemocyte bactericidal activity than its MSHA mutant; in fact, the percentages of killed bacteria after 120 min of incubation were 60 and 34%, respectively. The addition of
d
-mannose abolished the serum-mediated increase in adherence, association, and sensitivity to killing of the wild-type strain without affecting the interactions of the mutant. A similar increase in N16961 adherence to hemocytes was observed when serum was adsorbed with MSHA-deficient bacteria. In contrast, serum adsorbed with either wild-type
V. cholerae
El Tor or wild-type
Escherichia coli
carrying type 1 fimbriae was no longer able to increase adherence of N16961 to hemocytes. The results indicate that hemolymph-soluble factors are involved in interactions between hemocytes and mannose-sensitive adhesins.
Dispersal and Phylogenetic Diversity of Nonmarine Picocyanobacteria, Inferred from 16S rRNA Gene and
cpcBA
-Intergenic Spacer Sequence Analyses
Authors: Crosbie Nicholas D. , Pöckl Matthias , Weisse Thomas
Published: September 2003

More than 20
Synechococcus
and
Cyanobium
isolates were obtained from central European subalpine lakes and sequenced for their 16S rRNA gene and part of the phycocyanin operon (
cpc
), specifically the intergenic spacer (IGS) between
cpcB
and
cpcA
, and corresponding flanking regions (
cpcBA
-IGS). Maximum-likelihood analyses revealed the existence of at least six to seven clusters of nonmarine picocyanobacteria within the picophytoplankton clade and support the conjecture of global dispersal for some closely related picocyanobacterial genotypes.
Precipitation of Barite by
Myxococcus xanthus
: Possible Implications for the Biogeochemical Cycle of Barium
Authors: González-Muñoz Maria Teresa , Fernández-Luque Belén , Martínez-Ruiz Francisca , Ben Chekroun Kaoutar , Arias José María , Rodríguez-Gallego Manuel , Martínez-Cañamero Magdalena , de Linares Concepción , Paytan Adina
Published: September 2003

Bacterial precipitation of barite (BaSO
4
) under laboratory conditions is reported for the first time. The bacterium
Myxococcus xanthus
was cultivated in a solid medium with a diluted solution of barium chloride. Crystallization occurred as a result of the presence of live bacteria and the bacterial metabolic activity. A phosphorous-rich amorphous phase preceded the more crystalline barite formation. These experiments may indicate the involvement of bacteria in the barium biogeochemical cycle, which is closely related to the carbon cycle.
Molecular Detection and Quantitation of the Red TideDinoflagellate
Karenia brevis
in the MarineEnvironment
Authors: Gray M. , Wawrik B. , Paul J. , Casper E.
Published: September 2003

A
real-time reverse transcription-PCR method targeting the
rbcL
gene was developed for the detection and quantitation of
the Florida red tide organism,
Karenia brevis.
The assay was
sensitive to less than 1 cell per reaction, did not detect
rbcL
from 38 nontarget taxa, and accurately quantitated
K.
brevis
organisms in red tide samples from around Florida. These
studies have resulted in a sensitive and specific method for
K.
brevis
detection in the marine
environment.
Selective Plating Underestimates Abundance and Shows Differential Recovery of Bifidobacterial Species from Human Feces
Authors: Apajalahti Juha H. A. , Kettunen Anu , Nurminen Päivi H. , Jatila Hanna , Holben William E.
Published: September 2003

The aim of the present work was to compare the efficacies and levels of selectivity of different culture-dependent and -independent methods for analyzing bifidobacteria in human stool samples. The three different culture media used here significantly differed from each other, particularly with regard to the recovery of
Bifidobacterium adolescentis
.
Bifidobacterium
medium failed to recover
B. adolescentis
; Beerens medium recovered some
B. adolescentis
organisms (17% of total bifidobacteria), whereas tomato-Eugon medium recovered mainly
B. adolescentis
organisms (58% of total bifidobacteria). A culture-independent method that combines GC fractionation of bacterial community DNA and 16S rRNA sequencing indicated that
B. adolescentis
organisms accounted for 85% of all bifidobacteria. Methodological biases, such as those described in this paper, should be taken into account in interpreting earlier studies and designing future experiments.
Development and Clinical Evaluation of an Internally Controlled, Single-Tube Multiplex Real-Time PCR Assay for Detection of
Legionella pneumophila
and Other
Legionella
Species
Authors: Templeton Kate E. , Scheltinga Sitha A. , Sillekens Peter , Crielaard Jantine W. , van Dam Alje P. , Goossens Herman , Claas Eric C. J.
Published: September 2003

