bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization and Mutational Analysis of Three Allelic
lsc
Genes Encoding Levansucrase in
Pseudomonas syringae
Authors: Li Hongqiao , Ullrich Matthias S.
Published: July 2002

In the plant pathogen
Pseudomonas syringae
pv. glycinea PG4180 and other bacterial species, synthesis of the exopolysaccharide levan is catalyzed by the extracellular enzyme levansucrase. The results of Southern blotting and PCR analysis indicated the presence of three levansucrase-encoding genes in strain PG4180:
lscA
,
lscB
, and
lscC
. In this study,
lscB
and
lscC
were cloned from a genomic library of strain PG4180. Sequence analysis of the two
lsc
genes showed that they were virtually identical to each other and highly similar to the previously characterized
lscA
gene.
lscA
and
lscC
had a chromosomal location, whereas
lscB
resided on an indigenous plasmid of PG4180. Mutants with impaired expression of individual
lsc
genes and double mutants were generated by marker exchange mutagenesis. Determination of levansucrase activities in these mutants revealed that the
lscB
gene product was secreted but not that of
lscA
or
lscC
. Our results indicated that
lscB
and
lscC
but not
lscA
contributed to periplasmic levan synthesis of PG4180. The
lscB lscC
double mutant was completely defective in levan formation and could be complemented by either
lscB
or
lscC.
Our data suggested a compartment-specific localization of two
lsc
gene products, with LscB being the secreted, extracellular enzyme and LscC being the predominantly periplasmic levansucrase. Results of Western blot analyses indicated that
lscA
was not expressed and that
lscA
was not associated with levansucrase activities in any particular protein fraction. LscA could be detected in PG4180 only when transcribed from the vector-borne P

lac

promoter. PCR screening in various
P. syringae
strains with primers derived from the three characterized
lsc
genes demonstrated the presence of multiple Lsc isoenzymes in other
P. syringae
pathovars.
DNA Microarray-Mediated Transcriptional Profiling of the
Escherichia coli
Response to Hydrogen Peroxide
Authors: Zheng Ming , Wang Xunde , Templeton Lori J. , Smulski Dana R. , LaRossa Robert A. , Storz Gisela
Published: July 2002

The genome-wide transcription profile of
Escherichia coli
cells treated with hydrogen peroxide was examined with a DNA microarray composed of 4,169
E. coli
open reading frames. By measuring gene expression in isogenic wild-type and
oxyR
deletion strains, we confirmed that the peroxide response regulator OxyR activates most of the highly hydrogen peroxide-inducible genes. The DNA microarray measurements allowed the identification of several new OxyR-activated genes, including the
hemH
heme biosynthetic gene; the six-gene
suf
operon, which may participate in Fe-S cluster assembly or repair; and four genes of unknown function. We also identified several genes, including
uxuA,
encoding mannonate hydrolase, whose expression might be repressed by OxyR, since their expression was elevated in the Δ
oxyR
mutant strain. In addition, the induction of some genes was found to be OxyR independent, indicating the existence of other peroxide sensors and regulators in
E. coli
. For example, the
isc
operon, which specifies Fe-S cluster formation and repair activities, was induced by hydrogen peroxide in strains lacking either OxyR or the superoxide response regulators SoxRS. These results expand our understanding of the oxidative stress response and raise interesting questions regarding the nature of other regulators that modulate gene expression in response to hydrogen peroxide.
Three Target Genes for the Transcriptional Activator Cat8p of
Kluyveromyces lactis
: Acetyl Coenzyme A Synthetase Genes
KlACS1
and
KlACS2
and Lactate Permease Gene
KlJEN1
Authors: Lodi Tiziana , Saliola Michele , Donnini Claudia , Goffrini Paola
Published: July 2002

The aerobic yeast
Kluyveromyces lactis
and the predominantly fermentative
Saccharomyces cerevisiae
share many of the genes encoding the enzymes of carbon and energy metabolism. The physiological features that distinguish the two yeasts appear to result essentially from different organization of regulatory circuits, in particular glucose repression and gluconeogenesis. We have isolated the
KlCAT8
gene (a homologue of
S. cerevisiae CAT8
, encoding a DNA binding protein) as a multicopy suppressor of a
fog1
mutation. The Fog1 protein is a homologue of the Snf1 complex components Gal83p, Sip1p, and Sip2p of
S. cerevisiae
. While
CAT8
controls the key enzymes of gluconeogenesis in
S. cerevisiae
,
KlCAT8
of
K. lactis
does not (I. Georis, J. J. Krijger, K. D. Breunig, and J. Vandenhaute, Mol. Gen. Genet. 264:193–203, 2000). We therefore examined possible targets of KlCat8p. We found that the acetyl coenzyme A synthetase genes,
KlACS1
and
KlACS2
, were specifically regulated by
KlCAT8
, but very differently from the
S. cerevisiae
counterparts.
KlACS1
was induced by acetate and lactate, while
KlACS2
was induced by ethanol, both under the control of
KlCAT8.
Also,
KlJEN1
, encoding the lactate-inducible and glucose-repressible lactate permease, was found under a tight control of
KlCAT8
.
Stringent Response Activates Quorum Sensing and Modulates Cell Density-Dependent Gene Expression in
Pseudomonas aeruginosa
Authors: van Delden Christian , Comte Rachel , Bally And Marc
Published: July 2002

