bluuebunny/crossref_metadata_2025 huggingface dataset:
Allelic Variation of Polymorphic Locus
lytB
, Encoding a Choline-Binding Protein, from Streptococci of the Mitis Group
Authors: Moscoso Miriam , Obregón Virginia , López Rubens , García José L. , García Ernesto
Published: December 2005

The choline-binding protein LytB, an
N
-acetylglucosaminidase of
Streptococcus pneumoniae
, is the key enzyme for daughter cell separation and is believed to play a critical pathogenic role, facilitating bacterial spreading during infection. Because of these peculiarities LytB is a putative vaccine target. To determine the extent of LytB polymorphism, the
lytB
alleles from seven typical, clinical pneumococcal isolates of various serotypes and from 13 additional streptococci of the mitis group (12 atypical pneumococci and the
Streptococcus mitis
type strain) were sequenced. Sequence alignment showed that the main differences among alleles were differences in the number of repeats (range, 12 to 18) characteristic of choline-binding proteins. These differences were located in the region corresponding to repeats 11 to 17. Typical pneumococcal strains contained either 14, 16, or 18 repeats, whereas all of the atypical isolates except strains 1283 and 782 (which had 14 and 16 repeats, respectively) and the
S. mitis
type strain had only 12 repeats; atypical isolate 10546 turned out to be a Δ
lytB
mutant. We also found that there are two major types of alternating repeats in
lytB
, which encode 21 and 23 amino acids. Choline-binding proteins are linked to the choline-containing cell wall substrate through choline residues at the interface of two consecutive choline-binding repeats that create a choline-binding site. The observation that all strains contained an even number of repeats suggests that the duplication events that gave rise to the choline-binding repeats of LytB involved two repeats simultaneously, an observation that is in keeping with previous crystallographic data. Typical pneumococcal isolates usually grew as diplococci, indicating that an active LytB enzyme was present. In contrast, most atypical isolates formed long chains of cells that did not disperse after addition of purified LytB, suggesting that in these strains chains were produced through mechanisms unrelated to LytB.
Identification and Localization of Extraradicular Biofilm-Forming Bacteria Associated with Refractory Endodontic Pathogens
Authors: Noguchi Nobuo , Noiri Yuichiro , Narimatsu Masahiro , Ebisu Shigeyuki
Published: December 2005

Bacterial biofilms have been found to develop on root surfaces outside the apical foramen and be associated with refractory periapical periodontitis. However, it is unknown which bacterial species form extraradicular biofilms. The present study aimed to investigate the identity and localization of bacteria in human extraradicular biofilms. Twenty extraradicular biofilms, used to identify bacteria using a PCR-based 16S rRNA gene assay, and seven root-tips, used to observe immunohistochemical localization of three selected bacterial species, were taken from 27 patients with refractory periapical periodontitis. Bacterial DNA was detected from 14 of the 20 samples, and 113 bacterial species were isolated.
Fusobacterium nucleatum
(14 of 14),
Porphyromonas gingivalis
(12 of 14), and
Tannellera forsythensis
(8 of 14) were frequently detected. Unidentified and uncultured bacterial DNA was also detected in 11 of the 14 samples in which DNA was detected. In the biofilms,
P. gingivalis
was immunohistochemically detected in all parts of the extraradicular biofilms. Positive reactions to anti-
F. nucleatum
and anti-
T. forsythensis
sera were found at specific portions of the biofilm. These findings suggested that
P. gingivalis
,
T. forsythensis
, and
F. nucleatum
were associated with extraradicular biofilm formation and refractory periapical periodontitis.
Culture-Independent Characterization of the Microbiota of the Ant LionMyrmeleon mobilis(Neuroptera: Myrmeleontidae)
Authors: Dunn Anne K. , Stabb Eric V.
Published: December 2005

Ant lions are insect larvae that feed on the liquefied internal components of insect prey. Prey capture is assisted by the injection of toxins that are reportedly derived from both the insect and bacterial symbionts. These larvae display interesting gut physiology where the midgut is not connected to the hindgut, preventing elimination of solid waste until adulthood. The presence of a discontinuous gut and the potential involvement of bacteria in prey paralyzation suggest an interesting microbial role in ant lion biology; however, the ant lion microbiota has not been described in detail. We therefore performed culture-independent 16S rRNA gene sequence analysis of the bacteria associated with tissues of an ant lion,Myrmeleon mobilis. All 222 sequences were identified asProteobacteriaand could be subdivided into two main groups, the α-Proteobacteriawith similarity toWolbachiaspp. (75 clones) and the γ-Proteobacteriawith similarity to the familyEnterobacteriaceae(144 clones). TheEnterobacteriaceae-like 16S rRNA gene sequences were most commonly isolated from gut tissue, andWolbachia-like sequences were predominant in the head and body tissue. Fluorescence in situ hybridization analyses supported the localization of enterics to gut tissue andWolbachiato nongut tissue. The diversity of sequences isolated from freshly caught, laboratory-fed, and laboratory-starved ant lions were qualitatively similar, although the libraries from each treatment were significantly different (P= 0.05). These results represent the first culture-independent analysis of the microbiota associated with a discontinuous insect gut and suggest that the ant lion microbial community is relatively simple, which may be a reflection of the diet and gut physiology of these insects.
Modification of Surface Properties of Biomaterials Influences the Ability of
Candida albicans
To Form Biofilms
Authors: Chandra Jyotsna , Patel Jasmine D. , Li Jian , Zhou Guangyin , Mukherjee Pranab K. , McCormick Thomas S. , Anderson James M. , Ghannoum Mahmoud A.
Published: December 2005

