bluuebunny/crossref_metadata_2025 huggingface dataset:
Urkinase: Structure of Acetate Kinase, a Member of the ASKHA Superfamily of Phosphotransferases
Authors: Buss Kathryn A. , Cooper David R. , Ingram-Smith Cheryl , Ferry James G. , Sanders David Avram , Hasson Miriam S.
Published: July 2002

Acetate kinase, an enzyme widely distributed in the
Bacteria
and
Archaea
domains, catalyzes the phosphorylation of acetate. We have determined the three-dimensional structure of
Methanosarcina thermophila
acetate kinase bound to ADP through crystallography. As we previously predicted, acetate kinase contains a core fold that is topologically identical to that of the ADP-binding domains of glycerol kinase, hexokinase, the 70-kDa heat shock cognate (Hsc70), and actin. Numerous charged active-site residues are conserved within acetate kinases, but few are conserved within the phosphotransferase superfamily. The identity of the points of insertion of polypeptide segments into the core fold of the superfamily members indicates that the insertions existed in the common ancestor of the phosphotransferases. Another remarkable shared feature is the unusual, epsilon conformation of the residue that directly precedes a conserved glycine residue (Gly-331 in acetate kinase) that binds the α-phosphate of ADP. Structural, biochemical, and geochemical considerations indicate that an acetate kinase may be the ancestral enzyme of the ASKHA (acetate and sugar kinases/Hsc70/actin) superfamily of phosphotransferases.
In Vivo Expression from the RpoS-Dependent P1 Promoter of the Osmotically Regulated
proU
Operon in
Escherichia coli
and
Salmonella enterica
Serovar Typhimurium: Activation by
rho
and
hns
Mutations and by Cold Stress
Authors: Rajkumari K. , Gowrishankar J.
Published: July 2002

Unlike the ς
70
-controlled P2 promoter for the osmotically regulated
proU
operon of
Escherichia coli
and
Salmonella enterica
serovar Typhimurium, the ς
s
-controlled P1 promoter situated further upstream appears not to contribute to expression of the
proU
structural genes under ordinary growth conditions. For
S
.
enterica proU
P1, there is evidence that promoter crypticity is the result of a transcription attenuation phenomenon which is relieved by the deletion of a 22-base C-rich segment in the transcript. In this study, we have sought to identify growth conditions and
trans
-acting mutations which activate in vivo expression from
proU
P1. The cryptic
S
.
enterica proU
P1 promoter was activated, individually and additively, in a
rho
mutant (which is defective in the transcription termination factor Rho) as well as by growth at 10°C. The
E
.
coli proU
P1 promoter was also cryptic in constructs that carried 1.2 kb of downstream
proU
sequence, and in these cases activation of in vivo expression was achieved either by a
rho
mutation during growth at 10°C or by an
hns
null mutation (affecting the nucleoid protein H-NS) at 30°C. The
rho
mutation had no effect at either 10 or 30°C on in vivo expression from two other ς
s
-controlled promoters tested, those for
osmY
and
csiD
. In cells lacking the RNA-binding regulator protein Hfq, induction of
E
.
coli proU
P1 at 10°C and by
hns
mutation at 30°C was still observed, although the
hfq
mutation was associated with a reduction in the absolute levels of P1 expression. Our results suggest that expression from
proU
P1 is modulated both by nucleoid structure and by Rho-mediated transcription attenuation and that this promoter may be physiologically important for
proU
operon expression during low-temperature growth.
Structural and Immunochemical Characterization of the Lipooligosaccharides Expressed byNeisseria subflava44
Authors: Tong Yanhong , Reinhold Vernon , Reinhold Bruce , Brandt Brenda , Stein Daniel C.
Published: July 2002

Neisserial lipooligosaccharides (LOSs) are a family of complex cell surface glycolipids. We used mass spectrometry techniques (electrospray ionization, collision-induced dissociation, and multiple step), combined with fluorophore-assisted carbohydrate electrophoresis monosaccharide composition analysis, to determine the structure of the two low-molecular-mass LOS molecules (LOSI and LOSII) expressed byNeisseria subflava44. We determined that LOSI contains one glucose on both the α and β chains. LOSII is structurally related to LOSI and differs from it by the addition of a hexose (either glucose or galactose) on the α chain. LOSI and LOSII were able to bind monoclonal antibody (MAb) 25-1-LC1 when analyzed by Western blotting experiments. We used a set of genetically definedNeisseria gonorrhoeaemutants that expressed single defined LOS epitopes and a group ofNeisseria meningitidisstrains that expresses chemically defined LOS components to determine the structures recognized by MAb 25-1-LC1. We found that extensions onto the β-chain glucose of LOSI block the recognition by this MAb, as does further elongation from the LOSII α chain. The LOSI structure was determined to be the minimum structure that is recognized by MAb 25-1-LC1.
Formation of Chromosomal Tandem Arrays of the SXT Element and R391, Two Conjugative Chromosomally Integrating Elements That Share an Attachment Site
Authors: Hochhut Bianca , Beaber John W. , Woodgate Roger , Waldor Matthew K.
Published: July 2002