During nutrient starvation,
Escherichia coli
elicits a stringent response involving the ribosome-associated protein RelA. Activation of RelA results in a global change in the cellular metabolism including enhanced expression of the stationary-phase sigma factor RpoS. In the human pathogen
Pseudomonas aeruginosa
, a complex quorum-sensing circuitry, linked to RpoS expression, is required for cell density-dependent production of many secreted virulence factors, including LasB elastase. Quorum sensing relies on the activation of specific transcriptional regulators (LasR and RhlR) by their corresponding autoinducers (3-oxo-C
12
-homoserine lactone [HSL] and C
4
-HSL), which function as intercellular signals. We found that overexpression of
relA
activated the expression of
rpoS
in
P. aeruginosa
and led to premature, cell density-independent LasB elastase production. We therefore investigated the effects of the stringent response on quorum sensing. Both
lasR
and
rhlR
gene expression and autoinducer synthesis were prematurely activated during the stringent response induced by overexpression of
relA
. Premature expression of
lasR
and
rhlR
was also observed when
relA
was overexpressed in a PAO1
rpoS
mutant. The stringent response induced by the amino acid analogue serine hydroxamate (SHX) also led to premature production of the 3-oxo-C
12
-HSL autoinducer. This response to SHX was absent in a PAO1
relA
mutant. These findings suggest that the stringent response can activate the two quorum-sensing systems of
P. aeruginosa
independently of cell density.
Infrequent Genetic Exchange and Recombination in the Mitochondrial Genome of
Candida albicans
Authors: Anderson James B. , Wickens Claire , Khan Mustafa , Cowen Leah E. , Federspiel Nancy , Jones Ted , Kohn Linda M.
Published: July 2002

Previous analyses of diploid nuclear genotypes have concluded that recombination has occurred in populations of the yeast
Candida albicans
. To address the possibilities of clonality and recombination in an effectively haploid genome, we sequenced seven regions of mitochondrial DNA (mtDNA) in 45 strains of
C. albicans
from human immunodeficiency virus-positive patients in Toronto, Canada, and 3 standard reference isolates of
C. albicans
, CA, CAI4, and WO-1. Among a total of 2,553 nucleotides in the seven regions, 62 polymorphic nucleotide sites and seven indels defined nine distinct mtDNA haplotypes among the 48 strains. Five of these haplotypes occurred in more than one strain, indicating clonal proliferation of mtDNA. Phylogenetic analysis of mtDNA haplotypes resulted in one most-parsimonious tree. Most of the nucleotide sites undergoing parallel change in this tree were clustered in blocks that corresponded to sequenced regions. Because of the existence of these blocks, the apparent homoplasy can be attributed to infrequent, past genetic exchange and recombination between individuals and cannot be attributed to parallel mutation. Among strains sharing the same mtDNA haplotypes, multilocus nuclear genotypes were more similar than expected from a random comparison of nuclear DNA genotypes, suggesting that clonal proliferation of the mitochondrial genome was accompanied by clonal proliferation of the nuclear genome.
Conserved Gene Cluster at Replication Origins of the α-Proteobacteria
Caulobacter crescentus
and
Rickettsia prowazekii
Authors: Brassinga Ann Karen C. , Siam Rania , Marczynski Gregory T.
Published: July 2002

A 30-kb region surrounding the replication origin in
Caulobacter crescentus
was analyzed. Comparison to the genome sequence of another α-proteobacterium,
Rickettsia prowazekii
, revealed a conserved cluster of genes (RP001,
hemE, hemH
, and RP883) that overlaps the established origin of replication in
C. crescentus
and the putative origin of replication in
R. prowazekii
. The genes flanking this cluster differ between these two organisms. We therefore propose that this conserved gene cluster can be used to identify the origin of replication in other α-proteobacteria.
Gene Expression in
Pseudomonas aeruginosa
: Evidence of Iron Override Effects on Quorum Sensing and Biofilm-Specific Gene Regulation
Authors: Bollinger Nikki , Hassett Daniel J. , Iglewski Barbara H. , Costerton J. William , McDermott Timothy R.
Published: July 2002

Prior studies established that the
Pseudomonas aeruginosa
oxidative stress response is influenced by iron availability, whereas more recent evidence demonstrated that it was also controlled by quorum sensing (QS) regulatory circuitry. In the present study,
sodA
(encoding manganese-cofactored superoxide dismutase [Mn-SOD]) and Mn-SOD were used as a reporter gene and endogenous reporter enzyme, respectively, to reexamine control mechanisms that govern the oxidative stress response and to better understand how QS and a nutrient stress response interact or overlap in this bacterium. In cells grown in Trypticase soy broth (TSB), Mn-SOD was found in wild-type stationary-phase planktonic cells but not in a
lasI
or
lasR
mutant. However, Mn-SOD activity was completely suppressed in the wild-type strain when TSB was supplemented with iron. Reporter gene studies indicated that
sodA
transcription could be variably induced in iron-starved cells of all three strains, depending on growth stage. Iron starvation induction of
sodA
was greatest in the wild-type strain and least in the
lasR
mutant and was maximal in stationary-phase cells. Reporter experiments in the wild-type strain showed increased
lasI
::
lacZ
transcription in response to iron limitation, whereas the expression level in the
las
mutants was minimal and iron starvation induction of
lasI
::
lacZ
did not occur. Studies comparing Mn-SOD activity in
P. aeruginosa
biofilms and planktonic cultures were also initiated. In wild-type biofilms, Mn-SOD was not detected until after 6 days, although in iron-limited wild-type biofilms Mn-SOD was detected within the initial 24 h of biofilm establishment and formation. Unlike planktonic bacteria, Mn-SOD was constitutive in the
lasI
and
lasR
mutant biofilms but could be suppressed if the growth medium was amended with 25 μM ferric chloride. This study demonstrated that (i) the nutritional status of the cell must be taken into account when one is evaluating QS-based gene expression; (ii) in the biofilm mode of growth, QS may also have negative regulatory functions; (iii) QS-based gene regulation models based on studies with planktonic cells must be modified in order to explain biofilm gene expression behavior; and (iv) gene expression in biofilms is dynamic.
Role for a Phage Promoter in Shiga Toxin 2 Expression from a Pathogenic
Escherichia coli
Strain
Authors: Wagner Patrick L. , Neely Melody N. , Zhang Xiaoping , Acheson David W. K. , Waldor Matthew K. , Friedman David I.
Published: July 2002