Candida albicans
biofilms form on indwelling medical devices (e.g., denture acrylic or intravenous catheters) and are associated with both oral and invasive candidiasis. Here, we determined whether surface modifications of polyetherurethane (Elasthane 80A [E80A]), polycarbonateurethane, and poly(ethyleneterephthalate) (PET) can influence fungal biofilm formation. Polyurethanes were modified by adding 6% polyethylene oxide (6PEO), 6% fluorocarbon, or silicone, while the PET surface was modified to generate hydrophilic, hydrophobic, cationic, or anionic surfaces. Formation of biofilm was quantified by determining metabolic activity and total biomass (dry weight), while its architecture was analyzed by confocal scanning laser microscopy (CSLM). The metabolic activity of biofilm formed by
C. albicans
on 6PEO-E80A was significantly reduced (by 78%) compared to that of biofilm formed on the nonmodified E80A (optical densities of 0.054 ± 0.020 and 0.24 ± 0.10, respectively;
P
= 0.037). The total biomass of
Candida
biofilm formed on 6PEO-E80A was 74% lower than that on the nonmodified E80A surface (0.46 ± 0.15 versus 1.76 ± 0.32 mg, respectively;
P
= 0.003). Fungal cells were easily detached from the 6PEO-E80A surface, and we were unable to detect
C. albicans
biofilm on this surface by CSLM. All other surface modifications allowed formation of
C. albicans
biofilm, with some differences in thearchitecture. Correlation between contact angle and biofilm formation was observed for polyetherurethane substrates (
r
= 0.88) but not for PET biomaterials (
r
= −0.40). This study illustrates that surface modification is a viable approach for identifying surfaces that have antibiofilm characteristics. Investigations into the clinical utility of the identified surfaces are warranted.
Endosymbiotic
Bacteroidales
Bacteria of the Flagellated Protist
Pseudotrichonympha grassii
in the Gut of the Termite
Coptotermes formosanus
Authors: Noda Satoko , Iida Toshiya , Kitade Osamu , Nakajima Hideaki , Kudo Toshiaki , Ohkuma Moriya
Published: December 2005

A unique lineage of bacteria belonging to the order
Bacteroidales
was identified as an intracellular endosymbiont of the protist
Pseudotrichonympha grassii
(
Parabasalia
,
Hypermastigea
) in the gut of the termite
Coptotermes formosanus
. We identified the 16S rRNA,
gyrB
, elongation factor Tu, and
groEL
gene sequences in the endosymbiont and detected a very low level of sequence divergence (<0.9% of the nucleotides) in the endosymbiont population within and among protist cells. The
Bacteroidales
endosymbiont sequence was affiliated with a cluster comprising only sequences from termite gut bacteria and was not closely related to sequences identified for members of the
Bacteroidales
attached to the cell surfaces of other gut protists. Transmission electron microscopy showed that there were numerous rod-shaped bacteria in the cytoplasm of the host protist, and we detected the endosymbiont by fluorescence in situ hybridization (FISH) with an oligonucleotide probe specific for the 16S rRNA gene identified. Quantification of the abundance of the
Bacteroidales
endosymbiont by sequence-specific cleavage of rRNA with RNase H and FISH cell counting revealed, surprisingly, that the endosymbiont accounted for 82% of the total bacterial rRNA and 71% of the total bacterial cells in the gut community. The genetically nearly homogeneous endosymbionts of
Pseudotrichonympha
were very abundant in the gut symbiotic community of the termite.
Development and Characterization of a Subtilin-Regulated Expression System in
Bacillus subtilis
: Strict Control of Gene Expression by Addition of Subtilin
Authors: Bongers Roger S. , Veening Jan-Willem , Van Wieringen Maarten , Kuipers Oscar P. , Kleerebezem Michiel
Published: December 2005

A system for subtilin-regulated gene expression (SURE) in
Bacillus subtilis
that is based on the regulatory module involved in cell-density-dependent control of the production of subtilin is described. An integration vector for introduction of the essential sensor-regulator couple
spaRK
into the
amyE
locus of the
B. subtilis
chromosome and a
B. subtilis
168-derived production host in which the
spaRK
genes were functionally introduced were constructed. Furthermore, several expression plasmids harboring the subtilin-inducible wild-type
spaS
promoter or a mutated derivative of this promoter were constructed, which facilitated both transcriptional and translational promoter-gene fusions. Functional characterization of both
spaS
promoters and the cognate expression host could be performed by controlled overproduction of the β-glucuronidase (GusA) and green fluorescent protein (GFP) reporters. Both
spaS
promoters exhibited very low levels of basal expression, while extremely high levels of expression were observed upon induction with subtilin. Moreover, the level of expression depended directly on the amount of inducer (subtilin) used. The wild-type
spaS
promoter appeared to be more strictly controlled by the addition of subtilin, while the highest levels of expression were obtained when the mutated
spaS
promoter was used. Induction by subtilin led to 110- and 80-fold increases in GusA activity for the
spaS
promoter and its mutant derivative, respectively. Since the SURE system has attractive functional characteristics, including promoter silence under noninducing conditions and a controlled and high level of expression upon induction, and since it is not subject to catabolite control, we anticipate that it can provide a suitable expression system for various scientific and industrial applications.
Development and Evaluation of Genome-Probing Microarrays for Monitoring Lactic Acid Bacteria
Authors: Bae Jin-Woo , Rhee Sung-Keun , Park Ja Ryeong , Chung Won-Hyong , Nam Young-Do , Lee Insun , Kim Hongik , Park Yong-Ha
Published: December 2005