A multiplex real-time PCR assay for detection of
Legionella pneumophila
and
Legionella
spp. and including an internal control was designed.
Legionella
species,
L. pneumophila
, and the internal control were detected simultaneously by probes labeled with 6-carboxy-fluorescein, hexachlorofluorescein, and indodicarbocyanine, respectively. Therefore, no postamplification analysis was required in order to distinguish the targets. The sensitivity of both assays was 2.5 CFU/ml, and from analysis of 10 culture-positive and 74 culture-negative samples from patients investigated for legionellosis, 100% agreement was observed by both assays in comparison to culture. Four additional positives were found by the multiplex real-time PCR assay in the
Legionella
culture-negative samples.
Comparison between Prototype Hybrid Capture 3 and Hybrid Capture 2 Human Papillomavirus DNA Assays for Detection of High-Grade Cervical Intraepithelial Neoplasia and Cancer
Authors: Castle Philip E. , Lorincz Attila T. , Scott David R. , Sherman Mark E. , Glass Andrew G. , Rush Brenda B. , Wacholder Sholom , Burk Robert D. , Manos M. Michele , Schussler John E. , Macomber Paul , Schiffman Mark
Published: September 2003

We compared the performance of a prototype version of the Hybrid Capture 3 (HC3) human papillomavirus (HPV) DNA assay to the current generation Hybrid Capture 2 (HC2) assay, both of which target 13 oncogenic HPV types, for the detection of cervical intraepithelial neoplasia grade 3 and cancer (CIN3+) with cervicovaginal lavage specimens collected at enrollment into a 10-year cohort study at Kaiser Permanente (Portland, Oreg.). HC3 results for a risk-stratified sample (
n
= 4,364) were compared to HC2 results for the entire cohort (
n
= 20,810) with receiver operating characteristics curves, and the optimal cut points for both tests (relative light units [RLU]/positive control [PC]) for the detection of CIN3+ were determined. Specimens were also tested for HPV16 and HPV18 with separate HC3 type-specific probes. The optimal cut point for detecting CIN3+ was 1.0 RLU/PC for HC2, as previously shown, and was 0.6 RLU/PC for HC3. At the optimal cut points, HC3 and HC2 had similar screening performance characteristics for CIN3+ diagnosed at the enrollment visit. In analyses that included cases CIN3+ at enrollment and those diagnosed during early follow-up, HC3 had nonsignificantly higher sensitivity and equal specificity for the detection of CIN3+ compared to HC2; this increase in sensitivity was primarily the result of increased detection of CIN3+ in women who were 30 years of age or older and were cytologically negative (
P
= 0.006). We also compared the performance of the hybrid capture tests to MY09/11 L1 consensus primer PCR results (
n
= 1,247). HC3 was less likely than HC2 to test positive for specimens that tested positive by PCR for any untargeted types (
P
< 0.001). HC3 was less likely than HC2 to test positive for untargeted PCR-detected single infections with HPV53 (
P
= 0.001) and HPV66 (
P
= 0.01). There was good agreement between test positivity by PCR and by single type-specific HC3 probes for HPV16 (kappa = 0.76; 95% confidence interval [CI] = 0.71 to 0.82) and for HPV18 (kappa = 0.73; 95% CI = 0.68 to 0.79). In conclusion, we suggest that HC3 (≥0.6 RLU/PC) may be slightly more sensitive than and equally specific test as HC2 (≥1.0 RLU/PC) for the detection of CIN3+ over the duration of typical screening intervals.
Epidemiology and Molecular Characterization of
Clostridium difficile
Strains from Patients with Diarrhea: Low Disease Incidence and Evidence of Limited Cross-Infection in a Swedish Teaching Hospital
Authors: Svenungsson Bo , Burman Lars G. , Jalakas-Pörnull Kirsti , Lagergren Åsa , Struwe Johan , Åkerlund Thomas
Published: September 2003