Shiga toxins (Stxs), encoded by the
stxA
and
stxB
genes, are important contributors to the virulence of
Escherichia coli
O157:H7 and other Stx-producing
E. coli
(STEC) strains. The
stxA
and
stxB
genes in STEC strains are located on the genomes of resident prophages of the λ family immediately downstream of the phage late promoters (
p
R′
). The phage-encoded Q proteins modify RNA polymerase initiating transcription at the cognate
p
R′
promoter which creates transcription complexes that transcend a transcription terminator immediately downstream of
p
R′
as well as terminator kilobases distal to
p
R′
. To test if this Q-directed processive transcription plays a role in
stx
2
AB
expression, we constructed a mutant prophage in an O157:H7 clinical isolate from which
p
R′
and part of Q were deleted but which has an intact
p
Stx, the previously described
stx
2
AB
-associated promoter. We report that production of significant levels of Stx2 in this O157:H7 isolate depends on the
p
R′
promoter. Since transcription initiating at
p
R′
ultimately requires activation of the phage lytic cascade, expression of
stx
2
AB
in STEC depends primarily on prophage induction. By showing this central role for the prophage in
stx
2
AB
expression, our findings contradict the prevailing assumption that phages serve merely as agents for virulence gene transfer.
glnD
and
mviN
Are Genes of an Essential Operon in
Sinorhizobium meliloti
Authors: Rudnick Paul A. , Arcondéguy Tania , Kennedy Christina K. , Kahn Daniel
Published: July 2002

To evaluate the role of uridylyl-transferase, the
Sinorhizobium meliloti glnD
gene was isolated by heterologous complementation in
Azotobacter vinelandii
. The
glnD
gene is cotranscribed with a gene homologous to
Salmonella mviN
.
glnD1
::Ω or
mviN1
::Ω mutants could not be isolated by a powerful sucrose counterselection procedure unless a complementing cosmid was provided, indicating that
glnD
and
mviN
are members of an indispensable operon in
S. meliloti
.
PehN, a Polygalacturonase Homologue with a Low Hydrolase Activity, Is Coregulated with the Other
Erwinia chrysanthemi
Polygalacturonases
Authors: Hugouvieux-Cotte-Pattat Nicole , Shevchik Vladimir E. , Nasser William
Published: July 2002

Erwinia chrysanthemi
3937 secretes an arsenal of pectinolytic enzymes, including at least eight endo-pectate lyases encoded by
pel
genes, which play a major role in the soft-rot disease caused by this bacterium on various plants.
E. chrysanthemi
also produces some hydrolases that cleave pectin. Three adjacent hydrolase genes,
pehV
,
pehW
, and
pehX,
encoding exo-poly-α-
d
-galacturonosidases, have been characterized. These enzymes liberate digalacturonides from the nonreducing end of pectin. We report the identification of a novel gene, named
pehN,
encoding a protein homologous to the glycosyl hydrolases of family 28, which includes mainly polygalacturonases. PehN has a low hydrolase activity on polygalacturonate and on various pectins. PehN action favors the activity of the secreted endo-pectate lyases, mainly PelB and PelC, and that of the periplasmic exo-pectate lyase PelX. However, removal of the
pehN
gene does not significantly alter the virulence of
E. chrysanthemi
. Regulation of
pehN
transcription was analyzed by using gene fusions. Like other pectinase genes,
pehN
transcription is dependent on several environmental conditions. It is induced by pectic catabolic products and is affected by growth phase, catabolite repression, osmolarity, anaerobiosis, nitrogen starvation, and the presence of calcium ions. The transcription of
pehN
is modulated by the repressor KdgR, which controls almost all the steps of pectin catabolism, and by cyclic AMP receptor protein (CRP), the global activator of sugar catabolism. The regulator PecS, which represses the transcription of the
pel
genes but activates that of
pehV
,
pehW
, and
pehX
, also activates transcription of
pehN
. The three regulators KdgR, PecS, and CRP act by direct interaction with the
pehN
promoter region. The sequences involved in the binding of these three regulators and of RNA polymerase have been precisely defined. Analysis of the simultaneous binding of these proteins indicates that CRP and RNA polymerase bind cooperatively and that the binding of KdgR could prevent
pehN
transcription. In contrast, the activator effect of PecS is not linked to competition with KdgR or to cooperation with CRP or RNA polymerase. This effect probably results from competition between PecS and an unidentified repressor involved in
peh
regulation.
New Type of Osmoregulated Solute Transporter Identified in Halophilic Members of theBacteriaDomain: TRAP Transporter TeaABC Mediates Uptake of Ectoine and Hydroxyectoine inHalomonas elongataDSM 2581T
Authors: Grammann Katrin , Volke Angela , Kunte Hans Jörg
Published: July 2002

The halophilic bacteriumHalomonas elongatasynthesizes as its main compatible solute the aspartate derivative ectoine. We constructed a deletion mutant ofH. elongata, KB1, defective in ectoine synthesis and tolerating elevated salt concentrations only in the presence of external compatible solutes. The dependency of KB1 on solute uptake for growth in high-salt medium was exploited to select insertion mutants unable to accumulate external solutes via osmoregulated transporters. One insertion mutant out of 7,200 failed to accumulate the osmoprotectants ectoine and hydroxyectoine. Genetic analysis of the insertion site proved that the mutation affected an open reading frame (ORF) of 1,281 bp (teaC). The nucleotide sequence upstream ofteaCwas determined, and two further ORFs of 603 bp (teaB) and 1,023 bp (teaA) were identified. Deletion ofteaAandteaBproved that all three genes are mandatory for ectoine uptake. Sequence comparison showed significant identity of TeaA, TeaB, and TeaC to the transport proteins of the recently identified tripartite ATP-independent periplasmic transporter family (TRAP-T). The affinity of the cells for ectoines was determined (Ks= 21.7 μM), suggesting that the transporter TeaABC exhibits high affinity for ectoines. An elevation of the external osmolarity resulted in a strong increase in ectoine uptake via TeaABC, demonstrating that this transporter is osmoregulated. Deletion ofteaCandteaBCin the wild-type strain led to mutants which excreted significant amounts of ectoine into the medium when cultivated at high salt concentrations. Therefore, the physiological role of TeaABC may be primarily to recover ectoine leaking through the cytoplasmic membrane.
Differential Regulation of Multiple Proteins ofEscherichia coliandSalmonella entericaSerovar Typhimurium by the Transcriptional Regulator SlyA
Authors: Spory Andrea , Bosserhoff Armin , von Rhein Christine , Goebel Werner , Ludwig Albrecht
Published: July 2002