The SXT element, a conjugative, self-transmissible, integrating element (a constin) originally derived from a
Vibrio cholerae
O139 isolate from India, and IncJ element R391, originally derived from a South African
Providencia rettgeri
isolate, were found to be genetically and functionally related. Both of these constins integrate site specifically into the
Escherichia coli
chromosome at an identical attachment site within the 5′ end of
prfC
. They encode nearly identical integrases, which are required for chromosomal integration, excision, and extrachromosomal circularization of these elements, and they have similar
tra
genes. Therefore, these closely related constins have virtually identical mechanisms for chromosomal integration and dissemination. The presence of either element in a recipient cell did not significantly reduce its ability to acquire the other element, indicating that R391 and SXT do not encode surface exclusion determinants. In cells harboring both elements, SXT and R391 were integrated in tandem fashion on the chromosome, and homologous recombination appeared to play little or no role in the formation of these arrays. Interference between R391 and SXT was detected by measuring the frequency of loss of an unselected resident element upon introduction of a second selected element. In these assays, R391 was found to have a stronger effect on SXT stability than vice versa. The level of expression and/or activity of the donor and recipient integrases may play a role in the interference between these two related constins.
Regulation of Metalloprotease Gene Expression in
Vibrio vulnificus
by a
Vibrio harveyi
LuxR Homologue
Authors: Shao Chung-Ping , Hor Lien-I
Published: July 2002

Expression of the
Vibrio vulnificus
metalloprotease gene,
vvp
, was turned up rapidly when bacterial growth reached the late log phase. A similar pattern of expression has been found in the metalloprotease gene of
Vibrio cholerae
, and this has been shown to be regulated by a
Vibrio harveyi
LuxR-like transcriptional activator. To find out whether a LuxR homologue exists in
V. vulnificus
, a gene library of this organism was screened by colony hybridization using a probe derived from a sequence that is conserved in various
luxR
-like genes of vibrios. A gene containing a 618-bp open reading frame was identified and found to be identical to the
smcR
gene of
V. vulnificus
reported previously. An isogenic SmcR-deficient (RD) mutant was further constructed by an in vivo allelic exchange technique. This mutant exhibited an extremely low level of
vvp
transcription compared with that of the parent strain. On the other hand, the cytolysin gene,
vvhA
, was expressed at a higher level in the RD mutant than in the parent strain during the log phase of growth. These data suggested that SmcR might not only be a positive regulator of the protease gene but might also be involved in negative regulation of the cytolysin gene. Virulence of the RD mutant in either normal or iron-overloaded mice challenged by intraperitoneal injection was comparable to that of the parent strain, indicating that SmcR is not required for
V. vulnificus
virulence in mice.
Physiological Basis for Conservation of the Signal Recognition Particle Targeting Pathway in
Escherichia coli
Authors: Bernstein Harris D. , Hyndman Janine B.
Published: July 2002

The
Escherichia coli
signal recognition particle (SRP) is a ribonucleoprotein complex that targets nascent inner membrane proteins (IMPs) to transport sites in the inner membrane (IM). Since SRP depletion only partially inhibits IMP insertion under some growth conditions, however, it is not clear why the particle is absolutely essential for viability. Insights into this question emerged from experiments in which we analyzed the physiological consequences of reducing the intracellular concentration of SRP below the wild-type level. We found that even moderate SRP deficiencies that have little effect on cell growth led to the induction of a heat shock response. Genetic manipulations that suppress the heat shock response were lethal in SRP-deficient cells, indicating that the elevated synthesis of heat shock proteins plays an important role in maintaining cell viability. Although it is conceivable that the heat shock response serves to increase the capacity of cells to target IMPs via chaperone-based mechanisms, SRP-deficient cells did not show an increased dependence on either GroEL or DnaK. By contrast, the heat shock-regulated proteases Lon and ClpQ became essential for viability when SRP levels were reduced. These results suggest that the heat shock response protects SRP-deficient cells by increasing their capacity to degrade mislocalized IMPs. Consistent with this notion, a model IMP that was mislocalized in the cytoplasm as the result of SRP depletion appeared to be more stable in a
Δlon ΔclpQ
strain than in control cells. Taken together, the data provide direct evidence that SRP is essential in
E. coli
and possibly conserved throughout prokaryotic evolution as well partly because efficient IMP targeting prevents a toxic accumulation of aggregated proteins in the cytoplasm.
Interruption of thecydBLocus inBrucella abortusAttenuates Intracellular Survival and Virulence in the Mouse Model of Infection
Authors: Endley Seema , McMurray David , Ficht Thomas A.
Published: July 2002

Brucellosis is characterized by abortion in ruminants and a protracted undulant fever in humans, which often results in severe pathological manifestations. Scant information exists about the molecular mechanisms employed byBrucella abortusto combat host defenses or to persist and replicate within host cells. Transposon (Tn5) mutagenesis ofB. abortusand the subsequent screening of mutants for sensitivity to killing in murine macrophages and in the mouse model led to the identification of mutants which were severely attenuated for intracellular survival. One group of mutants was interrupted incydB, a gene that is part of thecydABoperon encoding cytochromebdoxidase, which catalyzes an alternate terminal electron transport step in bacterial respiration. The elevated affinity for molecular oxygen of this enzyme inEscherichia colihas suggested that it is involved in the protection of sensitive enzymatic activities such as those of hydrogenases and nitrogenases from damage.B. abortus cydB::Tn5strains exhibited heightened sensitivity to the respiratory inhibitors zinc and azide, highly reactive oxygen species such as hydrogen peroxide, low pH, and attenuated virulence in the mouse model of infection. Virulence was restored by an intact copy ofcydABor byB. abortusgenes encoding the oxidative radical-scavenging enzyme Cu/Zn superoxide dismutase or catalase. These results suggest a bifunctional role for the products of thecydABoperon, both in preventing the buildup of oxidative free radicals and in detoxifying the intracellular compartment, thus indicating the importance of these products in preventing intracellular destruction. Intracellular conditions that favor expression of thecydABoperon are under investigation and may be linked to the acid sensitivity also observed in this strain.
Characterization of the
Plesiomonas shigelloides
Genes Encoding the Heme Iron Utilization System
Authors: Henderson D. P. , Wyckoff E. E. , Rashidi C. E. , Verlei H. , Oldham A. L.
Published: July 2002