We prospectively studied the epidemiology of
Clostridium difficile-
associated diarrhea (CDAD) in a 900-bed hospital over the course of 12 months by PCR-ribotyping of
C. difficile
isolates. A total of 304 cases were diagnosed, corresponding to an overall incidence of 7/1,000 admissions, with higher rates in nephrology, hematology, and organ transplantation wards (37, 30, and 21/1,000), and 72% were classified as hospital associated (onset in hospital or onset at home but after a hospital stay within 2 months). All 382 isolates from 227 of 304 (75%) patients available for PCR-ribotyping were typeable, yielding 70 PCR-ribotypes. The three most common types comprised 30% of hospital-associated and 34% of community-associated cases, indicating import via admitted patients as a major source of
C. difficile
strains occurring in the hospital. Of the 227 patients studied, 38% each contributed 2 to 13 fecal samples positive for
C. difficile
over the course of the study period. Repeat isolates of the same PCR-ribotype as the first isolate were found in 79% of these patients and in 95% of specimens delivered within 30 days, compared to 63% of those obtained at 31 to 204 days. Nosocomial acquisition of CDAD, defined as the proportion of cases sharing
C. difficile
type and admitted to the same ward within 2 or 12 months, was 20% and 32% of hospital-associated cases and 14% and 23% of all cases, respectively. Thus, most CDAD cases diagnosed over the course of the study period, including those associated with hospitalization, appeared to be caused by endogenous
C. difficile
strains rather than by strains truly being acquired in the hospital.
Unexpected Decrease with Age of
Helicobacter pylori
Seroprevalence among Swedish Blood Donors
Authors: Sörberg M. , Nyrén O. , Granström M.
Published: September 2003

Blood donors are often used as proxies for the general population in studies of
Helicobacter pylori
epidemiology. Our aim was to test if the age-specific seroprevalence rates among blood donors match with the corresponding rates in a random population sample. This descriptive study was based on sera obtained from 3,502 blood donors representing all Swedish counties and cities. An age-stratified random population sample of 1,030 from Stockholm County served as comparison. Sera were analyzed by an in-house enzyme-linked immunosorbent assay for
H. pylori
immunoglobulin G antibodies. In the population sample, we found the expected increase with age in the seroprevalence of
H. pylori
infection. This was true also among young blood donors, while the prevalence-by-age curve showed a deflection downward among blood donors who are ≥ 50 years of age. In this age group, the probability of being seropositive was reduced by 73% (95% confidence interval [CI], 63 to 81%) relative to the population sample. Overall, the adjusted odds ratio for
H. pylori
seropositivity among blood donors was decreased by 43% (95% CI, 28 to 55%). Thus, it appears that blood donors who are
H. pylori
seropositive selectively disappear from the blood donor cohort. We speculate that
H. pylori
-seropositive blood donors may tolerate repeated bleedings less well than do noninfected individuals and/or that the general well-being among those who are infected may be somewhat impaired. Our unexpected observation indicates that blood donors may be less suitable as proxies for the general population in analytic studies of
H. pylori
infection and that the underlying cause needs further study.
Biofilm Formation by Group A Streptococci: Is There a Relationship with Treatment Failure?
Authors: Conley Joslyn , Olson Merle E. , Cook Linda S. , Ceri Howard , Phan Van , Davies H. Dele
Published: September 2003

Group A streptococcus (GAS) is the primary cause of bacterial pharyngitis in children and adults. Up to one-third of patients treated for GAS pharyngitis fail to respond to antibiotic therapy. The objective of this cohort study was to evaluate GAS biofilm formation as a mechanism for antibiotic treatment failure using previously collected GAS isolates and penicillin treatment outcome data. The minimum biofilm eradication concentration (MBEC) assay device was used to determine the biofilm-forming capabilities, efficiencies, and antibiotic susceptibilities of GAS isolates. The MBECs and MICs of several antibiotics for GAS were determined. All 99 GAS isolates available for this study formed biofilms, with various efficiencies. Antibiotic MBECs were consistently higher than MICs for all of the GAS isolates. MBECs indicated penicillin insensitivity in 60% of GAS isolates, producing the first report of in vitro GAS insensitivity to penicillin. Using MBECs to predict penicillin treatment failure had better sensitivity (56%) but lower specificity (36%) than the sensitivity (0%) and specificity (100%) when MICs were used. However, the positive predictive value of the MBEC was superior to that of the MIC (56 versus 0%), while the negative predictive values (42 and 47%) were similar. More studies are needed to understand the roles of biofilms and the MBEC assay in predicting GAS treatment failure. In addition, further investigations are necessary to determine if non-biofilm-forming strains of GAS exist and the roles of in vivo monospecies and multispecies biofilms in streptococcal pharyngitis treatment failure.
Importance of Methodology in Determination of
Chlamydia pneumoniae
Seropositivity in Healthy Subjects and in Patients with Coronary Atherosclerosis
Authors: Hoymans V. Y. , Bosmans J. M. , Van Renterghem L. , Mak R. , Ursi D. , Wuyts F. , Vrints C. J. , Ieven M.
Published: September 2003