SlyA is a transcriptional regulator ofEscherichia coli,Salmonella enterica, and other bacteria belonging to theEnterobacteriaceae. The SlyA protein has been shown to be involved in the virulence ofS. entericaserovar Typhimurium, but its role inE. coliis unclear. In this study, we employed the proteome technology to analyze the SlyA regulons of enteroinvasiveE. coli(EIEC) andSalmonellaserovar Typhimurium. In both cases, comparative analysis of the two-dimensional protein maps of a wild-type strain, a SlyA-overproducing derivative, and a correspondingslyAmutant revealed numerous proteins whose expression appeared to be either positively or negatively controlled by SlyA. Twenty of the putative SlyA-induced proteins and 13 of the putative SlyA-repressed proteins of the tested EIEC strain were identified by mass spectrometry. The former proteins included several molecular chaperones (GroEL, GroES, DnaK, GrpE, and CbpA), proteins involved in acid resistance (HdeA, HdeB, and GadA), the “starvation lipoprotein” (Slp), cytolysin ClyA (HlyE or SheA), and several enzymes involved in metabolic pathways, whereas most of the latter proteins proved to be biosynthetic enzymes. Consistently, the resistance of the EIECslyAmutant to heat and acid stress was impaired compared to that of the wild-type strain. Furthermore, the implication of SlyA in the regulation of several of the identifiedE. coliproteins was confirmed at the level of transcription withlacZfusions. Twenty-three of theSalmonellaserovar Typhimurium proteins found to be affected by SlyA were also identified by mass spectrometry. With the exception of GroEL these differed from those identified in the EIEC strain and included proteins involved in various processes. The data suggest that gene regulation by SlyA might be crucial for intracellular survival and/or replication of both EIEC andSalmonellaserovar Typhimurium in phagocytic host cells.
Characterization of
comQ
and
comX
, Two Genes Required for Production of ComX Pheromone in
Bacillus subtilis
Authors: Schneider Katherine Bacon , Palmer Tanya M. , Grossman Alan D.
Published: July 2002

Many microbes use secreted peptide-signaling molecules to stimulate changes in gene expression in response to high population density, a process called quorum sensing. ComX pheromone is a modified 10-amino-acid peptide used by
Bacillus subtilis
to modulate changes in gene expression in response to crowding.
comQ
and
comX
are required for production of ComX pheromone. We found that accumulation of ComX pheromone in culture supernatant paralleled cell growth, indicating that there was no autoinduction of production of ComX pheromone. We overexpressed
comQ
and
comX
separately and together and found that overexpression of
comX
alone was sufficient to cause an increase in production of ComX pheromone and early induction of a quorum-responsive promoter. These results indicate that the extracellular concentration of ComX pheromone plays a major role in determining the timing of the quorum response and that expression of
comX
is limiting for production of ComX pheromone. We made alanine substitutions in the residues that comprise the peptide backbone of ComX pheromone. Analysis of these mutants highlighted the importance of the modification for ComX pheromone function and identified three residues (T50, G54, and D55) that are unlikely to interact with proteins involved in production of or response to ComX pheromone. We have also identified and mutated a putative isoprenoid binding domain of ComQ. Mutations in this domain eliminated production of ComX pheromone, consistent with the hypothesis that ComQ is involved in modifying ComX pheromone and that the modification is likely to be an isoprenoid.
Comparison of In Vitro Activities of Voriconazole and Five Established Antifungal Agents against Different Species of Dermatophytes Using a Broth Macrodilution Method
Authors: Perea Sofia , Fothergill Annette W. , Sutton Deanna A. , Rinaldi Michael G.
Published: July 2002

The in vitro activities of voriconazole against 19 different species of dermatophytes were compared with those of terbinafine, itraconazole, ketoconazole, griseofulvin, and fluconazole. MICs were determined according to a National Committee for Clinical Laboratory Standards broth macrodilution method. Voriconazole appeared more active than ketoconazole, griseofulvin, and fluconazole and less active than itraconazole and terbinafine. Based on these results, voriconazole merits further investigation as a potentially useful agent for the treatment of dermatophytosis.
Heterogeneity of
Pseudomonas aeruginosa
in Brazilian Cystic Fibrosis Patients
Authors: Silbert Suzane , Barth Afonso Luis , Sader Hélio S.
Published: July 2002

The aim of this study was to assess the diversity and genomic variability of
Pseudomonas aeruginosa
isolates from cystic fibrosis (CF) patients being treated at a university hospital in Brazil. Ninety-seven isolates of
P. aeruginosa
from 43 CF patients were characterized by macrorestriction analysis of chromosomal DNA by pulsed-field gel electrophoresis (PFGE) and tested for susceptibility to 20 antimicrobial agents by broth microdilution. It was possible to evaluate single isolates from 20 patients and multiple isolates (two to seven) from 23 patients collected during a 22-month period. Among all of the unrelated patients, we detected only one pair of patients sharing a common strain. Among the 77 isolates from 23 patients who had multiple isolates analyzed, we identified 37 major types by PFGE, and five different colonization patterns were recognized. The isolates were susceptible to several antimicrobial agents, although consecutive isolates from the same patient may display differences in their susceptibilities. Mucoid isolates were more resistant (
P
< 0.001) than nonmucoid isolates to five antibiotics. Our results indicate that CF patients remain colonized by more than one strain of
P. aeruginosa
for long periods of time. In addition, the finding of several different genotypes in the same patient suggests that the colonizing strain may occasionally be replaced.
Rapid and Accurate Species-Level Identification of Coagulase-Negative Staphylococci by Using the
sodA
Gene as a Target
Authors: Poyart Claire , Quesne Gilles , Boumaila Claire , Trieu-Cuot Patrick
Published: July 2002