The genome-probing microarray (GPM) was developed for quantitative, high-throughput monitoring of community dynamics in lactic acid bacteria (LAB) fermentation through the deposit of 149 microbial genomes as probes on a glass slide. Compared to oligonucleotide microarrays, the specificity of GPM was remarkably increased to a species-specific level. GPM possesses about 10- to 100-fold higher sensitivity (2.5 ng of genomic DNA) than the currently used 50-mer oligonucleotide microarrays. Since signal variation between the different genomes was very low compared to that of cDNA or oligonucleotide-based microarrays, the capacity of global quantification of microbial genomes could also be observed in GPM hybridization. In order to assess the applicability of GPMs, LAB community dynamics were monitored during the fermentation of kimchi, a traditional Korean food. In this work, approximately 100 diverse LAB species could be quantitatively analyzed as actively involved in kimchi fermentation.
Shiga Toxin 2e-Producing
Escherichia coli
Isolates from Humans and Pigs Differ in Their Virulence Profiles and Interactions with Intestinal Epithelial Cells
Authors: Sonntag Anne-Katharina , Bielaszewska Martina , Mellmann Alexander , Dierksen Nadine , Schierack Peter , Wieler Lothar H. , Schmidt M. Alexander , Karch Helge
Published: December 2005

Thirteen
Escherichia coli
strains harboring
stx
2e
were isolated from 11,056 human stools. This frequency corresponded to
the presence of the
stx
2e
allele in 1.7% of all
Shiga toxin-producing
E. coli
(STEC) strains. The strains
harboring
stx
2e
were associated with mild diarrhea
(
n
= 9) or asymptomatic infections (
n
=
4). Because STEC isolates possessing
stx
2e
are porcine pathogens, we compared the human
STEC isolates with
stx
2e
-harboring
E.
coli
isolated from piglets with edema disease and postweaning
diarrhea. All pig isolates possessed the gene encoding the F18 adhesin,
and the majority possessed adhesin involved in diffuse adherence; these
adhesins were absent from all the human STEC isolates. In contrast, the
high-pathogenicity island encoding an iron uptake system was found only
in human isolates. Host-specific patterns of interaction with
intestinal epithelial cells were observed. All human isolates adhered
to human intestinal epithelial cell lines T84 and HCT-8 but not to pig
intestinal epithelial cell line IPEC-J2. In contrast, the pig isolates
completely lysed human epithelial cells but not IPEC-J2 cells, to which
most of them adhered. Our data demonstrate that
E. coli
isolates producing Shiga toxin 2e have imported specific virulence and
fitness determinants which allow them to adapt to the specific hosts in
which they cause various forms of
disease.
Directed Evolution of
Streptomyces clavuligerus
Deacetoxycephalosporin C Synthase for Enhancement of Penicillin G Expansion
Authors: Wei Chia-Li , Yang Yunn-Bor , Deng Chan-Hui , Liu Wen-Chi , Hsu Jyh-Shing , Lin Yu-Ching , Liaw Shwu-Huey , Tsai Ying-Chieh
Published: December 2005

The deacetoxycephalosporin C synthase from
Streptomyces clavuligerus
was directly modified for enhancement of penicillin G expansion into phenylacetyl-7-aminodeacetoxycephalosporanic acid, an important intermediate in the industrial manufacture of cephalosporin antibiotics. Nine new mutants, mutants M73T, T91A, A106T, C155Y, Y184H, M188V, M188I, H244Q, and L277Q with 1.4- to 5.7-fold increases in the
k
cat
/

K
m

ratio, were obtained by screening 6,364 clones after error-prone PCR-based random mutagenesis. Subsequently, DNA shuffling was carried out to screen possible combinations of substitutions, including previous point mutations. One quaternary mutant, the C155Y/Y184H/V275I/C281Y mutant, which had a
k
cat
/

K
m

ratio that was 41-fold higher was found after 10,572 clones were assayed. The distinct mutants obtained using different mutagenesis methods demonstrated the complementarity of the techniques. Interestingly, most of the mutated residues that result in enhanced activities are located within or near the unique small barrel subdomain, suggesting that manipulation of this subdomain may be a constructive strategy for improvement of penicillin expansion. Several mutations had very distinct effects on expansion of penicillins N and G, perhaps due to different penicillin-interacting modes within the enzyme. Thus, the present study provided not only promising enzymes for cephalosporin biosynthesis but also a large number of mutants, which provided new insights into the structure-function relationship of the protein that should lead to further rational engineering.
Detection ofMycobacterium aviumsubsp.paratuberculosisin Retail Cheeses from Greece and the Czech Republic
Authors: Ikonomopoulos John , Pavlik Ivo , Bartos Milan , Svastova Petra , Ayele Wuhib Yayo , Roubal Petr , Lukas John , Cook Nigel , Gazouli Maria
Published: December 2005

We investigated the presence ofMycobacterium aviumsubsp.paratuberculosisin retail cheeses from Greece and the Czech Republic. We found that 31.7% and 3.6% of our samples reacted positive by PCR and culture, respectively. Consumption of these cheeses is likely to result in human exposure toM. aviumsubsp.paratuberculosis, albeit at a low level for viable cells.
Novel Primers Reveal Wider Diversity among Marine Aerobic Anoxygenic Phototrophs
Authors: Yutin Natalya , Suzuki Marcelino T. , Béjà Oded
Published: December 2005

Aerobic anoxygenic phototrophic bacteria (AAnPs) were previously proposed to account for up to 11% of marine bacterioplankton and to potentially have great ecological importance in the world's oceans. Our data show that previously used primers based on the M subunit of anoxygenic photosynthetic reaction center genes (
pufM
) do not comprehensively identify the diversity of AAnPs in the ocean. We have designed and tested a new set of
pufM
-specific primers and revealed several new AAnP variants in environmental DNA samples and genomic libraries.
PCR-Induced Sequence Artifacts and Bias: Insights from Comparison of Two 16S rRNA Clone Libraries Constructed from the Same Sample
Authors: Acinas Silvia G. , Sarma-Rupavtarm Ramahi , Klepac-Ceraj Vanja , Polz Martin F.
Published: December 2005