Enzyme immunoassays (EIAs) for the detection of
Chlamydia pneumoniae
antibodies were compared to the microimmunofluorescence (MIF) test, the reference method. Furthermore, we assessed the hypothesis that a possible relationship between
Chlamydia pneumoniae
immunoglobulin G (IgG) antibodies and coronary artery disease is dependent on the type of EIA. Sera from 112 healthy men (mean age, 50.1 years) were tested for antibodies against
Chlamydia pneumoniae
by five commercial test kits: Focus Chlamydia MIF IgG test, Labsystems
Chlamydia pneumoniae
IgG EIA (LS EIA), R-Biopharm Elegance
Chlamydia pneumoniae
IgG EIA (RB EIA), Medac
Chlamydia pneumoniae
IgG sandwich enzyme-linked immunosorbent assay ELISA (MCp sELISA) and Medac Chlamydia IgG recombinant enzyme-linked immunosorbent assay ELISA (MC rELISA). Sera from 106 consecutive male patients (mean age, 63.6 years) undergoing diagnostic coronary angiography were also examined using the Focus MIF, LS EIA, MCp sELISA, and MC rELISA techniques. The agreement between LS EIA (65 to 83% [controls-patients]) or MC rELISA (49 to 61%) and Focus MIF (78 to 83%) was average to fair (κ = 0.597 and 0.234, respectively). MCp sELISA and RB EIA showed good agreement with MIF (κ = 0.686 and 0.665, respectively), with 80 to 89 and 79% of individuals reacting positively. A significant difference in seroprevalence between patients and healthy subjects was observed with the LS EIA, while seropositivities in the two study groups appeared equal when the Focus MIF assay was applied. The MC rELISA and MCp sELISA gave statistically significant differences in antibody seroprevalence in patients with two-vessel disease or when the patient group combined individuals with a two- or a three-vessel disease, respectively. The concordance between MIF and other commonly used serological assays for
C. pneumoniae
IgG antibody detection is good to fair. The choice of serological assay has important implications for
C. pneumoniae
antibody seroprevalence, as well as for the relationship between
C. pneumoniae
seropositivity and coronary artery disease.
Characterization of a Herpesvirus Associated with Tracheitis in Gouldian Finches (
Erythrura
[
Chloebia
]
gouldiae
)
Authors: Wellehan James F. X. , Gagea Mihai , Smith Dale A. , Taylor W. Michael , Berhane Yohannes , Bienzle Dorothee
Published: September 2003

Severe tracheitis and bronchitis were identified in two fatal cases of respiratory disease affecting a flock of Gouldian finches (
Erythrura
[
Chloebia
]
gouldiae
). Intranuclear inclusion bodies in epithelial cells of the upper respiratory tract were identified in samples from two birds. Electron microscopic examination showed that the inclusions consisted of viral particles consistent in appearance with
Herpesviridae
. Degenerate PCR primers targeting a conserved region of the herpesviral-DNA-dependent DNA polymerase were used to amplify a region of DNA isolated from tissues with lesions from each animal. Nucleotide sequencing of the PCR products yielded identical viral sequences that were distinct from known herpesviruses. An analysis of sequence homology indicated that these gene segments appear to belong to a member of the subfamily
Alphaherpesvirinae
.
Clonal Complexes of
Campylobacter jejuni
Identified by Multilocus Sequence Typing Correlate with Strain Associations Identified by Multilocus Enzyme Electrophoresis
Authors: Sails Andrew D. , Swaminathan Bala , Fields Patricia I.
Published: September 2003

Multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) with
Sma
I were used to subtype 55 isolates of
Campylobacter jejuni
from a diverse range of human and animal sources previously characterized by multilocus enzyme electrophoresis (MEE). MEE and MLST targeted 11 and 7 loci, respectively, and all loci were unique to each method. MEE, MLST, and PFGE identified 40, 37, and 48 discrete subtypes, respectively, with many of the subtypes occurring only once within the data set. Simpson's indices of diversity were calculated to be 0.979, 0.966, and 0.994 for MEE, MLST, and PFGE, respectively, demonstrating that MEE and MLST had similar discriminatory powers but that PFGE was more discriminatory. Allele diversity was higher in the MLST loci; individual single-locus diversities for the 11 MEE loci and the 7 MLST loci were 0.491 and 0.854, respectively. The clonal complexes recognized by MLST correlated with the strain associations previously recognized by MEE and contained some isolates indistinguishable by PFGE. Many clusters contained isolates from diverse geographical regions and from both humans and animals. These results demonstrate the usefulness of MLST for investigation of the global epidemiology of this important pathogen and illustrate its potential to identify indistinguishable strains or clones in geographically distinct regions.
Smallpox: Residual Antibody after Vaccination
Authors: Gallwitz Stephanie , Schutzbank Ted , Heberling Richard L. , Kalter S. S. , Galpin Jeffrey E.
Published: September 2003