Simple PCR and sequencing assays that utilize a single pair of degenerate primers were used to characterize a 429-bp-long DNA fragment internal (
sodA
int
) to the
sodA
gene encoding the manganese-dependent superoxide dismutase in 40 coagulase-negative staphylococcal (CNS) type strains. The topology of the phylogenetic tree obtained was in general agreement with that which was inferred from an analysis of their 16S rRNA or
hsp60
gene sequences. Sequence analysis revealed that the staphylococcal
sodA
genes exhibit a higher divergence than does the corresponding 16S ribosomal DNA. These results confirm that the
sodA
gene constitutes a highly discriminative target sequence for differentiating closely related bacterial species. Clinical isolates that could not be identified at the species level by phenotypical tests were identified by use of this database. These results demonstrate the usefulness of this method for rapid and accurate species identification of CNS isolates, although it does not allow discrimination of subspecies. The
sodA
sequence polymorphisms observed with staphylococcal species offer good opportunities for the development of assays based on DNA chip technologies.
Surveillance of Invasive
Streptococcus pneumoniae
Infection in the Province of Quebec, Canada, from 1996 to 1998: Serotype Distribution, Antimicrobial Susceptibility, and Clinical Characteristics
Authors: Jetté L. P. , Delage G. , Ringuette L. , Allard R. , De Wals P. , Lamothe F. , Loo V. ,   the Pneumococcus Study Group
Published: July 2002

In the province of Quebec, Canada, from 1996 to 1998, 3,650 invasive
Streptococcus pneumoniae
infections were reported. A total of 1,354 isolates were serotyped and tested for antimicrobial susceptibility. The distribution of serotypes remained stable over the 3 years, with serotypes 14, 6B, 4, 9V, 23F, and 19F accounting for 61% of the isolates. Overall, 90% of isolates were included in the current 23-valent vaccine and 67% were included in the 7-valent conjugate vaccine. We were able to determine that resistance to penicillin and to other antibiotics is increasing.
Clinical Evaluation of the Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test for Rapid Detection of
Mycobacterium tuberculosis
in Select Nonrespiratory Specimens
Authors: Woods Gail L. , Bergmann John S. , Williams-Bouyer Natalie
Published: July 2002

The performance of the Amplified Mycobacterium Tuberculosis Direct Test (MTD; Gen-Probe, Inc., San Diego, Calif.) for rapid diagnosis of extrapulmonary tuberculosis was evaluated by testing 178 nonrespiratory specimens from 158 patients. Criteria for specimen inclusion were (i) a positive smear for acid-fast bacilli (
n
= 54) and (ii) the source if the smear was negative (tissue biopsies and aspirates and abscess material were tested;
n
= 124). Results were compared to those of mycobacterial culture; clinical history was reviewed when MTD and culture results disagreed. Forty-eight specimens (27.0%) were positive for mycobacteria, including 23
Mycobacterium tuberculosis
complex specimens; of which 21 were smear positive. Twenty-five specimens were MTD positive; 20 of these grew
M. tuberculosis
complex. All of the five MTD-positive,
M. tuberculosis
complex culture-negative specimens were considered truly positive, based on review of the medical record. Of the three MTD-negative,
M. tuberculosis
complex culture-positive specimens, two contained inhibitory substances; one of the two was smear positive. Excluding the latter specimen from analysis, after chart review, the sensitivity, specificity, and positive and negative predictive values of the MTD were 92.6, 100, 100, and 98.7%, respectively, by specimen and 89.5, 100, 100, and 98.6% by patient. Given the few smear-negative samples from patients with extrapulmonary tuberculosis in our study, additional similar studies that include more smear-negative,
M. tuberculosis
complex culture-positive specimens to confirm our data are desirable.
Tsukamurella strandjordae
sp. nov., a Proposed New Species Causing Sepsis
Authors: Kattar Mireille M. , Cookson Brad T. , Carlson LaDonna C. , Stiglich Susan K. , Schwartz Margot A. , Nguyen Trang T. , Daza Riza , Wallis Carolyn K. , Yarfitz Stuart L. , Coyle Marie B.
Published: July 2002

We have isolated a gram-positive, weakly acid-alcohol-fast, irregular rod-shaped bacterium from cultures of blood from a 5-year-old girl with acute myelogenous leukemia. This isolate was compared with 14 other strains including reference strains of
Tsukamurella
species by a polyphasic approach based on physiological and biochemical properties, whole-cell short-chain fatty acid and mycolic acid analyses, DNA-DNA hybridization, and sequencing of the 16S rRNA gene. This isolate represents a new taxon within the genus
Tsukamurella
for which we propose the name
Tsukamurella strandjordae
sp. nov. Our study also revealed that
Tsukamurella paurometabola
ATCC 25938 represents a misnamed
Tsukamurella inchonensis
isolate and confirms that
Tsukamurella wratislaviensis
belongs to the genus
Rhodococcus
.
Quantitation of Bacteria in Bone Marrow from Patients with Typhoid Fever: Relationship between Counts and Clinical Features
Authors: Wain John , Bay Phan Van Be , Vinh Ha , Duong Nguyen M. , Diep To Song , Walsh Amanda L. , Parry Christopher M. , Hasserjian Robert P. , Ho Vo Anh , Hien Tran T. , Farrar Jeremy , White Nicholas J. , Day Nicholas P. J.
Published: July 2002