The contribution of PCR artifacts to 16S rRNA gene sequence diversity from a complex bacterioplankton sample was estimated.
Taq
DNA polymerase errors were found to be the dominant sequence artifact but could be constrained by clustering the sequences into 99% sequence similarity groups. Other artifacts (chimeras and heteroduplex molecules) were significantly reduced by employing modified amplification protocols. Surprisingly, no skew in sequence types was detected in the two libraries constructed from PCR products amplified for different numbers of cycles. Recommendations for modification of amplification protocols and for reporting diversity estimates at 99% sequence similarity as a standard are given.
Evaluation of New Broth Media for Microdilution Antibiotic Susceptibility Testing of Lactobacilli, Pediococci, Lactococci, and Bifidobacteria
Authors: Klare Ingo , Konstabel Carola , Müller-Bertling Sibylle , Reissbrodt Rolf , Huys Geert , Vancanneyt Marc , Swings Jean , Goossens Herman , Witte Wolfgang
Published: December 2005

Nine pure or mixed broth media were evaluated for their suitabilities to determine MICs in a microdilution test of 19 antibacterial agents for lactic acid bacteria (LAB) of the genera
Lactobacillus
,
Pediococcus
,
Lactococcus
, and
Bifidobacterium
. A mixed formulation of Iso-Sensitest broth (90%) and deMan-Rogosa-Sharpe broth (10%) with or without supplementation with
l
-cysteine, referred to as the LAB susceptibility test medium, provided the most optimal medium basis in terms of growth support of nonenterococcal LAB and correct indication of MICs of international control strains.
Cell-Cell Influences on Bacterial Community Development in Aquatic Biofilms
Authors: McLean Robert J. C. , Barnes Mary B. , Windham M. Katy , Merchant Mubina , Forstner Michael R. J. , Fuqua Clay
Published: December 2005

Dialysis tubing containing spent culture media, when placed in a lake, was colonized by a low diversity of bacteria, whereas abiotic controls had considerable diversity. Changes were seen in the presence and absence of acylated homoserine lactones, suggesting that these molecules and other factors may influence adherent-population composition.
Use of PCR Amplification of tRNA Gene-Linked Short Tandem Repeats for Genotyping
Entamoeba histolytica
Authors: Ali Ibne Karim M. , Zaki Mehreen , Clark C. Graham
Published: December 2005

We have developed a reliable method for PCR-based genotyping of
Entamoeba histolytica
based on variation in the numbers of short tandem repeats that are linked to tRNA genes in this species. Species-specific primer pairs were designed that differentiate
E. histolytica
from
E. dispar
as well as that reveal intraspecies PCR product length polymorphisms. The primers were tested with samples from different parts of the world, and DNA was extracted from cultured cells as well as liver abscess pus and feces by various methods. We now have the tools necessary to investigate a possible link between parasite genotype and the outcome of infection with
Entamoeba histolytica
, as well as other aspects of the organism's epidemiology.
Analysis of a Uropathogenic
Escherichia coli
Clonal Group by Multilocus Sequence Typing
Authors: Tartof Sara Y. , Solberg Owen D. , Manges Amee R. , Riley Lee W.
Published: December 2005

Although many strain typing methods exist for pathogenic
Escherichia coli
, most have drawbacks in terms of resolving power, interpretability, or scalability. For this reason, multilocus sequence typing (MLST) is an appealing alternative. However, its applicability to different pathogens in specific epidemiologic contexts is not well understood. Here, we applied a previously established MLST method based on housekeeping genes to a well-characterized collection of uropathogenic
E. coli
isolates to compare the discriminatory ability of this procedure with that of enterobacterial repeat intergenic consensus (ERIC2) PCR, serogrouping, and pulsed-field gel electrophoresis (PFGE). Among 45
E. coli
isolates studied, 17 different multilocus sequence types (ST) were identified. One MLST group (designated ST69 complex) was comprised of 22 isolates, all belonging to uropathogenic and bacteremic
E. coli
strains previously defined as clonal group A (CgA) by ERIC2 PCR. The ST69 strains contained five different serogroups and 14 PFGE types. ERIC2 PCR CgA strains belonging to different MLST groups were also identified. Interestingly, one cow
E. coli
isolate, previously shown by PFGE to be closely related to a human uropathogenic CgA strain, was found to cluster with the ST69 strains. All of the other animal and environmental CgA isolates had different MLST profiles. The discriminatory power of this MLST method based on housekeeping genes appears to be higher than that of ERIC2 PCR but lower than that of PFGE for epidemiologic study of uropathogenic
E. coli
.
Rapid Ped-2E9 Cell-Based Cytotoxicity Analysis and Genotyping of
Bacillus
Species
Authors: Gray Kristen M. , Banada Padmapriya P. , O'Neal Erin , Bhunia Arun K.
Published: December 2005

