bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence for Existence of “Mesotogas,” Members of the OrderThermotogalesAdapted to Low-Temperature Environments
Authors: Nesbø Camilla L. , Dlutek Marlena , Zhaxybayeva Olga , Doolittle W. Ford
Published: July 2006

All cultivated isolates of the bacterial orderThermotogalesare either thermophiles or hyperthermophiles, butThermotogales16S rRNA gene sequences have been detected in many mesophilic anaerobic and microaerophilic environments, particularly within communities involved in the remediation of pollutants. Here we provide metagenomic evidence for the existence ofThermotogaleslineages, which we informally call “mesotoga,” that are adapted to growth at lower temperatures. Two fosmid clones containing mesotoga DNA, originating from a low-temperature enrichment culture that degrades a polychlorinated biphenyl congener, were sequenced. Phylogenetic analysis clearly puts this bacterial lineage within theThermotogalesorder, with the rRNA gene trees and 21 of 58 open reading frames strongly supporting this relationship. An analysis of protein sequence composition showed that mesotoga proteins are adapted to function at lower temperatures than are their identifiable homologs from thermophilic and hyperthermophilic members of the orderThermotogales, supporting the notion that this bacterium lives and grows optimally at lower temperatures. The phylogenetic analysis suggests that the mesotoga lineage from which our fosmids derive has used both the acquisition of genes from its neighbors and the modification of existing thermophilic sequences to adapt to a mesophilic lifestyle.
Culture-Independent Characterization of the Digestive-Tract Microbiota of the Medicinal Leech Reveals a Tripartite Symbiosis
Authors: Worthen Paul L. , Gode Cindy J. , Graf Joerg
Published: July 2006

Culture-based studies of the microbial community within the gut of the medicinal leech have typically been focused on various
Aeromonas
species, which were believed to be the sole symbiont of the leech digestive tract. In this study, analysis of 16S rRNA gene clone libraries confirmed the presence of
Aeromonas veronii
and revealed a second symbiont, clone PW3, a novel member of the
Rikenellaceae
, within the crop, a large compartment where ingested blood is stored prior to digestion. The diversity of the bacterial community in the leech intestinum was determined, and additional symbionts were detected, including members of the α-, γ-, and δ-
Proteobacteria
,
Fusobacteria
,
Firmicutes
, and
Bacteroidetes
. The relative abundances of the clones suggested that
A. veronii
and the novel clone, PW3, also dominate the intestinum community, while other clones, representing transient organisms, were typically present in low numbers. The identities of these transients varied greatly between individual leeches. Neither time after feeding nor feeding on defibrinated blood caused a change in identity of the dominant members of the microbial communities. Terminal restriction fragment length polymorphism analysis was used to verify that the results from the clone libraries were representative of a larger data set. The presence of a two-member bacterial community in the crop provides a unique opportunity to investigate both symbiont-symbiont and symbiont-host interactions in a natural model of digestive-tract associations.
Purification and Characterization of an Arene
cis
-Dihydrodiol Dehydrogenase Endowed with Broad Substrate Specificity toward Polycyclic Aromatic Hydrocarbon Dihydrodiols
Authors: Jouanneau Yves , Meyer Christine
Published: July 2006

Initial reactions involved in the bacterial degradation of polycyclic aromatic hydrocarbons (PAHs) include a ring-dihydroxylation catalyzed by a dioxygenase and a subsequent oxidation of the dihydrodiol products by a dehydrogenase. In this study, the dihydrodiol dehydrogenase from the PAH-degrading
Sphingomonas
strain CHY-1 has been characterized. The
bphB
gene encoding PAH dihydrodiol dehydrogenase (PDDH) was cloned and overexpressed as a His-tagged protein. The recombinant protein was purified as a homotetramer with an apparent
M
r
of 110,000. PDDH oxidized the
cis
-dihydrodiols derived from biphenyl and eight polycyclic hydrocarbons, including chrysene, benz[
a
]anthracene, and benzo[
a
]pyene, to corresponding catechols. Remarkably, the enzyme oxidized pyrene 4,5-dihydrodiol, whereas pyrene is not metabolized by strain CHY-1. The PAH catechols produced by PDDH rapidly auto-oxidized in air but were regenerated upon reaction of the
o
-quinones formed with NADH. Kinetic analyses performed under anoxic conditions revealed that the enzyme efficiently utilized two- to four-ring dihydrodiols, with

K
m

values in the range of 1.4 to 7.1 μM, and exhibited a much higher Michaelis constant for NAD
+
(

K
m

of 160 μM). At pH 7.0, the specificity constant ranged from (1.3 ± 0.1) × 10
6
M
−1
s
−1
with benz[
a
]anthracene 1,2-dihydrodiol to (20.0 ± 0.8) × 10
6
M
−1
s
−1
with naphthalene 1,2-dihydrodiol. The catalytic activity of the enzyme was 13-fold higher at pH 9.5. PDDH was subjected to inhibition by NADH and by 3,4-dihydroxyphenanthrene, and the inhibition patterns suggested that the mechanism of the reaction was ordered Bi Bi. The regulation of PDDH activity appears as a means to prevent the accumulation of PAH catechols in bacterial cells.
Optimization of a Sampling System for Recovery and Detection of Airborne Porcine Reproductive and Respiratory Syndrome Virus and Swine Influenza Virus
Authors: Hermann J. R. , Hoff S. J. , Yoon K. J. , Burkhardt A. C. , Evans R. B. , Zimmerman J. J.
Published: July 2006