Enteric fever is the only bacterial infection of humans for which bone marrow examination is routinely recommended. A prospective study of the concentrations of bacteria in the bone marrow and their relationship to clinical features was conducted with 120 Vietnamese patients with suspected enteric fever, of whom 89 had confirmed typhoid fever. Ninety-three percent of the
Salmonella enterica
serovar Typhi samples isolated were resistant to ampicillin, chloramphenicol, and co-trimoxazole. For 81 patients with uncomplicated typhoid and satisfactory bone marrow aspirates, the number of serovar Typhi CFU in bone marrow aspirates was a median value of 9 (interquartile range [IQR], 1 to 85; range, 0.1 to 1,580) compared to 0.3 (IQR, 0.1 to 10; range, 0.1 to 399) CFU/ml in simultaneously sampled blood. The ratio of individual blood counts to bone marrow counts was 10 (IQR, 2.3 to 97.5). The number of bacteria in blood but not bone marrow was correlated inversely with the duration of preceding fever. Thus, with increasing duration of illness the ratio of bone marrow-to-blood bacterial concentrations increased; the median ratio was 4.8 (IQR, 1 to 27.5) during the first week compared with 158 (IQR, 60 to 397) during the third week. After lysing the host cells, the median ratio of viable bone marrow to blood increased, reflecting the higher concentration of intracellular serovar Typhi in the bone marrow. Effective antibiotic pretreatment had a significantly greater effect in reducing blood counts compared to bone marrow counts (
P
< 0.001). Thus, bacteria in the bone marrow of typhoid patients are less affected by antibiotic treatment than bacteria in the blood. The numbers of bacteria in bone marrow correlated negatively with the white blood cell (
R
= −0.3,
P
= 0.006) and platelet counts (
R
= −0.32,
P
= 0.01) and positively with fever clearance time after treatment (
R
= 0.4,
P
< 0.001). The bacterial load in bone marrow therefore may reflect the clinical course of the infection, and high levels may suppress neutrophil proliferation.
Genetic Variation among Human Isolates of Uninucleated Cyst-Producing
Entamoeba
Species
Authors: Verweij Jaco J. , Polderman Anton M. , Clark C. Graham
Published: July 2002

Twelve human infections with
Entamoeba
spp. producing uninucleated cysts were studied. DNA was extracted from infected feces and used to amplify part of the ameba small-subunit rRNA gene. Sequence analysis identified four distinct types of
Entamoeba
, all of which are related to
Entamoeba polecki
and
E. chattoni
and two of which have not been reported previously. Whether these genetic types represent different species is unclear. We propose that the agent of all human infections with uninucleated cyst-producing
Entamoeba
species be reported as “
E. polecki
-like.”
Utility of Paired BACTEC MYCO/F LYTIC Blood Culture Vials for Detection of Bacteremia, Mycobacteremia, and Fungemia
Authors: Archibald Lennox K. , Dobbie Hamish , Kazembe Peter , Nwanyanwu Okey , McKnight Celeste , Byrne Terry , Addison Rachel M. , Bell Michael , Reller L. Barth , Jarvis William R.
Published: July 2002

In previous bloodstream infection studies in Malawi, we inoculated blood from a single venesection into a single BACTEC MYCO/F LYTIC (MFL) vial. Inoculation of one vial, however, would be expected to reduce the sensitivity of bloodstream pathogen detection with MFL vials. To ascertain the degree of this loss of sensitivity, blood was drawn from each of 228 febrile, adult inpatients in Malawi and 5 ml of each blood sample was inoculated into each of two MFL vials. Of 228 paired vials, 51 (22%) were both positive, 172 (75%) were both negative, and 5 (3%) had discordant results. Bloodstream infection would have been detected in 11 (92%) of 12 patients with mycobacteremia and 38 (92%) of 41 patients with bacteremia had only one MFL vial been inoculated. Our study shows that a second MFL vial does not significantly increase diagnostic sensitivity.
Antifungal Susceptibility Testing of Fluconazole by Flow Cytometry Correlates with Clinical Outcome
Authors: Wenisch Christoph , Moore Caroline B. , Krause Robert , Presterl Elisabeth , Pichna Peter , Denning David W.
Published: July 2002

Susceptibility testing of fungi by flow cytometry (also called fluorescence-activated cell sorting [FACS]) using vital staining with FUN-1 showed a good correlation with the standard M27-A procedure for assessing MICs. In this study we determined MICs for blood culture isolates from patients with candidemia by NCCLS M27-A and FACS methods and correlated the clinical outcome of these patients with in vitro antifungal resistance test results. A total of 24 patients with candidemia for whom one or more blood cultures were positive for a
Candida
sp. were included. Susceptibility testing was performed by NCCLS M27-A and FACS methods. The correlation of MICs (NCCLS M27-A and FACS) and clinical outcome was calculated. In 83% of the cases, the MICs of fluconazole determined by FACS were within 1 dilution of the MICs determined by the NCCLS M27-A method. For proposed susceptibility breakpoints, there was 100% agreement between the M27-A and FACS methods. In the FACS assay, a fluconazole MIC of <1 μg/ml was associated with cure (
P
< 0.001) whereas an MIC of ≥1 μg/ml was associated with death (
P
< 0.001). The M27-A-derived fluconazole MICs did not correlate with outcome (
P
= 1 and
P
= 0.133).
Molecular Identification of Microorganisms from Endodontic Infections
Authors: Rolph H. J. , Lennon A. , Riggio M. P. , Saunders W. P. , MacKenzie D. , Coldero L. , Bagg J.
Published: July 2002