Plesiomonas shigelloides
is a gram-negative pathogen which can utilize heme as an iron source. In previous work,
P. shigelloides
genes which permitted heme iron utilization in a laboratory strain of
Escherichia coli
were isolated. In the present study, the cloned
P. shigelloides
sequences were found to encode ten potential heme utilization proteins: HugA, the putative heme receptor; TonB and ExbBD; HugB, the putative periplasmic binding protein; HugCD, the putative inner membrane permease; and the proteins HugW, HugX, and HugZ. Three of the genes,
hugA, hugZ
, and
tonB
, contain a Fur box in their putative promoters, indicating that the genes may be iron regulated. When the
P. shigelloides
genes were tested in
E. coli
K-12 or in a heme iron utilization mutant of
P. shigelloides, hugA
, the TonB system genes, and
hugW, hugX
, or
hugZ
were required for heme iron utilization. When the genes were tested in a
hemA entB
mutant of
E. coli, hugWXZ
were not required for utilization of heme as a porphyrin source, but their absence resulted in heme toxicity when the strains were grown in media containing heme as an iron source.
hugA
could replace the
Vibrio cholerae hutA
in a heme iron utilization assay, and
V. cholerae hutA
could complement a
P. shigelloides
heme utilization mutant, suggesting that HugA is the heme receptor. Our analyses of the TonB system of
P. shigelloides
indicated that it could function in
tonB
mutants of both
E. coli
and
V. cholerae
and that it was similar to the
V. cholerae
TonB1 system in the amino acid sequence of the proteins and in the ability of the system to function in high-salt medium.
Regulation of theacuFGene, Encoding Phosphoenolpyruvate Carboxykinase in the Filamentous FungusAspergillus nidulans
Authors: Hynes Michael J. , Draht Oliver W. , Davis Meryl A.
Published: July 2002

Phosphoenolpyruvate carboxykinase (PEPCK) is a key enzyme required for gluconeogenesis when microorganisms grow on carbon sources metabolized via the tricarboxylic acid (TCA) cycle.Aspergillus nidulans acuFmutants isolated by their inability to use acetate as a carbon source specifically lack PEPCK. TheacuFgene has been cloned and shown to encode a protein with high similarity to PEPCK from bacteria, plants, and fungi. The regulation ofacuFexpression has been studied by Northern blotting and by the construction oflacZfusion reporters. Induction by acetate is abolished in mutants unable to metabolize acetate via the TCA cycle, and induction by amino acids metabolized via 2-oxoglutarate is lost in mutants unable to form 2-oxoglutarate. Induction by acetate and proline is not additive, consistent with a single mechanism of induction. Malate and succinate result in induction, and it is proposed that PEPCK is controlled by a novel mechanism of induction by a TCA cycle intermediate or derivative, thereby allowing gluconeogenesis to occur during growth on any carbon source metabolized via the TCA cycle. It has been shown that thefacBgene, which mediates acetate induction of enzymes specifically required for acetate utilization, is not directly involved in PEPCK induction. This is in contrast toSaccharomyces cerevisiae, where Cat8p and Sip4p, homologs of FacB, regulate PEPCK as well as the expression of other genes necessary for growth on nonfermentable carbon sources in response to the carbon source present. This difference in the control of gluconeogenesis reflects the ability ofA.nidulansand other filamentous fungi to use a wide variety of carbon sources in comparison withS.cerevisiae. TheacuFgene was also found to be subject to activation by the CCAAT binding protein AnCF, a protein homologous to theS.cerevisiaeHap complex and the mammalian NFY complex.
The High-Molecular-Weight Cytochrome
c
Cyc2 of
Acidithiobacillus ferrooxidans
Is an Outer Membrane Protein
Authors: Yarzábal Andrés , Brasseur Gaël , Ratouchniak Jeanine , Lund Karen , Lemesle-Meunier Danielle , DeMoss John A. , Bonnefoy Violaine
Published: July 2002

A high-molecular-weight
c
-type cytochrome, Cyc2, and a putative 22-kDa
c
-type cytochrome were detected in the membrane fraction released during spheroplast formation from
Acidithiobacillus ferrooxidans
. This fraction was enriched in outer membrane components and devoid of cytoplasmic membrane markers. The genetics, as well as the subcellular localization of Cyc2 at the outer membrane level, therefore make it a prime candidate for the initial electron acceptor in the respiratory pathway between ferrous iron and oxygen.
Mutant Analysis and Cellular Localization of the AlgI, AlgJ, and AlgF Proteins Required for O Acetylation of Alginate in
Pseudomonas aeruginosa
Authors: Franklin Michael J. , Ohman Dennis E.
Published: July 2002