The objective of this research was to optimize sampling parameters for increased recovery and detection of airborne porcine reproductive and respiratory syndrome virus (PRRSV) and swine influenza virus (SIV). Collection media containing antifoams, activated carbons, protectants, and ethylene glycol were evaluated for direct effects on factors impacting the detection of PRRSV and SIV, including virus infectivity, viability of continuous cell lines used for the isolation of these viruses, and performance of reverse transcriptase PCR assays. The results showed that specific compounds influenced the likelihood of detecting PRRSV and SIV in collection medium. A subsequent study evaluated the effects of collection medium, impinger model, and sampling time on the recovery of aerosolized PRRSV using a method for making direct comparisons of up to six treatments simultaneously. The results demonstrated that various components in air-sampling systems, including collection medium, impinger model, and sampling time, independently influenced the recovery and detection of PRRSV and/or SIV. Interestingly, it was demonstrated that a 20% solution of ethylene glycol collected the greatest quantity of aerosolized PRRSV, which suggests the possibility of sampling at temperatures below freezing. Based on the results of these experiments, it is recommended that air-sampling systems be optimized for the target pathogen(s) and that recovery/detection results should be interpreted in the context of the actual performance of the system.
YtjE from
Lactococcus lactis
IL1403 Is a C-S Lyase with α,γ-Elimination Activity toward Methionine
Authors: Martínez-Cuesta M. Carmen , Peláez Carmen , Eagles John , Gasson Michael J. , Requena Teresa , Hanniffy Sean B.
Published: July 2006

Cheese microbiota and the enzymatic conversion of methionine to volatile sulfur compounds (VSCs) are important factors in flavor formation during cheese ripening and the foci in biotechnological approaches to flavor improvement. The product of
ytjE
of
Lactococcus lactis
IL1403, suggested to be a methionine-specific aminotransferase based on genome sequence analysis, was therefore investigated for its role in methionine catabolism. The
ytjE
gene from
Lactococcus lactis
IL1403 was cloned in
Escherichia coli
and overexpressed and purified as a recombinant protein. When tested, the YtjE protein did not exhibit a specific methionine aminotransferase activity. Instead, YtjE exhibited C-S lyase activity and shared homology with the MalY/PatC family of enzymes involved in the degradation of
l
-cysteine,
l
-cystine, and
l
-cystathionine. YtjE was also shown to exhibit α,γ-elimination activity toward
l
-methionine. In addition, gas chromatographic-mass spectrometry analysis showed that YtjE activity resulted in the formation of H
2
S from
l
-cysteine and methanethiol (and its oxidized derivatives dimethyl disulfide and dimethyl trisulfide) from
l
-methionine. Given their significance in cheese flavor development, VSC production by YtjE could offer an additional approach for the development of cultures with optimized aromatic properties.
Identification of Genes and Proteins Necessary for Catabolism of Acyclic Terpenes and Leucine/Isovalerate in
Pseudomonas aeruginosa
Authors: Förster-Fromme Karin , Höschle Birgit , Mack Christina , Bott Michael , Armbruster Wolfgang , Jendrossek Dieter
Published: July 2006

Geranyl-coenzyme A (CoA)-carboxylase (GCase; AtuC/AtuF) and methylcrotonyl-CoA-carboxylase (MCase; LiuB/LiuD) are characteristic enzymes of the catabolic pathway of acyclic terpenes (citronellol and geraniol) and of saturated methyl-branched compounds, such as leucine or isovalerate, respectively. Proteins encoded by two gene clusters (
atuABCDEFGH
and
liuRABCDE
) of
Pseudomonas aeruginosa
PAO1 were essential for acyclic terpene utilization (Atu) and for leucine and isovalerate utilization (Liu), respectively, as revealed by phenotype analysis of 10 insertion mutants, two-dimensional gel electrophoresis, determination of GCase and MCase activities, and Western blot analysis of wild-type and mutant strains. Analysis of the genome sequences of other pseudomonads (
P. putida
KT2440 and
P. fluorescens
Pf-5) revealed candidate genes for Liu proteins for both species and candidate genes for Atu proteins in
P. fluorescens
. This result concurred with the finding that
P. fluorescens
, but not
P. putida
, could grow on acyclic terpenes (citronellol and citronellate), while both species were able to utilize leucine and isovalerate. A regulatory gene,
atuR
, was identified upstream of
atuABCDEFGH
and negatively regulated expression of the
atu
gene cluster.
Molecular Epidemiology of Group A Rotaviruses in Water Sources and Selected Raw Vegetables in Southern Africa
Authors: van Zyl W. B. , Page N. A. , Grabow W. O. K. , Steele A. D. , Taylor M. B.
Published: July 2006

Group A rotaviruses (RVs) are the most important cause of acute viral gastroenteritis in infants and young children. In this study raw and treated drinking water supplies at plants in two geographic areas, as well as selected irrigation water and corresponding raw vegetables in three regions of southern Africa, were screened for the presence of RVs using molecular techniques. Group A RVs were detected in 11.8% of partially treated and 1.7% of finally treated drinking water samples and in 14% of irrigation water samples and 1.7% of corresponding raw vegetable samples. Type-specific reverse transcriptase-PCR and sequence analysis revealed the presence of multiple types (G1, G2, G8, and G9) in irrigation water and single types (G1 or G3) in raw and treated drinking water. Group A RVs detected in all samples consisted of mixed P types (P[4], P[6], P[8], and P[9]), with P[6] predominating. The detection of types G8, G9, and P[6] reflects the emergence of these types in clinical infections. The similarity of environmental types to those in patients with clinical RV infections confirms the value of wastewater screening as a tool for assessing RVs circulating in communities, with the benefit of detecting types that cause both clinical and subclinical infections. The results provide new information on RV types in water and related environments and identify the potential risk of waterborne transmission. In addition, the presence of RVs in drinking water underlines shortcomings in quality specifications. These data provide valuable information regarding the prevalence of RVs in environmental sources, with important implications for vaccine development.
Influences of Biofilm Structure and Antibiotic Resistance Mechanisms on Indirect Pathogenicity in a Model Polymicrobial Biofilm
Authors: O'Connell Heather A. , Kottkamp Greg S. , Eppelbaum James L. , Stubblefield Bryan A. , Gilbert Sarah E. , Gilbert Eric S.
Published: July 2006