A relatively wide range of bacteria have been isolated from root canals using standard culture techniques. However, only 50% of the bacteria in the oral cavity are cultivable (S. S. Socransky et al., Arch. Oral Biol. 8:278–280, 1963); hence, bacterial diversity in endodontic infections is underestimated. This study used a PCR-based 16S rRNA gene assay, followed by cloning and sequencing of 16S rRNA amplicons from a small subset of samples to assess the diversity of bacteria present in infected root canals. A total of 41 clinical samples from 15 de novo and 26 refractory cases of endodontic infections were assessed. Of these samples, 44% were positive by culture and 68% were positive by PCR. Eight samples were selected for further analysis. Of these, the two de novo cases yielded sequences related to those of the genera
Enterococcus, Lactobacillus, Propionibacterium
, and
Streptococcus
and two clones were related to previously uncultivated bacteria, while the sinus-associated, de novo case yielded sequences related to those of the genera
Lactobacillus, Pantoea, Prevotella
, and
Selenomonas
. The five refractory cases produced clones which were related to the genera
Capnocytophaga, Cytophaga, Dialister, Eubacterium, Fusobacterium, Gemella, Mogibacterium, Peptostreptococcus, Prevotella, Propionibacterium, Selenomonas, Solobacterium, Streptococcus
, and
Veillonella
and two clones representing previously uncultivated bacteria. The phylogenetic positions of several clones associated with the
Clostridiaceae
and
Sporomusa
subgroups of the
Firmicutes
grouping are also shown. This study demonstrates that molecular techniques can detect the presence of bacteria in endodontic infections when culture techniques yield a negative result and can be used to identify a wider range of endodontic-infection-related bacteria including the presence of previously unidentified or unculturable bacteria.
Genetic Diversity of
Streptococcus suis
Strains Isolated from Pigs and Humans as Revealed by Pulsed-Field Gel Electrophoresis
Authors: Berthelot-Hérault Florence , Marois Corinne , Gottschalk Marcelo , Kobisch Marylène
Published: July 2002

The genetic diversity of 123
Streptococcus suis
strains of capsular types 2, 1/2, 3, 7, and 9, isolated from pigs in France and from humans in different countries, was evaluated by pulsed-field gel electrophoresis (PFGE) of DNA restricted with
Sma
I. The method was highly discriminative (
D
= 0.98), results were reproducible, and the PFGE analysis was easy to interpret. Among all
S. suis
strains, 74 PFGE patterns were shown. At 60% homology, three groups (A, B, and C) were identified, and at 69% homology, eight subgroups (a to h) were observed. Strains isolated from diseased pigs or from humans were statistically clustered in group B, especially in subgroup d. By contrast,
S. suis
strains isolated from clinically healthy pigs were preferentially included in subgroup b of group A. Relationships could be established between capsular types 1/2, 3, and 9 and groups A, e, and B, respectively.
S. suis
strains isolated from humans were homogeneous, and a very high level of association between these strains and four DNA patterns was observed. The PFGE used in this study is a very useful tool for evaluating the genetic diversity of
S. suis
strains, and it would be used for epidemiological investigations.
PCR Detection and DNA Sequence Analysis of the Regulatory Region of Lymphotropic Papovavirus in Peripheral Blood Mononuclear Cells of an Immunocompromised Rhesus Macaque
Authors: Lednicky John A. , Halvorson Steven J. , Butel Janet S.
Published: July 2002

A lymphotropic papovavirus (LPV) archetypal regulatory region was amplified from DNA from the blood of an immunocompromised rhesus monkey. We believe this is the first nonserological evidence of LPV infection in rhesus monkeys.
Streptococcus sinensis
sp. nov., a Novel Species Isolated from a Patient with Infective Endocarditis
Authors: Woo Patrick C. Y. , Tam Dorothy M. W. , Leung Kit-Wah , Lau Susanna K. P. , Teng Jade L. L. , Wong Michelle K. M. , Yuen Kwok-Yung
Published: July 2002

A bacterium was isolated from the blood culture of a patient with infective endocarditis. The cells were facultative anaerobic, nonsporulating, gram-positive cocci arranged in chains. The bacterium grows on sheep blood agar as alpha-hemolytic, gray colonies of 0.5 to 1 mm in diameter after 24 h of incubation at 37°C in ambient air. Growth also occurs in 10 or 40% bile and on bile esculin agar but not in 6% NaCl. No enhancement of growth is observed in 5% CO
2
. It is nongroupable with Lancefield groups A, B, C, D, F, or G antisera and is resistant to optochin and bacitracin. The organism is aflagellated and is nonmotile at both 25 and 37°C. It is Voges-Proskauer test positive. It produces leucine arylamidase and β-glucosidase but not catalase, urease, lysine decarboxylase, or ornithine decarboxylase. It hydrolyzes esculin and arginine. It utilizes glucose, lactose, salicin, sucrose, pullulan, trehalose, cellobiose, hemicellulase, mannose, maltose, and starch. 16S rRNA gene sequencing showed that there were 3.6, 3.7, 4.3, 4.7, and 5.9% differences between the 16S rRNA gene sequence of the bacterium and those of
Streptococcus gordonii
,
Streptococcus intermedius
,
Streptococcus constellatus
,
Streptococcus sanguis
, and
Streptococcus anginosus
, respectively. The G+C content of it (mean ± standard deviation) was 53.0% ± 2.9%. Based on phylogenetic affiliation, it belongs to the mitis or anginosus group of
Streptococcus
. For these reasons a new species,
Streptococcus sinensis
sp. nov., is proposed, for which HKU4 is the type strain. Further studies should be performed to ascertain the potential of this bacterium to become an emerging cause of infective endocarditis.
Temperature-Mediated Heteroduplex Analysis Performed by Using Denaturing High-Performance Liquid Chromatography To Identify Sequence Polymorphisms in
Mycobacterium tuberculosis
Complex Organisms
Authors: Cooksey Robert C. , Morlock Glenn P. , Holloway Brian P. , Limor Josef , Hepburn Michael
Published: July 2002