Bacillus
species causing food-borne disease produce multiple toxins eliciting gastroenteritis. Toxin assays with mammalian cell cultures are reliable but may take 24 to 72 h to complete and also lack sensitivity. Here, a sensitive and rapid assay was developed using a murine hybridoma Ped-2E9 cell model.
Bacillus
culture supernatants containing toxins were added to a Ped-2E9 cell line and analyzed for cytotoxicity with an alkaline phosphatase release assay. Most
Bacillus cereus
strains produced positive cytotoxicity results within 1 h, and data were comparable to those obtained with the standard Chinese hamster ovary (CHO)-based cytotoxicity assay, which took about 72 h to complete. Moreover, the Ped-2E9 cell assay had 25- to 58-fold-higher sensitivity than the CHO assay. Enterotoxin-producing
Bacillus thuringiensis
also gave positive results with Ped-2E9 cells, while several other
Bacillus
species were negative. Eight isolates from food suspected of
Bacillus
contamination were also tested, and only one strain, which was later confirmed as
B. cereus
, gave a positive result. In comparison with two commercial diarrheal toxin assay kits (BDE-VIA and BCET-RPLA), the Ped-2E9 assay performed more reliably. Toxin fractions of >30 kDa showed the highest degree of cytotoxicity effects, and heat treatment significantly reduced the toxin activity, indicating the involvement of a heat-labile high-molecular-weight component in Ped-2E9 cytotoxicity. PCR results, in most cases, were in agreement with the cytotoxic potential of each strain. Ribotyping was used to identify cultures and indicated differences for several previously reported isolates. This Ped-2E9 cell assay could be used as a rapid (∼1-h) alternative to current methods for sensitive detection of enterotoxins from
Bacillus
species.
Novel Rotavirus VP7 Typing Assay Using a One-Step Reverse Transcriptase PCR Protocol and Product Sequencing and Utility of the Assay for Epidemiological Studies and Strain Characterization, Including Serotype Subgroup Analysis
Authors: DiStefano Daniel J. , Kraiouchkine Nikolai , Mallette Laura , Maliga Marianne , Kulnis Gregory , Keller Paul M. , Clark H. Fred , Shaw Alan R.
Published: December 2005

Rotavirus is the most common cause of severe dehydrating gastroenteritis in infants. To date, 10 different serotypes of rotavirus have been identified in human stools. While four or five serotypes dominate, serotype circulation varies with season and geography. Since our laboratory has been involved in the development of a multivalent rotavirus vaccine, it is important to identify the serotypes of rotavirus encountered during our clinical trials. We have developed methodologies for the molecular identification of rotavirus strains based on VP7 gene segment sequence. A 365-bp reverse transcriptase PCR product was generated from the VP7 gene segment using a pair of novel degenerate primers. All serotypes tested (both animal and human) yielded an identically sized product after amplification. Sequencing of these products is performed using truncated versions of the original primers. The sequence generated is compared against a database of rotavirus VP7 sequences, with the G type determined, based on the sequence homology. Using this assay, we have correctly identified human VP7 strains from a panel of available serotypes, as well as numerous animal strains. The assay was qualified using rotavirus positive stool samples, negative stool samples, and rotavirus-spiked stool samples. In addition, samples from cases of acute gastroenteritis collected at Children's Hospital of Philadelphia have been evaluated and indicate that the assay is able to discriminate subtle differences within serotypes. The assay has been utilized in the testing of >3,000 antigen-positive (enzyme immunoassay) samples collected during clinical trials of a rotavirus vaccine (RotaTeq) and identified a serotype in ∼92% of samples (3, 17, 19).
Use of Recombinant Antigens for Early Postnatal Diagnosis of Congenital Toxoplasmosis
Authors: Buffolano Wilma , Beghetto Elisa , Del Pezzo Mariassunta , Spadoni Andrea , Di Cristina Manlio , Petersen Eskild , Gargano Nicola
Published: December 2005

The main objective of this work was to improve the early serologic diagnosis of toxoplasmosis in children at risk of congenital infection by using recombinant antigens. Serum samples from 104 infants born to mothers with primary
Toxoplasma gondii
infection acquired during pregnancy, of which 35 were congenitally infected and 22 had clinical silent toxoplasmosis at birth, were included. Immunoglobulin M (IgM), IgG, and IgG subtype antibodies against epitopes carried by fragments of
T. gondii MIC2
,
MIC3
,
MIC4
,
M2AP
,
AMA1
, and
SAG1
gene products were measured by performing parallel enzyme immunoassays (Rec-ELISAs). Recombinant antigens preferentially reacted with IgG antibodies from infected infants compared to uninfected subjects (
P
< 0.0001), indicating that sera from infected children recognized a more diverse repertoire of antigens than sera transferred over the placenta from the mothers. Using two serial samples collected within 3 months of life, it was possible to demonstrate a neosynthesis of specific anti-MIC2 and anti-SAG1 immunoglobulin G, mainly of the IgG2 subtype, in 13 out of 20 infants with congenital toxoplasmosis. IgM antibodies in 97% of infected infants reacted with at least one of the recombinant antigens, confirming the diagnosis of congenital infection as soon as 2 months after birth (
P
< 0.0001). The use of recombinant antigens is effective in distinguishing
T. gondii
-infected from uninfected infants and shows that assays based on recombinant antigens improve the diagnosis of newborns with congenital toxoplasmosis.
Genetic Diversity and Recombination of Porcine Sapoviruses
Authors: Wang Qiu-Hong , Han Myung Guk , Funk Julie A. , Bowman Gary , Janies Daniel A. , Saif Linda J.
Published: December 2005

Sapoviruses (SaVs) are emerging enteric pathogens that cause diarrhea in humans and animals. Human SaVs are genetically variable and have been classified into four genogroups (GI, -II, -IV, and -V). To date, only two genetically similar porcine SaV strains have been reported that belong to GIII. To investigate the genetic diversity of porcine SaVs and their genetic relatedness to human strains, we sequenced 286 nucleotides (nt) of the RNA-dependent RNA polymerase (RdRp) region of nine porcine SaVs detected from field pig fecal samples collected in U.S. swine farms during the period from 1999 to 2003. One strain (Po/SaV/MI-QW19/2002/US) was most closely related to human GII SaVs. We also sequenced 3 kb of the viral genome, including the partial RdRp (766 to 790 nt), the complete capsid, the ORF2 and the 3′-untranslated region of four strains representative for the positive farms or for the distinct genetic clusters. From the sequence analysis of the complete capsid, we identified a potential new genogroup of porcine SaVs, with Po/SaV/OH-JJ681/00/US as the representative strain. Furthermore, two potential porcine SaV recombinants were identified. To our knowledge this is the first report of a porcine SaV strain more closely related genetically to human SaVs and the occurrence of porcine SaV recombinants. The presence of porcine SaVs more similar to human SaVs is a significant finding because of the potential for zoonotic infections or generation of porcine/human recombinants if intragenogroup human strains exist.
Identification of 43StreptococcusSpecies by Pyrosequencing Analysis of thernpBGene
Authors: Innings Åsa , Krabbe Margareta , Ullberg Måns , Herrmann Björn
Published: December 2005