Indirect pathogenicity (IP), the commensal protection of antibiotic-sensitive pathogens by resistant microorganisms of low intrinsic virulence, can prevent the eradication of polymicrobial infections. The contributions of antibiotic resistance mechanisms and biofilm structure to IP within polymicrobial biofilms were investigated using a model two-member consortium.
Escherichia coli
ATCC 33456 was transformed with vectors conferring either ampicillin or spectinomycin resistance, creating two distinct populations with different resistance mechanisms. Each strain alone or the consortium was grown as biofilms in flow cells and planktonically in chemostats. Comparisons in survival and activity were made on the basis of MICs and minimum biofilm preventative concentrations, a newly introduced descriptor. In ampicillin-containing medium, commensal interactions were evident during both modes of cultivation, but the sensitive strain experienced a greater benefit in the chemostat, indicating that the biofilm environment limited the commensal interaction between the Amp
r
and Spt
r
strains. In spectinomycin-containing medium, growth of the sensitive strain in chemostats and biofilms was not aided by the resistant strain. However, green fluorescent protein expression by the sensitive strain was greater in mixed-population biofilms (9% ± 1%) than when the strain was grown alone (2% ± 0%). No comparable benefit was evident during growth in the chemostat, indicating that the biofilm structure contributed to enhanced activity of the sensitive strain.
Use of a Nonmedicated Dietary Supplement Correlates with Increased Prevalence of Streptomycin-Sulfa-Tetracycline-Resistant
Escherichia coli
on a Dairy Farm
Authors: Khachatryan Artashes R. , Besser Thomas E. , Hancock Dale D. , Call Douglas R.
Published: July 2006

We examined how a dietary supplement affects the prevalence of antibiotic-resistant
Escherichia coli
on a dairy farm in Washington State. Between 2001 and 2004 the prevalence of fecal
E. coli
strains resistant to streptomycin, sulfadiazine, and tetracycline (SSuT strains) declined from 59.2% to 26.1% in the calf population. In 2003 the dairy discontinued use of a dietary supplement, and we hypothesized that the decline in prevalence of SSuT strains was related to this change in management. To test this we established three treatments in which calves received no supplement, the dietary supplement with oxytetracycline, or the dietary supplement without oxytetracycline. Calves receiving either dietary supplement had a significantly higher prevalence of SSuT
E. coli
than the no-supplement control group (≈37% versus 20%, respectively;
P
= 0.03). Importantly, there was no evidence that oxytetracycline contributed to an increased prevalence of fecal SSuT
E. coli
. We compared the growth characteristics of SSuT and non-SSuT
E. coli
in LB broth enriched with either the complete dietary supplement or its individual constituents. Both the complete dietary supplement and its vitamin D component supported a significantly higher cell density of SSuT strains (
P
= 0.003 and
P
= 0.001, respectively). The dry milk and vitamin A components of the dietary supplement did not support different cell densities. These results were consistent with selection and maintenance of SSuT
E. coli
due to environmental components independent of antibiotic selection.
Purification and Characterization of Two Enantioselective α-Ketoglutarate-Dependent Dioxygenases, RdpA and SdpA, fromSphingomonas herbicidovoransMH
Authors: Müller Tina A. , Fleischmann Thomas , van der Meer Jan Roelof , Kohler Hans-Peter E.
Published: July 2006

α-Ketoglutarate-dependent (R)-dichlorprop dioxygenase (RdpA) and α-ketoglutarate-dependent (S)-dichlorprop dioxygenase (SdpA), which are involved in the degradation of phenoxyalkanoic acid herbicides inSphingomonas herbicidovoransMH, were expressed and purified as His6-tagged fusion proteins fromEscherichia coliBL21(DE3)(pLysS). RdpA and SdpA belong to subgroup II of the α-ketoglutarate-dependent dioxygenases and share the specific motif HXDX24TX131HX10R. Amino acids His-111, Asp-113, and His-270 and amino acids His-102, Asp-104, and His 257 comprise the 2-His-1-carboxylate facial triads and were predicted to be involved in iron binding in RdpA and SdpA, respectively. RdpA exclusively transformed the (R) enantiomers of mecoprop [2-(4-chloro-2-methylphenoxy)propanoic acid] and dichlorprop [2-(2,4-dichlorophenoxy)propanoic acid], whereas SdpA was specific for the (S) enantiomers. The apparentKmvalues were 99 μM for (R)-mecoprop, 164 μM for (R)-dichlorprop, and 3 μM for α-ketoglutarate for RdpA and 132 μM for (S)-mecoprop, 495 μM for (S)-dichlorprop, and 20 μM for α-ketoglutarate for SdpA. Both enzymes had high apparentKmvalues for oxygen; these values were 159 μM for SdpA and >230 μM for RdpA, whose activity was linearly dependent on oxygen at the concentration range measured. Both enzymes had narrow cosubstrate specificity; only 2-oxoadipate was able to replace α-ketoglutarate, and the rates were substantially diminished. Ferrous iron was necessary for activity of the enzymes, and other divalent cations could not replace it. Although the results of growth experiments suggest that strain MH harbors a specific 2,4-dichlorophenoxyacetic acid-converting enzyme,tfdA-,tfdAα-, orcadAB-like genes were not discovered in a screening analysis in which heterologous hybridization and PCR were used.
Modification of Sexual Development and Carotene Production by Acetate and Other Small Carboxylic Acids in
Blakeslea trispora
and
Phycomyces blakesleeanus
Authors: Kuzina Vera , Cerdá-Olmedo Enrique
Published: July 2006