PCR products containing sequence polymorphisms were prepared from six mycobacterial genes, denatured, mixed with reference PCR products, and reannealed; the mixtures were then examined with a denaturing high-performance liquid chromatography system (WAVE) equipped with a temperature-controlled alkalated polystyrene divinyl benzene column. Mismatching of bases in heteroduplexes of the PCR products causes elution patterns of the DNA from the column to be altered. The six mycobacterial genes studied were
oxyR
, in which a specific polymorphism (G
1031
A) is found only in certain species of the
Mycobacterium tuberculosis
complex, and five genes in which mutations associated with antituberculosis drug resistance have been found. The resistance genes (with affected drug and PCR product sizes given parenthetically) were
rpoB
(rifampin; 258 bp),
katG
(isoniazid; 205 bp),
pncA
(pyrazinamide; 579 bp);
rpsL
(streptomycin; 196 bp), and
embB
(ethambutol; 185 bp). Elution patterns of heteroduplexes of all 20 polymorphisms studied shifted detectably at column temperatures ranging from 65.3 to 68°C and elution times of 3.5 to 6 min. These results show that temperature-mediated heteroduplex analysis is a potentially useful genotypic screen for mutations associated with antituberculosis drug resistance and for the G
1031
A polymorphism in
oxyR
. The method may allow users to detect novel as well as heterogeneous mutations without using expensive kits or detection labels.
Detection of Plasmid-Mediated AmpC β-Lactamase Genes in Clinical Isolates by Using Multiplex PCR
Authors: Pérez-Pérez F. Javier , Hanson Nancy D.
Published: July 2002

Therapeutic options for infections caused by gram-negative organisms expressing plasmid-mediated AmpC β-lactamases are limited because these organisms are usually resistant to all the β-lactam antibiotics, except for cefepime, cefpirome, and the carbapenems. These organisms are a major concern in nosocomial infections and should therefore be monitored in surveillance studies. Six families of plasmid-mediated AmpC β-lactamases have been identified, but no phenotypic test can differentiate among them, a fact which creates problems for surveillance and epidemiology studies. This report describes the development of a multiplex PCR for the purpose of identifying family-specific AmpC β-lactamase genes within gram-negative pathogens. The PCR uses six sets ofampC-specific primers resulting in amplicons that range from 190 bp to 520 bp and that are easily distinguished by gel electrophoresis.ampCmultiplex PCR differentiated the six plasmid-mediatedampC-specific families in organisms such asKlebsiella pneumoniae,Escherichia coli,Proteus mirabilis, andSalmonella entericaserovar Typhimurium. Family-specific primers did not amplify genes from the other families ofampCgenes. Furthermore, this PCR-based assay differentiated multiple genes within one reaction. In addition, WAVE technology, a high-pressure liquid chromatography-based separation system, was used as a way of decreasing analysis time and increasing the sensitivity of multiple-gene assays. In conclusion, a multiplex PCR technique was developed for identifying family-specificampCgenes responsible for AmpC β-lactamase expression in organisms with or without a chromosomal AmpC β-lactamase gene.
Efficient Particle Production by Minimal Gag Constructs Which Retain the Carboxy-Terminal Domain of Human Immunodeficiency Virus Type 1 Capsid-p2 and a Late Assembly Domain
Authors: Accola Molly A. , Strack Bettina , Göttlinger Heinrich G.
Published: July 2002

The human immunodeficiency virus type 1 (HIV-1) Gag precursor Pr55

gag

by itself is capable of assembling into retrovirus-like particles (VLP). In the present study, we attempted to identify the minimal Gag sequences required for the formation of VLP. Our results show that about 80% of Pr55

gag

can be either deleted or replaced by heterologous sequences without significantly compromising VLP production. The smallest chimeric molecule still able to efficiently form VLP was only about 16 kDa. This minimal Gag construct contained the leucine zipper domain of the yeast transcription factor GCN4 to substitute for the assembly function of nucleocapsid (NC), followed by a P-P-P-P-Y motif to provide late budding (L) domain function, and retained only the myristylation signal and the C-terminal capsid-p2 domain of Pr55

gag

. We also show that the L domain function of HIV-1 p6

gag

is not dependent on the presence of an active viral protease and that the NC domain of Pr55

gag

is dispensable for the incorporation of Vpr into VLP.
Conserved Surface-Exposed K/R-X-K/R Motifs and Net Positive Charge on Poxvirus Complement Control Proteins Serve as Putative Heparin Binding Sites and Contribute to Inhibition of Molecular Interactions with Human Endothelial Cells: a Novel Mechanism for Evasion of Host Defense
Authors: Smith Scott A. , Mullin Nick P. , Parkinson John , Shchelkunov Sergei N. , Totmenin Alexei V. , Loparev V. N. , Srisatjaluk Ratchapin , Reynolds David N. , Keeling Kristen L. , Justus David E. , Barlow Paul N. , Kotwal Girish J.
Published: July 2002

Vaccinia virus complement control protein (VCP) has been shown to possess the ability to inhibit both classical and alternative complement pathway activation. The newly found ability of this protein to bind to heparin has been shown in previous studies to result in uptake by mast cells, possibly promoting tissue persistence. It has also been shown to reduce chemotactic migration of leukocytes by blocking chemokine binding. In addition, this study shows that VCP—through its ability to bind to glycosaminoglycans (heparin-like molecules) on the surface of human endothelial cells—is able to block antibody binding to surface major histocompatibility complex class I molecules. Since heparin binding is critical for many functions of this protein, we have attempted to characterize the molecular basis for this interaction. Segments of this protein, generated by genetic engineering of the DNA encoding VCP into thePichia pastorisexpression system, were used to localize the regions with heparin binding activity. These regions were then analyzed to more specifically define their properties for binding. It was found that the number of putative binding sites (K/R-X-K/R), the overall positive charge, and the percentage of positively charged amino acids within the protein were responsible for this interaction.