Alginate is an extracellular polysaccharide produced by mucoid strains of
Pseudomonas aeruginosa
that are typically isolated from the pulmonary tracts of chronically infected cystic fibrosis patients. Alginate is a linear polymer of
d
-mannuronate and
l
-guluronate with
O
-acetyl ester linkages on the O-2 and/or O-3 position of the mannuronate residues. The presence of
O
-acetyl groups plays an important role in the ability of the polymer to act as a virulence factor, and the
algF
,
algJ
, and
algI
genes are known to be essential for the addition of
O
-acetyl groups to alginate. To better understand the mechanism of O acetylation of alginate, the cellular locations of the AlgI, AlgJ, and AlgF proteins were determined. For these studies, defined nonpolar
algI
,
algJ
, and
algF
deletion mutants of
P. aeruginosa
strain FRD1 were constructed, and each mutant produced alginate lacking
O
-acetyl groups. Expression of
algI
,
algJ
, or
algF
in
trans
in the corresponding mutant complemented each O acetylation defect. Random
phoA
(alkaline phosphatase [AP] gene) fusions to
algF
,
algJ
, and
algI
were constructed. All in-frame fusions to
algF
and
algJ
had AP activity, indicating that both AlgF and AlgJ were exported to the periplasm. Immunoblot analysis of spheroplasts and periplasmic fractions showed that AlgF was released with the periplasmic contents but that AlgJ remained with the spheroplast fraction. An N-terminal sequence analysis of AlgJ showed that its putative AlgJ signal peptide was not cleaved, suggesting that AlgJ is anchored to the cytoplasmic membrane by its uncleaved signal peptide. AP gene fusions were also used to map the membrane topology of AlgI, and the results suggest that it is an integral membrane protein with seven transmembrane domains. These results suggest that AlgI-AlgJ-AlgF may form a complex in the membrane that is the reaction center for O acetylation of alginate.
Spa32 Regulates a Switch in Substrate Specificity of the Type III Secreton of
Shigella flexneri
from Needle Components to Ipa Proteins
Authors: Magdalena Juana , Hachani Abderrahman , Chamekh Mustapha , Jouihri Noureddine , Gounon Pierre , Blocker Ariel , Allaoui Abdelmounaaïm
Published: July 2002

Type III secretion systems (TTSS) are essential virulence determinants of many gram-negative bacteria and serve, upon physical contact with target cells, to translocate bacterial proteins directly across eukaryotic cell membranes. The
Shigella
TTSS is encoded by the
mxi/spa
loci located on its virulence plasmid. By electron microscopy secretons are visualized as tripartite with an external needle, a transmembrane domain, and a cytoplasmic bulb. In the present study, we generated a
Shigella spa32
mutant and studied its phenotype. The
spa32
gene shows low sequence homology to
Salmonella
TTSS1
invJ/spaN
and to flagellar
fliK
. The
spa32
mutant, like the wild-type strain, secreted the Ipas and IpgD, which are normally secreted via the TTSS, at low levels into the growth medium. However, unlike the wild-type strain, the
spa32
mutant could neither be induced to secrete the Ipas and IpgD instantaneously upon addition of Congo red nor penetrate HeLa cells in vitro. Additionally, the Spa32 protein is secreted in large amounts by the TTSS during exponential growth but not upon Congo red induction. Interestingly, electron microscopy analysis of the
spa32
mutant revealed that the needle of its secretons were up to 10 times longer than those of the wild type. In addition, in the absence of induction, the
spa32
mutant secreted normal levels of MxiI but a large excess of MxiH. Taken together, our data indicate that the
spa32
mutant presents a novel phenotype and that the primary defect of the mutant may be its inability to regulate or control secretion of MxiH.
Direct Quantitation of the Numbers of Individual Penicillin-Binding Proteins per Cell in
Staphylococcus aureus
Authors: Pucci Michael J. , Dougherty Thomas J.
Published: July 2002

The penicillin-binding proteins (PBPs) are a set of enzymes that participate in bacterial peptidoglycan assembly. The absolute numbers of each PBP were determined by direct measurement and have been reported for two
Staphylococcus aureus
strains, RN4220 (methicillin-sensitive
S. aureus
) and RN450M (methicillin-resistant
S. aureus
). From the specific activity of the labeled penicillin and the absolute number of disintegrations per minute, and from the number of CFU per milliliter calculated from proteins and optical density, a determination of the number of PBPs per cell was made. These numbers ranged from approximately 150 to 825 PBPs/cell and represent the first direct determination of absolute numbers of PBPs in
S. aureus
.
Origin Binding Activity of the Mycobacterial Plasmid pAL5000 Replication Protein RepB Is Stimulated through Interactions with Host Factors and Coupled Expression of
repA
Authors: Basu Abhijit , Chawla-Sarkar Mamta , Chakrabarti Santanu , Das Gupta Sujoy K.
Published: July 2002

The minimal replication region of the mycobacterial plasmid pAL5000 encompasses the replication origin (
ori
) and two tandemly organized replication genes,
repA
and
repB,
the functions of which are not clearly known. It was observed that when the
repA
and
repB
genes were expressed in
Escherichia coli,
a strong
ori
binding activity was generated in the host cells. Inactivation of
repB
led to a complete loss of activity, whereas inactivation of
repA
had a partial effect, indicating that while
repB
plays an important role in the process, its activity is stimulated through coexpression of
repA
. However, this stimulatory effect could be demonstrated only when expression of
repA
and that of
repB
were coupled. At a relatively high concentration (1,000 nM), the purified RepB protein was found to form an
ori
complex with low specificity, which was sensitive to high salt concentrations and challenge by a nonspecific competitor. In contrast, the complex formed by an extract of
repA-repB
-expressing cells was highly specific and was resistant to both types of challenges. At a 10-fold-lower concentration, RepB did not exhibit
ori
binding activity, but it could nevertheless form a salt-resistant
ori
complex in vitro, provided that host factors were present. Antibody supershift experiments indicated that RepB is a key component of the specific complex formed by extracts prepared from
E. coli
cells expressing the
repA
and
repB
genes and also from mycobacterial cells harboring pAL5000-derived vectors. The results indicate that in vivo RepB interacts with host factors and forms an
ori
complex, but this activity is maximal only when there is coupled expression of
repA
.
Simultaneous Detection and Typing of Influenza Viruses A and B by a Nested Reverse Transcription-PCR: Comparison to Virus Isolation and Antigen Detection by Immunofluorescence and Optical Immunoassay (FLU OIA)
Authors: Herrmann Björn , Larsson Christine , Zweygberg Benita Wirgart
Published: July 2002