In
Phycomyces blakesleeanus
and
Blakeslea trispora
(order Mucorales, class Zygomycetes), sexual interaction on solid substrates leads to zygospore development and to increased carotene production (sexual carotenogenesis). Addition of small quantities of acetate, propionate, lactate, or leucine to mated cultures on minimal medium stimulated zygospore production and inhibited sexual carotenogenesis in both
Phycomyces
and
Blakeslea
. In
Blakeslea
, the threshold acetate concentration was <1 mmol/liter for both effects, and the concentrations that had one-half of the maximal effect were <2 mmol/liter for carotenogenesis and >7 mmol/liter for zygosporogenesis. The effects on
Phycomyces
were similar, but the concentrations of acetate had to be multiplied by ca. 3 to obtain the same results. Inhibition of sexual carotenogenesis by acetate occurred normally in
Phycomyces
mutants that cannot use acetate as a carbon source and in mutants whose dormant spores cannot be activated by acetate. Small carboxylic acids may be signals that, independent of their ability to trigger spore germination in
Phycomyces
, modify metabolism and development during the sexual cycle of
Phycomyces
and
Blakeslea
, uncoupling two processes that were thought to be linked and mediated by a common mechanism.
Suspension Array Analysis of 16S rRNA from Fe- and SO
4
2
-Reducing Bacteria in Uranium-Contaminated Sediments Undergoing Bioremediation
Authors: Chandler Darrell P. , Jarrell Ann E. , Roden Eric R. , Golova Julia , Chernov Boris , Schipma Matthew J. , Peacock Aaron D. , Long Philip E.
Published: July 2006

A 16S rRNA-targeted tunable bead array was developed and used in a retrospective analysis of metal- and sulfate-reducing bacteria in contaminated subsurface sediments undergoing in situ U(VI) bioremediation. Total RNA was extracted from subsurface sediments and interrogated directly, without a PCR step. Bead array validation studies with total RNA derived from 24 isolates indicated that the behavior and response of the 16S rRNA-targeted oligonucleotide probes could not be predicted based on the primary nucleic acid sequence. Likewise, signal intensity (absolute or normalized) could not be used to assess the abundance of one organism (or rRNA) relative to the abundance of another organism (or rRNA). Nevertheless, the microbial community structure and dynamics through time and space and as measured by the rRNA-targeted bead array were consistent with previous data acquired at the site, where indigenous sulfate- and iron-reducing bacteria and near neighbors of
Desulfotomaculum
were the organisms that were most responsive to a change in injected acetate concentrations. Bead array data were best interpreted by analyzing the relative changes in the probe responses for spatially and temporally related samples and by considering only the response of one probe to itself in relation to a background (reference) environmental sample. By limiting the interpretation of the data in this manner and placing it in the context of supporting geochemical and microbiological analyses, we concluded that ecologically relevant and meaningful information can be derived from direct microarray analysis of rRNA in uncharacterized environmental samples, even with the current analytical uncertainty surrounding the behavior of individual probes on tunable bead arrays.
Helicobacter anseris
sp. nov. and
Helicobacter brantae
sp. nov., Isolated from Feces of Resident Canada Geese in the Greater Boston Area
Authors: Fox James G. , Taylor Nancy S. , Howe Shelly , Tidd Michele , Xu Shilu , Paster Bruce J. , Dewhirst Floyd E.
Published: July 2006

Numbers of nonmigratory Canada geese have increased substantially in the past decade, and they have become a nuisance in some urban areas. Because of their close contact with humans in parks and areas adjacent to surface waterways, contact with their feces poses a zoonotic risk. A total of 97 geese from 10 separate geographic locales in the greater Boston area had their feces sampled for detection of
Helicobacter
spp. Identification of
Helicobacter
spp. based on 16S rRNA genus-specific helicobacter primers was noted in 39 of 97 (40.2%) DNA fecal extracts. Twenty-seven (27.8%) of these geese had helicobacters isolated from their feces. A urease-positive novel species,
Helicobacter anseris
, based on phenotypic, biochemical, and 16S rRNA analyses, was isolated from 20 geese from seven different flocks. A second, novel, urease-negative
Helicobacter
sp.,
H. brantae
, was identified in seven geese. Four geese had both novel
Helicobacter
spp. cultured from their feces. Whether these two novel helicobacters pose a zoonotic risk, similar to other enteric helicobacters (e.g.,
H. canadensis
, previously isolated from diarrheic and bacteremic humans and from geese in Europe), will require further studies.
Intrinsic Properties of So-Called Dormant Probiotic Bacteria, Determined by Flow Cytometric Viability Assays
Authors: Lahtinen Sampo J. , Ouwehand Arthur C. , Reinikainen Johanna P. , Korpela Jaakko M. , Sandholm Jouko , Salminen Seppo J.
Published: July 2006