Pyrosequencing technology was evaluated for identification of species within theStreptococcusgenus. Two variable regions in thernpBgene, which encodes the RNA subunit of endonuclease P, were sequenced in two reactions. Of 43 species, all could be identified to the species level except strains of the species pairsStreptococcus anginosus/S. constellatusandS. infantis/S. peroris. A total of 113 blood culture isolates were identified by pyrosequencing analysis of partialrnpBsequences. All but eight isolates could be unambiguously assigned to a specific species when the first 30 nucleotides of the two regions were compared to anrnpBdatabase comprising 107 streptococcal strains. Principal coordinate analysis of sequence variation of strains from viridans group streptococci resulted in species-specific clusters for the mitis and the salivarius groups but not for the anginosus group. The identification capacity of pyrosequencing was compared to the biochemical test systems VITEK 2 and Rapid ID 32 Strep. The concordance between pyrosequencing and VITEK 2 was 75%, and for Rapid ID 32 Strep the corresponding figure was 77%. Isolates with discrepant identifications in the three methods were subjected to entirernpBDNA sequence analysis that confirmed the identifications by pyrosequencing. In conclusion, pyrosequencing analysis of thernpBgene can reliably identifyStreptococcusspecies with high resolution.
Frequency and Possible Infection Control Implications of Gastrointestinal Colonization with Methicillin-Resistant
Staphylococcus aureus
Authors: Boyce John M. , Havill Nancy L. , Maria Benedicte
Published: December 2005

Methicillin-resistant
Staphylococcus aureus
(MRSA) is a major cause of health care-associated infections. Multiple factors, including transmission from unrecognized reservoirs of MRSA, are responsible for failure to control the spread of MRSA. We conducted prospective surveillance to determine the frequency of gastrointestinal colonization with MRSA among patients and its possible impact on nosocomial transmission of MRSA. Stool specimens submitted for
Clostridium difficile
toxin A/B assays were routinely inoculated on colistin-naladixic acid agar plates, and
S. aureus
was identified by using standard methods. Methicillin resistance was confirmed by growth on oxacillin-salt screening agar. For patients whose stool yielded MRSA, information regarding any previous cultures positive for MRSA or other organisms that would require contact precautions was obtained from the laboratory's computer system. During a 1-year period, 151 (9.8%) of 1,543 patients who had one or more stool specimens screened had MRSA in their stool. Ninety-three (62%) of the 151 patients had no previous history of MRSA colonization or infection. Of these 93, 75 were inpatients. Sixty (80%) of the 75 inpatients with no previous history of MRSA were not under “contact precautions.” The 60 patients would have spent an estimated total of 267 days without being placed under contact precautions if their positive stool cultures had not resulted in their being isolated. Placing patients under contact precautions based on their positive stool cultures prevented an estimated 35 episodes of MRSA transmission. We conclude that gastrointestinal colonization with MRSA may serve as an unrecognized reservoir from which transmission of MRSA may occur in health care facilities.
Clinical Significance of Hepatitis B Virus (HBV) Genotypes and Precore and Core Promoter Mutations Affecting HBV e Antigen Expression in Taiwan
Authors: Chen Chien-Hung , Lee Chuan-Mo , Lu Sheng-Nan , Changchien Chi-Sin , Eng Hock-Liew , Huang Chao-Min , Wang Jing-Houng , Hung Chao-Hung , Hu Tsung-Hui
Published: December 2005

To assess the prevalence and clinical significance of hepatitis B virus (HBV) genotypes and precore and core promoter mutations in Taiwan, a cohort of 200 Taiwanese chronic hepatitis B patients was analyzed. The HBV genotypes and sequences of the precore and the core promoter regions were determined in 66 asymptomatic carriers and 134 patients who had liver biopsy-verified chronic hepatitis and liver cirrhosis. The HBV e-antigen (HBeAg)-negative patients had a higher frequency of mutations at core promoter nucleotides 1753 and 1773 and precore nucleotides 1846, 1896, and 1899 than HBeAg-positive patients. Among the 200 patients, the frequencies of genotype C, T1762 and A1764, C1753, T1766 and A1768, and A1896 mutations increased and the frequencies of T or G1752, T1773, G1799, and C1858 mutations decreased with advancing liver diseases. These factors were different between those with HBeAg-positive status and those with HBeAg-negative status. Based on multiple logistic regression analysis, the risk factors of liver cirrhosis for 200 patients were the presence of T1762 and A1764 mutations (odds ratio [OR] = 11.11; 95% confidence interval [CI] = 3.91 to 31.25;P< 0.001), age ≥35 years (OR = 3.42; 95% CI = 1.33 to 8.77;P= 0.011), and genotype C (OR = 2.87; 95% CI = 1.21 to 6.81;P= 0.017). Further categorical analysis found that 62.1% of patients with genotype C, T1762 and A1764 mutations and age ≥35 years had liver cirrhosis. None of the 55 patients infected with the genotype B, A1762 and G1764 wild type and age <35 years showed liver cirrhosis. In conclusion, our data suggest that pathogenic differences between HBeAg-positive and -negative patients may exist. In Taiwan, HBV genotype C and the T1762 and A1764 mutations may play a role in HBV-related liver cirrhosis, and these could serve as molecular markers for prediction of the clinical outcomes of chronic HBV patients.
Recurrent Melioidosis in Patients in Northeast Thailand Is Frequently Due to Reinfection Rather than Relapse
Authors: Maharjan Bina , Chantratita Narisara , Vesaratchavest Mongkol , Cheng Allen , Wuthiekanun Vanaporn , Chierakul Wirongrong , Chaowagul Wipada , Day Nicholas P. J. , Peacock Sharon J.
Published: December 2005