Of all the microorganisms and toxins, poxviruses (
Orthopoxvirus
) have the greatest potential for use by terrorists. These viruses can spread rapidly through the environment following initial infection. In 1980, the World Health Organization Eradication Program discontinued vaccination for smallpox and declared that the disease had been eliminated. With the threat of smallpox virus as a bioterrorism weapon, questions have been asked about the persistence of protection (as offered by antibodies) following vaccination with vaccinia virus vaccine. To address this, sera from 204 adults vaccinated as children were tested by enzyme immunoassay (EIA) for the presence of vaccinia virus antibody. Of the 204 individuals whose sera were examined for the presence of vaccinia antibody, 165 (80.9%) had been vaccinated once and 39 (19.1%) had been vaccinated at least twice. Of the 165 sera from individuals vaccinated once, 112 (67.9%) were positive. Of the 39 sera from individuals vaccinated more than once, 31 (79.5%) were positive. The presence of a vaccination scar at the time of blood collection was not determined. Fifty-six nonvaccinated individuals, under 30 years of age, were tested by EIA; four of these (7.1%) were positive for vaccinia virus antibody by EIA. Forty-four EIA-positive and 16 EIA-negative sera were also tested by serum neutralization (SN) as a comparison with the EIA test results; one serum (negative by EIA) was SN positive. No attempt was made to ascertain any demographics other than age (date of birth) and “remembered” times of vaccination.
Microarray-BasedIdentification of Thermophilic
Campylobacter jejuni
,
C.coli
,
C. lari
, and
C.upsaliensis
Authors: Volokhov Dmitriy , Chizhikov Vladimir , Chumakov Konstantin , Rasooly Avraham
Published: September 2003

DNA
microarrays are an excellent potential tool for clinical microbiology,
since this technology allows relatively rapid identification and
characterization of microbial and viral pathogens. In the present
study, an oligonucleotide microarray was developed and used for the
analysis of thermophilic
Campylobacter
spp., the primary
food-borne pathogen in the United States. We analyzed four
Campylobacter
species:
Campylobacter jejuni
,
C.
coli
,
C. lari
, and
C. upsaliensis.
Our assay
relies on the PCR amplification of specific regions in five target
genes (
fur
,
glyA
,
cdtABC
,
ceuB-C
,
and
fliY
) as a first step, followed by microarray-based
analysis of amplified DNAs. Alleles of two genes,
fur
and
glyA
, which are found in all tested thermophilic
Campylobacter
spp., were used for identification and
discrimination among four bacterial species, the
ceuB-C
gene
was used for discrimination between
C. jejuni
and
C.
coli
, and the
fliY
and
cdt
genes were used as
additional genetic markers specific either for
C. upsaliensis
and
C. lari
or for
C. jejuni.
The array was developed
and validated by using 51 previously characterized
Campylobacter
isolates. All isolates were unambiguously
identified on the basis of hybridization patterns with 72 individual
species-specific oligoprobes. Microarray identification of
C.
jejuni
and
C. coli
was confirmed by PCR amplification of
other genes used for identification (
hipO
and
ask
).
Our results demonstrate that oligonucleotide microarrays are suitable
for rapid and accurate simultaneous differentiation among
C.
jejuni
,
C. coli
,
C. lari
, and
C.
upsaliensis
.
Detection of Infectious Human Immunodeficiency Virus Type 1 in Female Genital Secretions by a Short-Term Culture Method
Authors: Cummins James E. , Villanueva Julie M. , Evans-Strickfaden Tammy , Sesay Shekou M. , Abner Sheila R. , Bush Timothy J. , Green Timothy A. , Lennox Jeffrey L. , Wright Thomas , Folks Thomas M. , Hart Clyde E. , Dezzutti Charlene S.
Published: September 2003

Infectious human immunodeficiency virus type 1 (HIV-1) is difficult to detect in female genital secretions by standard virus culture techniques. To improve detection of cell-free HIV-1 in female genital secretions, we adapted a short-term assay that uses the multinuclear-activation galactosidase indicator (MAGI) assay. When vaginal lavages from HIV-1-infected women were tested with the adapted MAGI assay, 25 (64%) of 39 lavages with detectable, cell-free HIV-1 RNA were shown to have infectious virus. No infectious virus was found in 10 vaginal lavages from HIV-1-infected women with undetectable vaginal viral loads. Significantly (
P
< 0.01) more lavages from HIV-1-infected women tested positive for infectious virus by the MAGI assay than by standard peripheral blood mononuclear cell (PBMC) coculture, which detected infectious virus in only 6 (17%) of 35 vaginal lavages. Lavages with viral loads of >10,000 copies per lavage yielded significantly (
P
< 0.01) more positive cultures than those with <10,000 copies by using the MAGI assay. Detection of infectious HIV-1 in vaginal lavages was not associated with the presence of genital tract infections or CD4
+
-T-cell counts. However, although the results were not significant (
P
= 0.08), the MAGI assay detected infectious virus from more vaginal lavages at a vaginal pH of ≥4.5 than at a pH of <4.5. These results indicate that the MAGI assay is more sensitive than PBMC culture methods for detecting infectious virus in female genital secretions. Accurate measurements of infectious virus in genital secretions will improve studies that evaluate sexual transmission of HIV-1.
Multiplex PCR Assay for Simultaneous Detection of Nine Clinically Relevant Antibiotic Resistance Genes inStaphylococcus aureus
Authors: Strommenger Birgit , Kettlitz Christiane , Werner Guido , Witte Wolfgang
Published: September 2003