Plate counting and four culture-independent flow cytometric assays were used to determine the viability and intrinsic properties of three probiotic strains during storage. The strains showed reduction in plate counts but were able to maintain esterase activity, intact cytoplasmic membrane, and pH gradient. The apparently uncultivable probiotic cells were active and stress resistant.
Analysis of the Life Cycle of the Soil Saprophyte
Bacillus cereus
in Liquid Soil Extract and in Soil
Authors: Vilain Sébastien , Luo Yun , Hildreth Michael B. , Brözel Volker S.
Published: July 2006

Bacillus
is commonly isolated from soils, with organisms of
Bacillus cereus
sensu lato being prevalent. Knowledge of the ecology of
B. cereus
and other
Bacillus
species in soil is far from complete. While the older literature favors a model of growth on soil-associated organic matter, the current paradigm is that
B. cereus
sensu lato germinates and grows in association with animals or plants, resulting in either symbiotic or pathogenic interactions. An in terra approach to study soil-associated bacteria is described, using filter-sterilized soil-extracted soluble organic matter (SESOM) and artificial soil microcosms (ASM) saturated with SESOM.
B. cereus
ATCC 14579 displayed a life cycle, with the ability to germinate, grow, and subsequently sporulate in both the liquid SESOM extract and in ASM inserted into wells in agar medium. Cells grew in liquid SESOM without separating, forming multicellular structures that coalesced to form clumps and encasing the ensuing spores in an extracellular matrix.
Bacillus
was able to translocate from the point of inoculation through soil microcosms as shown by the emergence of outgrowths on the surrounding agar surface. Microscopic inspection revealed bundles of parallel chains inside the soil. The motility inhibitor
l
-ethionine failed to suppress outgrowth, ruling out translocation by a flagellar-mediated mechanism such as swimming or swarming.
Bacillus subtilis
subsp.
subtilis
Marburg and four
Bacillus
isolates taken at random from soils also displayed a life cycle in SESOM and ASM and were all able to translocate through ASM, even in presence of
l
-ethionine. These data indicate that
B. cereus
is a saprophytic bacterium that is able to grow in soil and furthermore that it is adapted to translocate by employing a multicellular mode of growth.
Simian Immunodeficiency Virus (SIV)/Immunoglobulin G Immune Complexes in SIV-Infected Macaques Block Detection of CD16 but Not Cytolytic Activity of Natural Killer Cells
Authors: Wei Qing , Stallworth Jackie W. , Vance Patricia J. , Hoxie James A. , Fultz Patricia N.
Published: July 2006

Natural killer cells are components of the innate immune system that play an important role in eliminating viruses and malignant cells. Using simian immunodeficiency virus (SIV) infection of macaques as a model, flow cytometry revealed a gradual loss of CD16+NK cell numbers that was associated with disease progression. Of note, the apparent loss of NK cells was detected in whole-blood samples but not in isolated peripheral blood mononuclear cells (PBMC), suggesting that an inhibitor(s) of the antibody used to detect CD16, the low-affinity immunoglobulin G (IgG) receptor, was present in blood but was removed during PBMC isolation. (Actual decreases in CD16+cell numbers in PBMC generally were not detected until animals became lymphopenic.) The putative decrease in CD16+cell numbers in whole blood correlated with increasing SIV-specific antibody titers and levels of plasma virion RNA. With the addition of increasing amounts of plasma from progressor, but not nonprogressor, macaques to PBMC from an uninfected animal, the apparent percentage of CD16+cells and the mean fluorescence intensity of antibodies binding to CD16 declined proportionately. A similar decrease was observed with the addition of monomeric IgG (mIgG) and IgG immune complexes (IgG-ICs) purified from the inhibitory plasma samples; some of the ICs contained SIV p27gagantigen and/or virions. Of interest, addition of purified IgG/IgG-ICs to NK cell lytic assays did not inhibit killing of K562 cells. These results indicate that during progressive SIV and, by inference, human immunodeficiency virus disease, CD16+NK cell numbers can be underestimated, or the cells not detected at all, when one is using a whole-blood fluorescence-activated cell sorter assay and a fluorochrome-labeled antibody that can be blocked by mIgG or IgG-ICs. Although this blocking had no apparent effect on NK cell activity in vitro, the in vivo effects are unknown.
Different Levels of Immunogenicity of Two Strains of Fowlpox Virus as Recombinant Vaccine Vectors Eliciting T-Cell Responses in Heterologous Prime-Boost Vaccination Strategies
Authors: Cottingham Matthew G. , van Maurik Andre , Zago Manola , Newton Angela T. , Anderson Richard J. , Howard M. Keith , Schneider Jörg , Skinner Michael A.
Published: July 2006

The FP9 strain ofFowlpox virushas been described as a more immunogenic recombinant vaccine vector than the Webster FPV-M (FPW) strain (R. J. Anderson et al., J. Immunol. 172:3094-3100, 2004). This study expands the comparison to include two separate recombinant antigens and multiple, rather than single, independent viral clones derived from the two strains. Dual-poxvirus heterologous prime-boost vaccination regimens using individual clones of recombinant FP9 or FPW in combination with recombinant modifiedVaccinia virusAnkara expressing the same antigen were evaluated for their ability to elicit T-cell responses against recombinant antigens fromPlasmodium berghei(circumsporozoite protein) or human immunodeficiency virus type 1 (a Gag-Pol-Nef fusion protein). Gamma interferon enzyme-linked immunospot assay and fluorescence-activated cell sorting assays of the responses to specific epitopes confirmed the approximately twofold-greater cellular immunogenicity of FP9 compared to FPW, when given as the priming or boosting immunization. Equality of transgene expression in mouse cells infected with the two strains in vitro was verified by Western blotting. Directed partial sequence analysis and PCR analysis of FPW and comparison to available whole-genome sequences revealed that many loci that are mutated in the highly attenuated and culture-adapted FP9 strain are wild type in FPW, including the seven multikilobase deletions. These “passage-specific” alterations are hypothesized to be involved in determining the immunogenicity ofFowlpox virusas a recombinant vaccine vector.
Tuberculosis in Elephants: Antibody Responses to Defined Antigens of
Mycobacterium tuberculosis
, Potential for Early Diagnosis, and Monitoring of Treatment
Authors: Lyashchenko Konstantin P. , Greenwald Rena , Esfandiari Javan , Olsen John H. , Ball Ray , Dumonceaux Genevieve , Dunker Freeland , Buckley Carol , Richard Michael , Murray Suzan , Payeur Janet B. , Andersen Peter , Pollock John M. , Mikota Susan , Miller Michele , Sofranko Denise , Waters W. Ray
Published: July 2006