Human melioidosis is associated with a high rate of recurrent disease, despite adequate antimicrobial treatment. Here, we define the rate of relapse versus the rate of reinfection in 116 patients with 123 episodes of recurrent melioidosis who were treated at Sappasithiprasong Hospital in Northeast Thailand between 1986 and 2005. Pulsed-field gel electrophoresis was performed on all isolates; isolates from primary and recurrent disease for a given patient different by one or more bands were examined by a sequence-based approach based on multilocus sequence typing. Overall, 92 episodes (75%) of recurrent disease were caused by the same strain (relapse) and 31 episodes (25%) were due to infection with a new strain (reinfection). The interval to recurrence differed between patients with relapse and reinfection; those with relapses had a median time to relapse of 228 days (range, 15 to 3,757 days; interquartile range [IQR], 99.5 to 608 days), while those with reinfection had a median time to reinfection of 823 days (range, 17 to 2,931 days; IQR, 453 to 1,211 days) (
P
= 0.0001). A total of 64 episodes (52%) occurred within 12 months of the primary infection. Relapse was responsible for 57 of 64 (89%) episodes of recurrent infection within the first year after primary disease, whereas relapse was responsible for 35 of 59 (59%) episodes after 1 year (
P
< 0.0001). Our data indicate that in this setting of endemicity, reinfection is responsible for one-quarter of recurrent cases. This finding has important implications for the clinical management of melioidosis patients and for antibiotic treatment studies that use recurrent disease as a marker for treatment failure.
Relationship between the Original Multiply Resistant South African Isolates ofStreptococcus pneumoniaefrom 1977 to 1978 and Contemporary International Resistant Clones
Authors: Reinert Ralf René , Jacobs Michael R. , Appelbaum Peter C. , Bajaksouzian Saralee , Cordeiro Soraia , van der Linden Mark , Al-Lahham Adnan
Published: December 2005

High-level penicillin G-resistant as well as multidrug-resistantStreptococcus pneumoniaeisolates were first described in South Africa in 1977. The relationship between these original multidrug-resistant South African isolates and other resistant clones was investigated. Twenty-six representative isolates isolated from initial outbreaks in South Africa from 1977 to 1978 were characterized by multilocus sequence typing and pulsed-field gel electrophoresis. Twenty-one isolates were penicillin resistant and five were penicillin intermediate, with variable susceptibilities to macrolides, clindamycin, chloramphenicol, tetracycline, and trimethoprim-sulfamethoxazole. Fourteen isolates were serotype 19A, 11 were serotype 6A, and one was serotype 14. Penicillin-resistant serotype 19A isolates belonged to three closely related sequence types (STs), ST 41 (n= 6), ST 1605 (n= 3), and ST 1656 (n= 1). Penicillin-resistant serotype 6A isolates belonged to two closely related STs, ST 1094 (n= 10) and ST 1607 (n= 1), and were not closely related to other international clones. The serotype 14 penicillin-intermediate isolate was not closely related to the other isolates from South Africa but was a predicted founder of a clonal group with 41 different STs. Five new STs, ST 1605, ST 1607, ST 1608, ST 1610, and ST 1656, are described for the first time in this study. New molecular methods have characterized the original multiply resistant South African pneumococcal isolates from 1977 to 1978 and have shown the relationships of these clones to major pneumococcal clones.
Distribution of Insertion- and Deletion-Associated Genetic Polymorphisms among Four
Mycobacterium tuberculosis
Phospholipase C Genes and Associations with Extrathoracic Tuberculosis: a Population-Based Study
Authors: Kong Y. , Cave M. D. , Yang D. , Zhang L. , Marrs C. F. , Foxman B. , Bates J. H. , Wilson F. , Mukasa L. N. , Yang Z. H.
Published: December 2005

The
Mycobacterium tuberculosis
genome contains four phospholipase C (PLC)-encoding genes, designated
plcA
,
plcB
,
plcC
, and
plcD
, respectively. Each of the four genes contributes to the overall PLC activity of
M. tuberculosis.
PLC is hypothesized to contribute to
M. tuberculosis
virulence. Infection of
M. tuberculosis
strains carrying a truncated
plcD
gene is associated with the occurrence of extrathoracic tuberculosis. However, whether the other three
plc
genes are also associated with extrathoracic tuberculosis remains to be assessed. We investigated the insertion- and deletion-associated genetic diversity in all four
plc
genes among 682 epidemiologically and clinically well-characterized
M. tuberculosis
clinical isolates using PCR, DNA sequencing, and Southern hybridization. Two hundred sixty-six (39%) of the 682 isolates had an interruption in at least one of the four
plc
genes, most often associated with an IS
6110
insertion. The
plcD
gene interruption was the most common: it was observed in 233 (34%) of the isolates, compared to 4.7%, 4.1%, and 5.9% for
plcA
,
plcB
, and
plcC
gene interruption, respectively. The association between the
plc
gene genotypes and disease presentation was adjusted for clustering using generalized estimating equations for both bivariate and multivariate analyses. After controlling for the genotypes of the
plcABC
genes and the host-related risk factors, interruption in the
plcD
gene remained significantly associated with extrathoracic tuberculosis (odds ratio, 3.27; 95% confidence interval, 1.32 to 8.14). The data suggest that the
plcD
gene might play a more important role in the pathogenesis of thoracic TB than it does in the pathogenesis of extrathoracic TB.
Comparison of the Sodium Hydroxide Specimen Processing Method with the C
18
-Carboxypropylbetaine Specimen Processing Method Using Independent Specimens with Auramine Smear, the MB/BacT Liquid Culture System, and the COBAS AMPLICOR MTB Test
Authors: Padilla Eduardo , Manterola José M. , González Victoria , Thornton Charles G. , Quesada M. Dolores , Sánchez M. Dolores , Pérez Miguel , Ausina Vicente
Published: December 2005