In this study we describe a multiplex PCR assay for the detection of nine clinically relevant antibiotic resistance genes ofStaphylococcus aureus.Conditions were optimized to amplify fragments ofmecA(encoding methicillin resistance),aacA-aphD(aminoglycoside resistance),tetK,tetM(tetracycline resistance),erm(A),erm(C) (macrolide-lincosamide-streptogramin B resistance),vat(A),vat(B), andvat(C) (streptogramin A resistance) simultaneously in one PCR amplification. An additional primer pair for the amplification of a fragment of the staphylococcal 16S rDNA was included as a positive control. The multiplex PCR assay was evaluated on 30 differentS. aureusisolates, and the PCR results correlated with the phenotypic antibiotic resistance data obtained by the broth microdilution assay. The multiplex PCR assay offers a rapid, simple, and accurate identification of antibiotic resistance profiles and could be used in clinical diagnosis as well as for the surveillance of the spread of antibiotic resistance determinants in epidemiological studies.
Evaluation of a Multiplex PCR Test for Simultaneous Identification and Serotyping of
Actinobacillus pleuropneumoniae
Serotypes 2, 5, and 6
Authors: Jessing Stine G. , Angen Øystein , Inzana Tomas J.
Published: September 2003

Serotype-specific DNA regions involved in the biosynthesis of capsular polysaccharides (
cps
region) were used to develop a multiplex PCR test for the simultaneous species identification and serotyping of
Actinobacillus pleuropneumoniae
serotypes 2, 5, and 6. Primers specific for serotypes 2, 5, and 6 were combined with the already existing species-specific primers used in a PCR test based on the
omlA
gene. The PCR test was evaluated with serotype reference strains of
A. pleuropneumoniae
as well as 182 Danish field isolates previously serotyped by latex agglutination or immunodiffusion. For all serologically typeable strains, a complete correspondence was found between the results obtained by the multiplex PCR test and the results obtained by the traditional serotyping methods. Six of eight serologically nontypeable strains could be allocated to a serotype on the basis of the multiplex PCR results. The species specificity of the assay was evaluated with a collection of 93 strains representing 29 different species within the family
Pasteurellaceae
, as well as species normally found in the respiratory tracts of swine. All of these strains were negative by the multiplex PCR test, including 50 field isolates of the phylogenetically closely related species
Actinobacillus lignieresii
. When the multiplex PCR test was used to test Danish field strains, it was able to identify the serotypes of approximately 94% of all strains isolated from swine with clinical disease. More than 90% of the isolates that cross-reacted by the latex agglutination test were of serotype 2, 5, or 6. Determination of the serotype by PCR represents a convenient and specific method for the serotyping of
A. pleuropneumoniae
in diagnostic laboratories.
Molecular Epidemiology of
Aspergillus fumigatus
Isolates Recovered from Water, Air, and Patients Shows Two Clusters of Genetically Distinct Strains
Authors: Warris Adilia , Klaassen Corné H. W. , Meis Jacques F. G. M. , de Ruiter Maaike T. , de Valk Hanneke A. , Abrahamsen Tore G. , Gaustad Peter , Verweij Paul E.
Published: September 2003