A nested reverse transcription (RT)-PCR was developed for simultaneous detection and typing of influenza viruses A and B. The detection limit for influenza virus A subtypes H1 and H3 and that for influenza virus B were between 1 and 4 target gene copies per reaction for each type. The clinical benefit of the RT-PCR method was evaluated by comparing the results with virus isolation and direct immunofluorescence (IF) assays on 215 nasopharyngeal aspirates from patients with suspected influenza virus infection. The RT-PCR detected 83 cases of influenza A, compared to 66 cases detected by virus isolation and 68 cases detected by IF assay. The corresponding figures for the detection of influenza B were 15, 12, and 11 cases, respectively. In total, 16 out of 98 RT-PCR-positive specimens were negative by virus isolation and IF. An optical immunoassay for rapid detection of influenza A and B (FLU OIA; Bio Star Inc., Boulder, Colo.) was compared to RT-PCR and IF on 105 nasopharyngeal aspirates and 79 swabs. The sensitivity for the OIA was 40.4% compared to PCR and 48.8% compared to IF assay, when nasopharyngeal aspirates were examined. The specificities were 94.3 and 93.9%, respectively. The sensitivity was higher for OIA on nasopharyngeal swabs, 77.5% and 86.6% compared to PCR and IF, respectively, while the specificity was lower, 82.0% and 75.5%, respectively. The RT-PCR provides a sensitive and specific method for detecting and typing influenza viruses A and B. The rapid OIA is useful as a complementary test, but it cannot replace established methods without further evaluation.
Contemporary Assessment of Antimicrobial Susceptibility Testing Methods for Polymyxin B and Colistin: Review of Available Interpretative Criteria and Quality Control Guidelines
Authors: Gales Ana C. , Reis Adriana O. , Jones Ronald N.
Published: July 2002

The emergence of infections caused by multidrug-resistant
Pseudomonas aeruginosa
and
Acinetobacter
spp. has necessitated the search for alternative parenteral agents such as the polymyxins. The National Committee for Clinical Laboratory Standards (NCCLS) documents do not currently provide interpretative criteria for the testing of the polymyxins, colistin and polymyxin B. Therefore, an evaluation of the antimicrobial activity of colistin and polymyxin B was initiated using 200 bloodstream infection pathogens collected through the SENTRY Antimicrobial Surveillance Program. All susceptibility tests were performed according to the NCCLS recommendations. Polymyxin B and colistin displayed a nearly identical spectrum of activity, exhibiting excellent potency against
P. aeruginosa
(MIC
90
, 2 μg/ml) and
Acinetobacter
sp. (MIC
90
, 2 μg/ml). In contrast, they showed limited activity against some other nonfermentative bacilli such as
Burkholderia cepacia
(MIC
90
, ≥128 μg/ml). Excellent correlation was achieved between broth microdilution and agar dilution tests (
r
= 0.96 to 0.98); 94.3% of the results were ±1 log
2
dilution between the methods used for both compounds. At a resistance breakpoint of ≥4 μg/ml for both agents, unacceptable false-susceptible or very major errors were noted for colistin (5%) and polymyxin B (6%). Modified zone criteria for colistin (≤11 and ≥14 mm) and polymyxin B (≤10 and ≥14 mm) were suggested, but some degree of error persisted (≥3.5%). It is recommended that all susceptible disk diffusion results be confirmed by MIC tests using the preferred reference NCCLS method. The quality control (QC) ranges listed in the product package insert require an adjusted range by approximately 3 mm for both NCCLS gram-negative quality control strains. This evaluation of in vitro susceptibility test methods for the polymyxin class drugs confirmed continued serious testing error with the disk diffusion method, the possible need for breakpoint adjustments, and the recalculation of disk diffusion QC ranges. Clinical laboratories should exclusively use MIC methods to assist the therapeutic application of colistin or polymyxin B until disk diffusion test modifications are sanctioned and published by the NCCLS.
Fecal Leukocyte Stain Has Diagnostic Value for Outpatients but Not Inpatients
Authors: Savola Kristen L. , Baron Ellen Jo , Tompkins Lucy S. , Passaro Douglas J.
Published: July 2002

The methylene blue stain for fecal leukocytes (FL) is widely used as an adjunct to slower but more accurate tests of diarrheal etiology, such as stool culture (SCx) or toxin assays for
Clostridium difficile
. Prior studies investigating the utility of FL for predicting SCx and
C. difficile
toxin assay (CDTA) results did not evaluate the importance of inpatient versus outpatient status. We conducted a study of patients who submitted a stool specimen to the Stanford Hospital Microbiology Laboratory between May 1998 and April 1999. The results for stool specimens that were tested by FL and by a confirmatory test (either SCx or CDTA) were used to determine whether the FL method helped to predict the results of these tests. Of 797 stools that were tested by FL method and at least one confirmatory test, 502 stools were tested by CDTA, and 473 stools were cultured. The FL test was 14% sensitive and 90% specific for
C. difficile
with a diagnostic threshold of one white blood cell/high-power field (WBC/HPF). The overall likelihood ratio (LR) for a positive CDTA was 1.4 with a 95% confidence interval (CI) of 0.5 to 3.7 (
P
= 0.5) and was similar among inpatients and outpatients. In contrast, the presence of ≥1 WBC/HPF was 52% sensitive and 88% specific for the 27 positive SCx results and helped to predict a positive SCx result (LR, 4.2; 95% CI, 2.7 to 6.5;
P
< 0.001). The sensitivity of ≥1 WBC/HPF was 57%, and its predictive value for SCx was higher among outpatients (outpatient LR, 5.0; 95% CI, 2.9 to 8.6;
P
< 0.001; inpatient LR, 1.9; 95% CI, 0.3 to 10.8;
P
= 0.5). Among inpatients, only 4 (1.5%) of the 273 SCx results were positive, and the presence of ≥1 WBC/HPF was insensitive (25%) and did not predict a positive SCx (LR, 1.9; 95% CI, 0.3 to 10.8;
P
= 0.5). When the data were reanalyzed using a diagnostic threshold of five WBC/HPF for FL, the predictive power of the FL method was similar. Thus, FL was of no value in predicting CDTA positivity, nor was it helpful in predicting SCx results for inpatients. Neither SCx nor the FL method should routinely be performed on samples from inpatients. Among outpatients, presence of FLs should suggest a bacterial diarrhea in clinically compatible cases.
TaqMan-Based Detection of
Trichomonas vaginalis
DNA from Female Genital Specimens
Authors: Jordan Jeanne A. , Lowery Donna , Trucco Massimo
Published: July 2002