Tuberculosis (TB) in elephants is a re-emerging zoonotic disease caused primarily by
Mycobacterium tuberculosis
. Current diagnosis relies on trunk wash culture, the only officially recognized test, which has serious limitations. Innovative and efficient diagnostic methods are urgently needed. Rapid identification of infected animals is a crucial prerequisite for more effective control of TB, as early diagnosis allows timely initiation of chemotherapy. Serology has diagnostic potential, although key antigens have not been identified and optimal immunoassay formats are not established. To characterize the humoral responses in elephant TB, we tested 143 serum samples collected from 15 elephants over time. These included 48 samples from five culture-confirmed TB cases, of which four were in Asian elephants infected with
M. tuberculosis
and one was in an African elephant with
Mycobacterium bovis
. Multiantigen print immunoassay (MAPIA) employing a panel of 12 defined antigens was used to identify serologic correlates of active disease. ESAT-6 was the immunodominant antigen recognized in elephant TB. Serum immunoglobulin G antibodies to ESAT-6 and other proteins were detected up to 3.5 years prior to culture of
M. tuberculosis
from trunk washes. Antibody levels to certain antigens gradually decreased in response to antitubercular therapy, suggesting the possibility of treatment monitoring. In addition to MAPIA, serum samples were evaluated with a recently developed rapid test (RT) based on lateral flow technology (ElephantTB STAT-PAK). Similarly to MAPIA, infected elephants were identified using the RT up to 4 years prior to positive culture. These findings demonstrate the potential for TB surveillance and treatment monitoring using the RT and MAPIA, respectively.
Evaluation of Two Enzyme Immunoassays for Detection of Immunoglobulin G Antibodies to Mumps Virus
Authors: Backhouse J. L. , Gidding H. F. , McIntyre P. B. , Gilbert G. L.
Published: July 2006

To determine suitability for national serosurveys, we compared two commercial enzyme-linked immunosorbent assays (ELISAs) for mumps antibody, Enzygnost Anti-Parotitis-Virus/IgG (which uses a whole-virus antigen) and Microimmune Mumps IgG Screen ELISA (which uses a recombinant nucleoprotein antigen), by testing 1,915 opportunistically collected sera submitted to diagnostic laboratories across Australia in 1997 to 1998. The proportion of positive results increased with age in both ELISAs but was significantly higher with the Microimmune than with the Enzygnost ELISA overall (88% versus 63%;
P
< 0.01) and in all age groups. However, the proportion of equivocal results was significantly higher with the Enzygnost than with the Microimmune ELISA (9% versus 4%;
P
< 0.01). Of the 572 sera with discrepant or equivocal results, 508 had sufficient sample remaining to perform the neutralization test (NT). A proportion with concordant results in both ELISAs were also tested by the NT. For sera with discrepant results, there was significantly better agreement between the NT and Microimmune than between the NT and Enzygnost (310/444 [70%] versus 135/348 [39%];
P
< 0.01). Of 64 sera with equivocal Microimmune results, 45 (70%) were positive in the NT compared with 140 of 160 (88%) equivocal Enzygnost results (
P
< 0.01). Compared with the NT, the Microimmune ELISA is more sensitive (96% versus 80%) but apparently less specific (36% versus 85%) than the Enzygnost ELISA. However, this is likely to be due to the generally lower sensitivity of the NT, since the Microimmune results reflect expected seroprevalence, based on vaccine uptake in the age groups studied. We conclude that the Microimmune ELISA is a more appropriate assay than the Enzygnost ELISA for estimation of mumps seroprevalence.
Evaluation of a PCR Melting Profile Technique for Bacterial Strain Differentiation
Authors: Krawczyk Beata , Samet Alfred , Leibner Justyna , Śledzińska Anna , Kur Józef
Published: July 2006

In search of an effective DNA typing technique for hospital epidemiology use, the performance and convenience of a PCR melting profile (PCR MP) technique based on using low denaturation temperatures during ligation-mediated PCR (LM PCR) of bacterial DNA was tested. A number of
Escherichia coli
isolates from patients of the Clinical Hospital in Gdańsk, Poland, were examined. We found that the PCR MP technique is a rapid method that offers good discriminatory power and excellent reproducibility and may be applied for epidemiological studies. The usefulness of the PCR MP for molecular typing was compared with the pulsed-field gel electrophoresis method, which is currently considered the gold standard for epidemiological studies of isolates recovered from patients and the environment. Clustering of PCR MP fingerprinting data matched pulsed-field gel electrophoresis data. The features of the PCR MP technique are discussed in comparison with conventional methods. Data presented here demonstrate the complexity of the epidemiological situation concerning
E. coli
that may occur in a hospital.
Septic Arthritis Caused by
Mycobacterium kansasii
in a Prosthetic Knee Joint
Authors: Neuberger A. , Sprecher H. , Oren I.
Published: July 2006