There has been an increase in data suggesting that besides air, hospital water is a potential source of transmission of filamentous fungi, and in particular
Aspergillus fumigatus
. Molecular characterization of environmental and clinical
A. fumigatus
isolates, collected prospectively during an 18-month period, was performed to establish if waterborne fungi play a role in the pathogenesis of invasive aspergillosis. Isolates recovered from water (
n
= 54) and air (
n
= 21) at various locations inside and outside the hospital and from 15 patients (
n
= 21) with proven, probable, or possible invasive aspergillosis were genotyped by amplified fragment length polymorphism analysis. Based on genomic fingerprints, the environmental
A. fumigatus
isolates could be grouped into two major clusters primarily containing isolates recovered from either air or water. The genotypic relatedness between clinical and environmental isolates suggests that patients with invasive aspergillosis can be infected by strains originating from water or from air. In addition, 12 clusters with genetically indistinguishable or highly related strains were differentiated, each containing two to three isolates. In two clusters, clinical isolates recovered from patients matched those recovered from water sources, while in another cluster the clinical isolate was indistinguishable from one cultured from air. This observation might open new perspectives in the development of infection control measures to prevent invasive aspergillosis in high-risk patients. The genetic variability found between airborne and waterborne
A. fumigatus
strains might prove to be a powerful tool in understanding the transmission of invasive aspergillosis and in outbreak control.
Analysis of Loci Required for Determination of Serotype Antigenicity in
Streptococcus mutans
and Its Clinical Utilization
Authors: Shibata Yukie , Ozaki Kazuhisa , Seki Mitsuko , Kawato Takayuki , Tanaka Hideki , Nakano Yoshio , Yamashita Yoshihisa
Published: September 2003

We recently identified the genes responsible for the serotype c-specific glucose side chain formation of rhamnose-glucose polysaccharide (RGP) in
Streptococcus mutans
. These genes were located downstream from the
rgpA
through
rgpF
locus that is involved in the synthesis of RGP. In the present study, the corresponding chromosomal regions were isolated from serotype e and f strains and characterized. The
rgpA
through
rgpF
homologs were well conserved among the three serotypes. By contrast, the regions downstream from the
rgpF
homolog differed considerably among the three serotypes. Replacement of these regions in the different serotype strains converted their serotypic phenotypes, suggesting that these regions participated in serotype-specific glucose side chain formation in each serotype strain. Based on the differences among the DNA sequences of these regions, a PCR method was developed to determine serotypes.
S. mutans
was isolated from 198 of 432 preschool children (3 to 4 years old). The serotypes of all but one
S. mutans
isolate were identified by serotyping PCR. Serotype c predominated (84.8%), serotype e was the next most common (13.3%), and serotype f occured rarely (1.9%) in Japanese preschool children. Caries experience in the group with a mixed infection by multiple serotypes of
S. mutans
was significantly higher than that in the group with a monoinfection by a single serotype.
Molecular Analysis of
Burkholderia cepacia
Complex Isolates from a Portuguese Cystic Fibrosis Center: a 7-Year Study
Authors: Cunha Mónica V. , Leitão Jorge H. , Mahenthiralingam Eshwar , Vandamme Peter , Lito Luís , Barreto Celeste , Salgado Maria José , Sá-Correia Isabel
Published: September 2003

This work reports results of a systematic molecular analysis involving 113
Burkholderia cepacia
complex isolates obtained from 23 cystic fibrosis (CF) patients under surveillance over a 7-year period at the major Portuguese CF center, the Santa Maria Hospital in Lisbon. The majority of the isolates were serial isolates from persistently infected patients (more than one-half of the population examined). In agreement with previous studies,
B. cenocepacia
(formerly genomovar III) was the most prevalent species; it was isolated from 52% of the patients infected with
B. cepacia
complex isolates. Contrasting with previous studies, a very significant percentage of the Portuguese CF subpopulation examined was infected with
B. cepacia
genomovar I (36%) and
B. stabilis
(18%).
B. multivorans
was recovered from two of the infected patients. All four of the species or genomovars were associated with poor clinical outcome, including the cepacia syndrome, and gave rise to chronic and transient infections, with the clinical condition depending on the patient and other still-unidentified factors. The
B. cepacia
epidemic strain marker region was found exclusively in genomovar III strains, while
cblA
was detected in genomovars I and III, only. There was no clear relation between the presence of these markers and transmissibility. Altogether, our results indicate that the use of these markers or the genomovar status in identifying patients at higher risk for infection is uncertain.
Evaluation of Real-Time PCR for Detection of and Discrimination between
Bordetella pertussis
,
Bordetella parapertussis
, and
Bordetella holmesii
for Clinical Diagnosis
Authors: Templeton Kate E. , Scheltinga Sitha A. , van der Zee Anneke , Diederen Bram M. W. , Kruijssen Anna M. , Goossens Herman , Kuijper Ed , Claas Eric C. J.
Published: September 2003