A double-labeled fluorescent probe was designed and evaluated for detecting
Trichomonas vaginalis
DNA in a 5′ nuclease (TaqMan) assay. The
T. vaginalis
-specific probe contains a 5′-fluorescein (5′-FAM) and a 3′-rhodamine (TAMRA) derivative. Female genital secretions were collected on Amplicor (Roche Molecular, Indianapolis, Ind.) swabs and by a transport system used for
Chlamydia trachomatis
and/or
Neisseria gonorrhoeae
DNA detection by PCR. Five hundred fifty-two female genital specimens, of which 248 (45%) were vaginal specimens and 304 (55%) were introital, were tested for both
T. vaginalis
DNA and viable microorganisms using the 5′ nuclease assay and broth culture, respectively. Of these, 304 of 552 (55%) were also evaluated by direct microscopic examination for the characteristic motile organism. After resolving discrepancies, the comparisons produced an analytical sensitivity and specificity for the TaqMan-based PCR assay of 97.8 and 97.4%, respectively. As a result, ΔRQ values (differences in fluorescence due to probe hybridization and resulting 5′-FAM cleavage from the specific PCR product) of ≥2.0 and ≤1.5 were established for
T. vaginalis
-positive and -negative cutoffs, respectively. ΔRQ values between 1.5 and 2.0 were considered indeterminate. Overall findings revealed a high level of agreement between PCR and culture for detecting
T. vaginalis
. Potential benefits of the 5′ nuclease assay include a greater sensitivity compared to direct microscopic examination and the ease of testing large numbers of clinical specimens in a significantly shorter turnaround time compared to culture.
Combined PCR-Heteroduplex Mobility Assay for Detection and Differentiation of Influenza A Viruses from Different Animal Species
Authors: Ellis Joanna S. , Zambon Maria C.
Published: July 2002

Transfer of influenza A viruses from animal hosts to man may lead to the emergence of new human pandemic strains. The early detection and identification of such events are therefore paramount in the surveillance of influenza viruses. To detect and partially characterize influenza A viruses from different animal species, a combined reverse transcription (RT)-PCR heteroduplex mobility assay (HMA) was designed. This M gene RT-PCR was shown to be sensitive and specific for the detection of human, avian, and swine influenza A viruses. PCR amplicons from human, avian, and swine viruses of 15 different subtypes, with between 1.9 and 21.4% nucleotide divergence, were differentiated by HMA. Sequencing of the amplicons showed that the heteroduplex mobility patterns correlated with the sequence divergence between test and reference DNA. The application of the RT-PCR HMA method for rapid screening of samples was assessed with a reference panel of viruses of human, avian, and swine origin. The avian H9N2 virus A/HongKong/1073/99, which crossed the species barrier to humans, was screened against the reference panel. It was found to be most closely related to the avian A/Quail/HongKong/G1/97 H9N2 reference PCR product. Sequence analysis showed a nucleotide divergence of 1.1% between the A/Quail/HongKong/G1/97 and A/HongKong/1073/99 amplicons. From the results of our work, we consider the RT-PCR HMA method described to offer a rapid and sensitive means for screening for novel or unusual influenza viruses.
Detection and Differentiation of Human Polyomaviruses JC and BK by LightCycler PCR
Authors: Whiley David M. , Mackay Ian M. , Sloots Theo P.
Published: July 2002

Human polyomaviruses JC and BK may cause several clinical manifestations in immunocompromised hosts, including progressive multifocal leukoencephalopathy and hemorrhagic cystitis. Molecular detection by PCR is recognized as a sensitive and specific method for detecting human polyomaviruses in clinical samples. In this study, a real-time PCR assay using the LightCycler platform was evaluated and compared to an “in-house” PCR assay using a conventional detection method. A total of 122 urine specimens were tested, and human polyomavirus was detected in 49 specimens (40%) by both conventional PCR and LightCycler PCR. The remaining 73 specimens (60%) were found negative by both assays. For 46 of the 49 positive specimens, LightCycler PCR and conventional PCR identified the same polyomavirus type. These samples included 30 samples with JC virus (JCV), 14 samples with BK virus (BKV), and 2 samples in which both viruses were detected. In the remaining three samples, both JCV and BKV were detected by the conventional assay, but only JCV was detected by the LightCycler assay. The results of this study show that the LightCycler PCR assay displays sensitivity and specificity similar to those of a conventional PCR assay. These data, combined with its rapid turnaround time for results and decreased hands-on time, make the LightCycler PCR assay highly suitable for the rapid detection and differentiation of JCV and BKV in the clinical laboratory.
Use of BACTEC 9240 Blood Culture System for Detection of
Brucella melitensis
in Synovial Fluid
Authors: Yagupsky Pablo , Peled Nechama , Press Joseph
Published: July 2002