Mycobacterium kansasii
is a relatively common cause of nontuberculous mycobacterial pulmonary infection. Septic arthritis caused by
Mycobacterium kansasii
, on the other hand, is rare. Reported here for the first time is the case of an 82-year-old patient with an infection of a prosthetic knee joint with
Mycobacterium kansasii
.
Clostridium aldenense
sp. nov. and
Clostridium citroniae
sp. nov. Isolated from Human Clinical Infections
Authors: Warren Yumi A. , Tyrrell Kerin L. , Citron Diane M. , Goldstein Ellie J. C.
Published: July 2006

One hundred eight isolates were previously identified in our laboratory as
Clostridium clostridioforme
by colonial and cellular morphology, as well as biochemical tests. Recent studies have indicated that there are actually three different species in this
C. clostridioforme
group:
C. hathewayi
,
C. bolteae
, and
C. clostridioforme
. Our isolates were reexamined using biochemical and enzymatic tests and molecular methods. Forty-six isolates were reidentified as
C. hathewayi
, 34 as
C. bolteae
, five as
C. clostridioforme
, and one as
C. symbiosum
. Twenty-two strains were identified only to the genus level by 16S rRNA gene sequencing, and although they are microscopically and morphologically indistinguishable from the above-mentioned three species, they are phenotypically different and only 96 to 98% similar by gene sequencing. Twenty of these 22 strains were indole positive and formed two novel species. We propose
Clostridium aldenense
sp. nov. and
Clostridium citroniae
sp. nov. as names for these new species. They are differentiated from each other by results for raffinose, rhamnose, α-galactosidase, and β-galactosidase: positive, negative, positive, and positive, respectively, for the former species and negative, positive, negative, and negative, respectively, for the latter species. The type strain of
C. aldenense
is RMA 9741 (ATCC BAA-1318; CCUG 52204), and the type strain of
C. citroniae
is RMA 16102 (ATCC BAA-1317; CCUG 52203).
Use of In Vitro Assays To Determine Effects of Human Serum on Biological Characteristics of
Acanthamoeba castellanii
Authors: Sissons James , Alsam Selwa , Stins Monique , Rivas Antonio Ortega , Morales Jacob Lorenzo , Faull Jane , Khan Naveed Ahmed
Published: July 2006

Normal human serum inhibits
Acanthamoeba
(encephalitis isolate) binding to and cytotoxicity of human brain microvascular endothelial cells, which constitute the blood-brain barrier. Zymographic assays revealed that serum inhibits extracellular protease activities of acanthamoebae. But it is most likely that inhibition of specific properties of acanthamoebae is a consequence of the initial amoebicidal-amoebistatic effects induced by serum. For example, serum exhibited amoebicidal effects; i.e., up to 50% of the exposed trophozoites were killed. The residual subpopulation, although viable, remained static over longer incubations. Interestingly, serum enhanced the phagocytic ability of acanthamoebae, as measured by bacterial uptake. Overall, our results demonstrate that human serum has inhibitory effects on
Acanthamoeba
growth and viability, protease secretions, and binding to and subsequent cytotoxicity for brain microvascular endothelial cells. Conversely,
Acanthamoeba
phagocytosis was stimulated by serum.
Comparisons of Community-Associated Methicillin-Resistant
Staphylococcus aureus
(MRSA) and Hospital-Associated MSRA Infections in Sacramento, California
Authors: Huang Hsin , Flynn Neil M. , King Jeff H. , Monchaud Caroline , Morita Margaret , Cohen Stuart H.
Published: July 2006

Methicillin-resistant
Staphylococcus aureus
(MRSA) has long been a common pathogen in healthcare facilities, but in the past decade, it has emerged as a problematic pathogen in the community setting as well. A retrospective case series study of patients from whom MRSA was isolated from December 1, 2003, through May 31, 2004, was conducted at the University of California, Davis, Medical Center. Patient data were collected from electronic medical records and traditional chart reviews to determine whether MRSA acquisition was likely to have been in the community or in the hospital. Antimicrobial susceptibility testing and pulsed-field gel electrophoresis (PFGE) were performed for all confirmed isolates. Skin and soft tissue were the most common infection sites for all MRSA patients. Among the 283 MRSA infections, 127 (44.9%) were defined as community-associated (CA)-MRSA. Ninety-six percent of the CA-MRSA isolates were susceptible to clindamycin. Double-disk diffusion tests were performed to examine inducible clindamycin resistance by erythromycin induction on both CA and hospital-associated (HA) clindamycin-susceptible and erythromycin-resistant isolates. Ten percent (17 of 183) were positive. Most CA-MRSA isolates were identified by PFGE as a unique strain, genotype USA300, which was not genetically related to the predominant genotype, USA100, in the HA-MRSA isolates. Injecting drug users accounted for 49% of CA-MRSA infections but only 19% of the HA-MRSA infections (odds ratio, 4.2; 95% confidence interval, 2.4 to 7.4). Our study shows that a single clone of CA-MRSA accounts for the majority of infections. This strain originated in the community and is not related to MRSA strains from healthcare settings. Injecting drug users could be a major reservoir for CA-MRSA transmission.
Molecular Evolution of Metallo-β-Lactamase-Producing
Pseudomonas aeruginosa
in a Nosocomial Setting of High-Level Endemicity
Authors: Lagatolla Cristina , Edalucci Elisabetta , Dolzani Lucilla , Riccio Maria Letizia , De Luca Filomena , Medessi Erica , Rossolini Gian Maria , Tonin Enrico Angelo
Published: July 2006