PCR is increasingly being used as a diagnostic test for the detection of
Bordetella pertussis
and
Bordetella parapertussis
DNA, as it has improved sensitivity and specificity in comparison to conventional techniques. The assay described here uses the two insertion sequences IS
481
and IS
1001
for
B. pertussis
and
B. parapertussis
, respectively, with detection by molecular beacons. The real-time PCR for IS
481
detects both
B. pertussis
and
Bordetella holmesii
, and the real-time PCR for IS
1001
detects both
B. parapertussis
and
B. holmesii
. By performing both assays discrimination between
B. pertussis
and
B. parapertussis
can be obtained. The sensitivity was 1 to 10 CFU/ml for
B. pertussis
, 10 CFU/ml for
B. parapertussis
, and 10 CFU/ml for
B. holmesii
in both assays. The clinical sensitivity of the
B. pertussis
assay was not affected by duplexing with an internal control PCR. Real-time PCR, conventional PCR, and culture were performed on 57 clinical samples. Eight of the 57 (14%) were found positive by culture, 19 of 57 (33%) were found positive by conventional PCR, and 22 of 57 (39%) were found positive by real-time PCR. One sample was inhibitory. When the
B. pertussis
assay was compared with a clinical standard for
B. pertussis
infection, sensitivity was 38, 83, and 100% and specificity was 100, 97, and 97% for culture, conventional PCR, and real-time PCR, respectively. The real-time PCR designed for
B. pertussis
and
B. parapertussis
provides sensitive and specific diagnosis of
B. pertussis
and
B. parapertussis
infections and is therefore suitable for implementation in the diagnostic laboratory.
Molecular Analysis of Cases of Italian Sheep Scrapie and Comparison with Cases of Bovine Spongiform Encephalopathy (BSE) and Experimental BSE in Sheep
Authors: Nonno Romolo , Esposito Elena , Vaccari Gabriele , Conte Michela , Marcon Stefano , Di Bari Michele , Ligios Ciriaco , Di Guardo Giovanni , Agrimi Umberto
Published: September 2003

Concerns have been raised about the possibility that the bovine spongiform encephalopathy (BSE) agent could have been transmitted to sheep populations via contaminated feedstuffs. The objective of our study was to investigate the suitability of molecular strain typing methods as a surveillance tool for studying scrapie strain variations and for differentiating PrP
Sc
from sheep scrapie, BSE, and sheep BSE. We studied 38 Italian sheep scrapie cases from 13 outbreaks, along with a British scrapie case, an experimental ovine BSE, and 3 BSE cases, by analyzing the glycoform patterns and the apparent molecular masses of the nonglycosylated forms of semipurified, proteinase-treated PrP
Sc
. Both criteria were able to clearly differentiate sheep scrapie from BSE and ovine experimental BSE. PrP
Sc
from BSE and sheep BSE showed a higher glycoform ratio and a lower molecular mass of the nonglycosylated form compared to scrapie PrP
Sc
. Scrapie cases displayed homogeneous PrP
Sc
features regardless of breed, flock, and geographic origin. The glycoform patterns observed varied with the antibody used, but either a monoclonal antibody (MAb) (F99/97.6.1) or a polyclonal antibody (P7-7) was able to distinguish scrapie from BSE PrP
Sc
. While more extensive surveys are needed to further corroborate these findings, our results suggest that large-scale molecular screening of sheep populations for BSE surveillance may be eventually possible.
Ribosomal DNA Sequencing for Identification of Aerobic Gram-Positive Rods in the Clinical Laboratory (an 18-Month Evaluation)
Authors: Bosshard P. P. , Abels S. , Zbinden R. , Böttger E. C. , Altwegg M.
Published: September 2003

We have evaluated over a period of 18 months the use of 16S ribosomal DNA (rDNA) sequence analysis as a means of identifying aerobic gram-positive rods in the clinical laboratory. Two collections of strains were studied: (i) 37 clinical strains of gram-positive rods well identified by phenotypic tests, and (ii) 136 clinical isolates difficult to identify by standard microbiological investigations, i.e., identification at the species level was impossible. Results of molecular analyses were compared with those of conventional phenotypic identification procedures. Good overall agreement between phenotypic and molecular identification procedures was found for the collection of 37 clinical strains well identified by conventional means. For the 136 clinical strains which were difficult to identify by standard microbiological investigations, phenotypic characterization identified 71 of 136 (52.2%) isolates at the genus level; 65 of 136 (47.8%) isolates could not be discriminated at any taxonomic level. In comparison, 16S rDNA sequencing identified 89 of 136 (65.4%) isolates at the species level, 43 of 136 (31.6%) isolates at the genus level, and 4 of 136 (2.9%) isolates at the family level. We conclude that (i) rDNA sequencing is an effective means for the identification of aerobic gram-positive rods which are difficult to identify by conventional techniques, and (ii) molecular identification procedures are not required for isolates well identified by phenotypic investigations.