Synovial fluid specimens aspirated from patients with arthritis were inoculated into an aerobic Peds Plus blood culture bottle and monitored by the BACTEC 9240 instrument for 4 weeks. A total of 1,072 synovial fluid cultures were processed, and 15 (0.14%) were positive for
Brucella melitensis
. A single culture, harboring 1.3 CFU of viable organisms per ml, was missed by the instrument. All 14 positive BACTEC cultures were detected within 3 to 7 days.
Isolation and Characterization of a
Salmonella enterica
Serotype Typhi Variant and Its Clinical and Public Health Implications
Authors: Woo Patrick C. Y. , Fung Ami M. Y. , Wong Samson S. Y. , Tsoi Hoi-Wah , Yuen Kwok-Yung
Published: July 2002

We report the isolation and characterization of a member of the family
Enterobacteriaceae
isolated from the gallbladder pus of a food handler. Conventional biochemical tests suggested
Salmonella enterica
serotype Typhi, but the isolate agglutinated with poly(O), 2O, 9O, and Vi
Salmonella
antisera but not with poly(H) or any individual H
Salmonella
antisera. 16S rRNA gene sequencing showed that there were two base differences between the isolate and
Salmonella enterica
serotype Montevideo, four base differences between the isolate and serotype Typhi, five base differences between the isolate and
Salmonella enterica
serotype Typhimurium, and six base differences between the isolate and
Salmonella enterica
serotype Dublin, indicating that the isolate was a strain of
S. enterica
. Electron microscopy confirmed that the isolate was aflagellated. The flagellin gene sequence of the isolate was 100% identical to that of the H1-d flagellin gene of serotype Typhi. Sequencing of the
rfbE
gene, which encoded the CDP-tyvelose epimerase of the isolate, showed that there was a point mutation at position +694 (G→T), leading to an amino acid substitution (Gly→Cys). This may have resulted in a protein of reduced catalytic activity and hence the presence of both 2O and 9O antigens. We therefore concluded that the isolate was a variant of serotype Typhi. Besides antibiotic therapy and cholecystectomy, removal of all stones in the biliary tree was performed for eradication of the carrier state.
Infection withBartonella weissiiand Detection ofNanobacteriumAntigens in a North Carolina Beef Herd
Authors: Breitschwerdt Edward B. , Sontakke Sushama , Cannedy Allen , Hancock Susan I. , Bradley Julie M.
Published: July 2002

Very recently,Bartonellaorganisms have been isolated from large ruminants (deer, elk, and dairy and beef cattle) located in the United States and in France. In this study, we report the serologic, microbiologic, and molecular findings related to the isolation of aBartonellaspecies in North Carolina beef cattle and the detection of nanobacterial antigen using a commercially available enzyme-linked immunosorbent assay. Between August 1998 and September 1999, blood was collected from 38 cattle ranging in age from 1 month to 6.5 years. After a 1-month incubation period, aBartonellasp. was isolated on a 5% rabbit blood agar plate from three of six EDTA blood samples. PCR amplification of the 16S rRNA gene from all three isolates resulted in a DNA sequence that was 100% identical to that ofB. weissii16S rRNA (GenBank no.AF199502). By IFA testing, 36 of 38 cattle had antibodies (≥1:64) toBartonella weissii(bovine origin) antigens. Nanobacterial antigen was detected in 22 of 22 serum samples. We conclude that infection with an organism similar or closely related toB. weissiican occur in North Carolina cattle and that although their actual existence is still controversialNanobacteriumantigens were detected with a commercially available test kit. The epidemiology, vector biology, and potential pathogenicity of these organisms in cattle deserve future consideration.
Adaptation ofEscherichia colito the Bovine Mammary Gland
Authors: Bradley A. J. , Green M. J.
Published: July 2002

Clinical mastitis in six Somerset dairy herds was monitored over a 12-month period.Escherichia coliwas implicated in 34.7% of all clinical cases. Forty-one percent of all clinicalE. colimastitis cases occurred in just 2.2% of the population. A total of 23.9% of clinicalE. colicases occurred in quarters suffering recurrent cases ofE. colimastitis. The genotypes of strains involved in recurrent cases of clinicalE. colimastitis were compared by DNA fingerprinting with enterobacterial repetitive intergenic consensus primers. In 85.7% of cases of recurrent quarterE. colimastitis, the same genotype was implicated as the cause of disease, suggesting persistence of the organism within the mammary environment. The same genotype as that in the original case was also implicated in 8.5% of recurrent cases occurring in different quarters of the same cow, suggesting spread between quarters. These findings challenge our current understanding of the epidemiology ofE. colimastitis and suggest that pathogen adaptation and host susceptibility may be playing a part in the changing pattern of clinical mastitis experienced in the modern dairy herd.
Development of a Real-Time PCR Assay for Detection ofToxoplasma gondiiin Pig and Mouse Tissues
Authors: Jauregui L. H. , Higgins J. , Zarlenga D. , Dubey J. P. , Lunney J. K.
Published: July 2002

A highly sensitive and specific method has been developed to reproducibly detect and quantitateToxoplasma gondiiburden in animal tissue samples usingT. gondiiITS1-derived primers and a fluorogenic probe via real-time PCR. Assay specificity was confirmed against a panel of DNA samples fromT. gondiiand other common protozoa as well as host animal tissue. This Toxo TaqMan assay was able to detect as little as 0.1 pg ofT. gondiigenomic DNA, which is equivalent to 1T. gondiibradyzoite, and has a dynamic range of detection of from 100 ng to 100 fg ofT. gondiiDNA. Tissues from experimentally infected mice and pigs as well as bradyzoite-spiked pig muscle samples were used to test and standardize this technique. Positive signals were obtained withT. gondiiparasite concentrations ranging from 4 to 3.7 × 105parasites per g of spiked pig tissue, with excellent linearity (R2= 0.9776). AllT. gondii-infected animals were correctly identified by this technique. Results indicate that this assay is applicable to swine carcasses and commercial pig products, is compatible with automation technology for potential slaughterhouse use, and will enable scientists to diagnose and quantitateT. gondiiin animal tissues.
New Immunochromatographic Rapid Test for Diagnosis of Acute Puumala Virus Infection
Authors: Hujakka Helena , Koistinen Vesa , Eerikäinen Pekka , Kuronen Ilpo , Mononen Ilkka , Parviainen Markku , Lundkvist Åke , Vaheri Antti , Närvänen Ale , Vapalahti Olli
Published: July 2002