An outbreak of multidrug-resistant
Pseudomonas aeruginosa
strains producing VIM-type metallo-β-lactamases (MBLs) has occurred in an Italian hospital since 2000 (C. Lagatolla, E. A. Tonin, C. Monti-Bragadin, L. Dolzani, F. Gombac, C. Bearzi, E. Edalucci, F. Gionechetti, and G. M. Rossolini, Emerg. Infect. Dis. 10:535-538, 2004). In this work, using molecular methods, we characterized 128 carbapenem-resistant isolates (including 98 VIM-positive isolates) collected from that hospital from 2000 to 2002 to investigate the dynamics of the dissemination of MBL producers in the clinical setting. Genotyping by random amplification of polymorphic DNA and pulsed-field gel electrophoresis showed that most VIM-positive isolates belonged to two different clonal lineages, producing either a VIM-1- or a VIM-2-like MBL, whose ancestors were detected for the first time in the hospital in 1999, suggesting that clonal expansion played a predominant role in the dissemination of these isolates. The 86 clonally related isolates carrying a
bla
VIM-1
-like gene on an In70-like integron were clearly related to a VIM-1-positive
P. aeruginosa
clone circulating in various Italian hospitals since the late 1990s. VIM-negative
P. aeruginosa
strains related to the VIM-1-positive clone were detected during the same period, suggesting that the latter strain was derived from a clonal lineage already circulating in the hospital. In the VIM-2-like positive clone, the MBL gene was carried by an unusual class 1 integron, named In71, lacking the 3′ conserved sequence region typical of
sul1
-associated integrons. A different class 1 integron with an original structure carrying a
bla
VIM-2
determinant, named In74, was detected in a sporadic isolate. A retrospective investigation did not reveal the presence of strains related to any of the VIM-producing isolates earlier than 1997.
Mutations Prevalent among Rifampin- and Isoniazid-Resistant
Mycobacterium tuberculosis
Isolates from a Hospital in Vietnam
Authors: Caws M. , Duy Phan Minh , Tho Dau Quang , Lan Nguyen Thi Ngoc , Hoa Dai Viet , Farrar Jeremy
Published: July 2006

Vietnam is ranked 13th among the WHO list of 22 high-burden countries, based upon estimated total number of tuberculosis cases. Despite having a model national tuberculosis program, consistently achieving and exceeding WHO targets for detection and cure, drug-resistant and multidrug-resistant tuberculosis cases continue to rise. Rapid multidrug-resistant tests applicable in this setting, coupled with effective treatment regimens, would be a useful tool in reversing this trend, allowing early identification of patients with multidrug-resistant tuberculosis and avoiding resistance-amplifying regimens. Sequencing of consecutive isolates identified by the National Tuberculosis Program showed 89% of isoniazid-resistant isolates could be detected by targeting just 2 codons,
katG
315 and −15C→T in the
inhA
promoter, while rifampin resistance will be more complex to detect, with many different mutation and insertion events in
rpoB
. The most prevalent rifampin resistance-conferring mutations, as in other countries, were in
rpoB
codons 531 (43%), 526 (31%), and 516 (15%). However, a hybridization-based resistance test with probes targeting the 5 most common mutations would only detect 78% of rifampin-resistant isolates. Overall, these data suggest that rifampin resistance may be used as a surrogate marker for multidrug-resistant tuberculosis and that a sensitivity of between 70 to 80% may be possible for rapid molecular detection of multidrug-resistant tuberculosis in this setting.
Molecular Epidemiologic Identification of
Escherichia coli
Genes That Are Potentially Involved in Movement of the Organism from the Intestinal Tract to the Vagina and Bladder
Authors: Xie Jingping , Foxman Betsy , Zhang Lixin , Marrs Carl F.
Published: July 2006

A first step in urinary tract infection (UTI) pathogenesis in the otherwise healthy host is the movement of uropathogenic
Escherichia coli
from the intestinal tract to the urinary tract. We conducted a genomic subtraction to isolate genetic regions associated with this movement. A representative UTI isolate present in the rectum, vagina, and bladder of a woman with UTI was chosen as the tester; the driver was a phylogenetically distant rectal isolate (based on pulsed-field gel electrophoresis analysis) with a profile of uropathogenic virulence genes similar to that of the tester. Tester-specific regions identified by the subtraction were screened, using DNA dot blot hybridization, against a collection of 88 uropathogens isolated from the rectum, urine, and/or vagina of women with UTIs and 54
E. coli
isolates from the same women that were found only in the rectum. Twelve genetic regions occurred more often in multisite isolates than in rectal site-only isolates. Eleven of these 12 genetic regions are homologous to regions in the sequenced uropathogenic
E. coli
CFT073 strain.
Use of BBL CHROMagar MRSA Medium for Identification of Methicillin-Resistant
Staphylococcus aureus
Directly from Blood Cultures
Authors: Pape John , Wadlin Jill , Nachamkin Irving
Published: July 2006

We evaluated the ability of BBL CHROMagar MRSA medium (Becton Dickinson, Sparks, MD) to identify methicillin-resistant
Staphylococcus aureus
(MRSA) directly upon subculture from positive blood culture bottles. There were 124 MRSA isolates recovered from blood cultures in the study. BBL CHROMagar MRSA medium was highly sensitive (97.6% [121/124] at 18 to 24 h of incubation and 100% [124/124] at 48 h) and 99.9% specific for identifying MRSA from positive blood cultures.