A study was performed to diagnose tuberculosis by smear, culture, and nucleic acid amplification. The study was comprised of two independent arms. Each arm used a different specimen processing method; in one arm, all specimens were processed with
N
-acetyl-
l
-cysteine-sodium hydroxide, and in the other arm, all specimens were processed with C
18
-carboxypropylbetaine and lytic enzymes. In each arm, all processed sediments were split for analysis by auramine smear, by culture using the MB/BacT liquid culture system and solid media, and by nucleic acid amplification using the COBAS AMPLICOR MTB test. In the
N
-acetyl-
l
-cysteine-sodium hydroxide arm, 1,468 specimens were analyzed: 65 were smear positive; 88 and 42 were culture positive for
Mycobacterium tuberculosis
and nontuberculous mycobacteria, respectively; and 103 were PCR positive. Relative to cultures positive for
M. tuberculosis
, the sensitivity and specificity of the smear were 68.2% and 99.6%, respectively, and those of PCR were 75.0% and 97.3%, respectively. In the C
18
-carboxypropylbetaine study arm, 1,423 specimens were analyzed: 44 were smear positive; 82 and 31 were culture positive for
M. tuberculosis
and nontuberculous mycobacteria, respectively; and 91 were PCR positive. The sensitivity and specificity of the smear were 48.8% and 99.7%, respectively, and those of PCR were 78.0% and 98.0%, respectively. When the two arms were compared, C
18
-carboxypropylbetaine specimen processing significantly increased the number of smear-negative and culture-positive specimens and significantly increased the PCR sensitivity among this same group of specimens while at the same time significantly reducing the inhibition rate.
DNA Polymorphism and Molecular Subtyping of the Capsular Gene Cluster of Group B
Streptococcus
Authors: Manning Shannon D. , Lacher David W. , Davies H. Dele , Foxman Betsy , Whittam Thomas S.
Published: December 2005

Serotyping and other phenotypic methods are often used to characterize the capsular polysaccharide of group B streptococci (GBS). We describe a capsular genotyping method that utilizes PCR of capsular polysaccharide synthesis genes (
cps
) and restriction enzyme digestion. This method facilitates the detection of DNA polymorphism in
cps
genes and correlates well with serotyping.
Frequency and Species Distribution of Gliotoxin-Producing
Aspergillus
Isolates Recovered from Patients at a Tertiary-Care Cancer Center
Authors: Lewis Russell E. , Wiederhold Nathan P. , Lionakis Michail S. , Prince Randall A. , Kontoyiannis Dimitrios P.
Published: December 2005

Aspergillus
isolates (
n
= 103) collected from cancer patients were screened to determine the taxonomic distribution and quantity of gliotoxin production. Gliotoxin was detected in 93% of
Aspergillus fumigatus
, 75% of
A. niger
, 25% of
A. terreus
, and 4% of
A. flavus
cultures. Gliotoxin concentrations were highest in cultures of
A. fumigatus
.
The Role of 16S rRNA Gene Sequencing in Identification of Microorganisms Misidentified by Conventional Methods
Authors: Petti C. A. , Polage C. R. , Schreckenberger P.
Published: December 2005

Traditional methods for microbial identification require the recognition of differences in morphology, growth, enzymatic activity, and metabolism to define genera and species. Full and partial 16S rRNA gene sequencing methods have emerged as useful tools for identifying phenotypically aberrant microorganisms. We report on three bacterial blood isolates from three different College of American Pathologists-certified laboratories that were referred to ARUP Laboratories for definitive identification. Because phenotypic identification suggested unusual organisms not typically associated with the submitted clinical diagnosis, consultation with the Medical Director was sought and further testing was performed including partial 16S rRNA gene sequencing. All three patients had endocarditis, and conventional methods identified isolates from patients A, B, and C as a
Facklamia
sp.,
Eubacterium tenue
, and a
Bifidobacterium
sp. 16S rRNA gene sequencing identified the isolates as
Enterococcus faecalis
,
Cardiobacterium valvarum
, and
Streptococcus mutans
, respectively. We conclude that the initial identifications of these three isolates were erroneous, may have misled clinicians, and potentially impacted patient care. 16S rRNA gene sequencing is a more objective identification tool, unaffected by phenotypic variation or technologist bias, and has the potential to reduce laboratory errors.
Evaluation of the LightCycler
Staphylococcus
M
GRADE
Kits on Positive Blood Cultures That Contained Gram-Positive Cocci in Clusters
Authors: Shrestha Nabin K. , Tuohy Marion J. , Padmanabhan Ravindran A. , Hall Gerri S. , Procop Gary W.
Published: December 2005

We evaluated the Roche LightCycler
Staphylococcus
M
GRADE
kits to differentiate between
Staphylococcus aureus
and coagulase-negative staphylococci in blood cultures growing clusters of gram-positive cocci. Testing 100 bottles (36 containing
S. aureus
), the assay was 100% sensitive and 98.44% specific for
S. aureus
and 100% sensitive and specific for coagulase-negative staphylococci.
Validation of Performance of Plastic versus Glass Bottles for Culturing Anaerobes from Blood in BacT/ALERT SN Medium
Authors: Mirrett Stanley , Joyce Maria J. , Reller L. Barth
Published: December 2005

To validate performance, we compared the new plastic BacT/ALERT (bioMérieux, Durham, NC) SN bottle to the current glass SN bottle with samples of blood obtained for culture from adults and found them comparable for both recovery and speed of detection of microorganisms. We conclude that the safety advantage of plastic bottles can be achieved without compromising performance.