A new immunochromatographic rapid test,
POC
PUUMALA (Erilab Ltd., Kuopio, Finland), for detection of acute-phase Puumala virus (PUUV) infection was developed based on a highly purified baculovirus-expressed PUUV nucleocapsid protein antigen and lateral immunodiffusion techniques. After addition of sample (5 μl of serum, plasma, or fingertip blood) and buffer, PUUV-specific immunoglobulin M (IgM) antibodies, if present, together with the gold-conjugated anti-human IgM, formed a specific colored line in 5 min. The sensitivity and specificity of the test were evaluated with 200 serum samples and 30 fingertip blood samples. The reference method for the serum samples was a μ-capture enzyme immunoassay (EIA) for IgM and an immunofluorescence assay (IFA) for IgG antibodies. The analytical sensitivity and specificity of the rapid test were 100 and 99%, respectively, for unfrozen serum samples (
n
= 103; 12 PUUV IgM-positive samples). When freeze-thawed serum samples were used, the sensitivity and specificity were each 97.1% (
n
= 70; 35 PUUV IgM-positive samples). The specificity of the test was 96.2% for 27 serum samples with nonspecific IgM antibodies or rheumatoid factor (RF). The fingertip blood samples (
n
= 30) were negative, but they gave clear positive results when spiked with IgM-positive sera (
n
= 20). The results were in good agreement with the standard diagnostic methods. The rapid performance, the lack of need for refined laboratory equipment, and the high specificity with fresh serum and fingertip blood samples indicate that the developed
POC
PUUMALA rapid test is a useful tool for fast diagnosis of acute PUUV infection.
Identification and Expression of a 50-Kilodalton Surface Antigen ofBabesia gibsoniand Evaluation of Its Diagnostic Potential in an Enzyme-Linked Immunosorbent Assay
Authors: Fukumoto Shinya , Xuan Xuenan , Nishikawa Yoshifumi , Inoue Noboru , Igarashi Ikuo , Nagasawa Hideyuki , Fujisaki Kozo , Mikami Takeshi
Published: July 2002

A cDNA expression library prepared fromBabesia gibsonimerozoite mRNA was screened withB. gibsoni-infected dog serum. cDNA encoding a 50-kDa protein was cloned and designated the P50 gene. The complete nucleotide sequence of the P50 gene was 1,922 bp. Computer analysis suggested that the sequence of the P50 gene contained an open reading frame of 1,401 bp with a coding capacity of approximately 50 kDa. The complete genomic nucleotide sequence of the P50 gene has been analyzed and shown to contain a single intron of 37 bp. Southern blotting analysis indicated that the P50 gene was present at a single copy in theB. gibsonigenome. The native P50 protein ofB. gibsoniwith a molecular mass of 50 kDa was identified by Western blotting with anti-recombinant P50 mouse serum. Confocal laser microscopic analysis showed that the P50 protein was located on the surface ofB. gibsonimerozoites. The recombinant P50 protein expressed by baculovirus in insect cells was used as the antigen in an enzyme-linked immunosorbent assay (ELISA). The ELISA was able to differentiate betweenB. gibsoni-infected dog serum andB. canis-infected dog serum or noninfected dog serum. Furthermore, the antibody response against the recombinant P50 protein was maintained until the chronic stage of infection in dogs experimentally infected withB. gibsoniwas developed. These results demonstrate that the recombinant P50 protein might be a useful diagnostic reagent for detection of antibodies toB. gibsoniin dogs.
Molecular and Epidemiological Analyses of Human Adenovirus Type 7 Strains Isolated from the 1995 Nationwide Outbreak in Japan
Authors: Noda Mamoru , Yoshida Tetsuya , Sakaguchi Takemasa , Ikeda Yoshifumi , Yamaoka Koji , Ogino Takeo
Published: July 2002

The adenovirus type 7 (Ad7) isolates from the 1995 nationwide outbreak in Japan were genetically and seroepidemiologically analyzed in comparison with Japanese Ad7 strains isolated before 1995 to determine their genome type and to speculate on their origin and causative factors of the outbreak. Twenty-six Ad7 isolates from the outbreak were identified by restriction enzyme analysis as the Ad7d2 genome type, while 22 Ad7 strains sporadically isolated in Japan before 1995 were identified as Ad7d. Partial nucleotide sequencing of the E3 region of Ad7d2 revealed a nucleotide substitution of G to A at position 265, resulting in the absence of the
Bst
EII site and making Ad7d2 distinct from Ad7d. In Hiroshima City, Japan, no Ad7 was isolated from 1982 to 1994, but 43 and 50 Ad7 strains were isolated in 1995 and 1996, respectively. A seroepidemiological study of 251 serum samples collected in 1989 in Hiroshima City showed that only 2.8% of the samples were positive for Ad7. These results indicate that the 1995 outbreak of Ad7 in Japan was caused by the Ad7d2 genome type, which might have been introduced from outside Japan. The results also suggest that the low mass immunity in Japan was critical for the outbreak and that the mutation in the E3 region in Ad7d2 may have influenced transmission.