bluuebunny/crossref_metadata_2025_split huggingface dataset:
Genetic Organization of the Region Encoding Regulation, Biosynthesis, and Transport of Rhizobactin 1021, a Siderophore Produced by
Sinorhizobium meliloti
Authors: Lynch Damien , O'Brien John , Welch Timothy , Clarke Paul , ÓCuı́v Páraic , Crosa Jorge H. , O'Connell Michael 
Published: July 2002

ABSTRACT

Eight genes have been identified that function in the regulation, biosynthesis, and transport of rhizobactin 1021, a hydroxamate siderophore produced under iron stress by
Sinorhizobium meliloti
. The genes were sequenced, and transposon insertion mutants were constructed for phenotypic analysis. Six of the genes, named
rhbABCDEF
, function in the biosynthesis of the siderophore and were shown to constitute an operon that is repressed under iron-replete conditions. Another gene in the cluster, named
rhtA
, encodes the outer membrane receptor protein for rhizobactin 1021. It was shown to be regulated by iron and to encode a product having 61% similarity to IutA, the outer membrane receptor for aerobactin. Transcription of both the
rhbABCDEF
operon and the
rhtA
gene was found to be positively regulated by the product of the eighth gene in the cluster, named
rhrA
, which has characteristics of an AraC-type transcriptional activator. The six genes in the
rhbABCDEF
operon have interesting gene junctions with short base overlaps existing between the genes. Similarities between the protein products of the biosynthesis genes and other proteins suggest that rhizobactin 1021 is synthesized by the formation of a novel siderophore precursor, 1,3-diaminopropane, which is then modified and attached to citrate in steps resembling those of the aerobactin biosynthetic pathway. The cluster of genes is located on the pSyma megaplasmid of
S. meliloti
2011. Reverse transcription-PCR with RNA isolated from mature alfalfa nodules yielded no products for
rhbF
or
rhtA
at a time when the
nifH
gene was strongly expressed, indicating that siderophore biosynthesis and transport genes are not strongly expressed when nitrogenase is being formed in root nodules. Mutants having transposon insertions in the biosynthesis or transport genes induced effective nitrogen-fixing nodules on alfalfa plants.
Functional Characterization and Regulation ofgadX, a Gene Encoding an AraC/XylS-Like Transcriptional Activator of theEscherichia coliGlutamic Acid Decarboxylase System
Authors: Tramonti Angela , Visca Paolo , De Canio Michele , Falconi Maurizio , De Biase Daniela 
Published: July 2002

ABSTRACTTheEscherichia colichromosome contains two distantly located genes,gadAandgadB, which encode biochemically undistinguishable isoforms of glutamic acid decarboxylase (Gad). The Gad reaction contributes to pH homeostasis by consuming intracellular H+and producing γ-aminobutyric acid. This compound is exported via the protein product of thegadCgene, which is cotranscribed withgadB. Here we demonstrate that transcription of bothgadAandgadBCis positively controlled bygadX, a gene downstream ofgadA, encoding a transcriptional regulator belonging to the AraC/XylS family. ThegadXpromoter encompasses the 67-bp region preceding thegadXtranscription start site and contains both RpoD and RpoS putative recognition sites. Transcription ofgadXoccurs in neutral rich medium upon entry into the stationary phase and is increased at acidic pH, paralleling the expression profile of thegadstructural genes. However, PT5lacO-controlledgadXexpression in neutral rich medium results in upregulation of target genes even in exponential phase, i.e., when thegadsystem is normally repressed. Autoregulation of the wholegadsystem is inferred by the positive effect of GadX on thegadApromoter andgadAXcotranscription. Transcription ofgadXis derepressed in anhnsmutant and strongly reduced in bothrpoSandhns rpoSmutants, consistent with the expression profile ofgadstructural genes in these genetic backgrounds. Gel shift and DNase I footprinting analyses with a MalE-GadX fusion protein demonstrate that GadX bindsgadAandgadBCpromoters at different sites and with different binding affinities.
Elevated Levels of Ketopantoate Hydroxymethyltransferase (PanB) Lead to a Physiologically Significant Coenzyme A Elevation in
Salmonella enterica
Serovar Typhimurium
Authors: Rubio Aileen , Downs D. M. 
Published: July 2002

ABSTRACT

Pantothenate is the product of the ATP-dependent condensation of pantoate and β-alanine and is a direct precursor of coenzyme A. A connection exists between pantothenate biosynthesis and thiamine biosynthesis in
Salmonella enterica
serovar Typhimurium since derivatives of a
purF
mutant that can grow (on glucose medium) in the absence of thiamine excrete pantothenate. We show here that the causative mutation in three such mutants was the addition of a CG base pair upstream of the
panB
gene. This base addition brings the spacing between the −10 and −35 hexamers of the promoter to a consensus spacing of 17 bp and results in increased transcription of the
pan
operon. Furthermore, overexpression of PanB caused by this mutation, or by other means, was necessary and sufficient to increase pantothenate production and allow PurF-independent thiamine synthesis on glucose medium.
Use of Plastic Vacutainer Tubes for Quantification of Human Immunodeficiency Virus Type 1 in Blood Specimens
Authors: Landry Marie L. , Garner Robin , Ferguson David 
Published: July 2002

ABSTRACT

Human immunodeficiency virus type 1 viral load results were compared for paired samples collected in plastic and glass Vacutainer tubes, using both standard (
n
= 60) and ultrasensitive (
n
= 66) assays. The results showed a strong correlation (
P
< 0.0001), and plastic tubes can be substituted for glass tubes.
Use of Penicillin MICs To Predict In Vitro Activity of Other β-Lactam Antimicrobial Agents against
Streptococcus pneumoniae
Authors: Brueggemann A. B. , Pfaller M. A. , Doern G. V. 
Published: July 2002

ABSTRACT

Linear regression analysis was used to compare penicillin MICs determined with 3,129 recent clinical isolates of
Streptococcus pneumoniae
to MICs obtained with nine other beta-lactam antimicrobial agents. A strong correlation between penicillin MICs and those obtained with other beta-lactams was demonstrated. It may be possible to test penicillin and use MICs obtained with penicillin to predict MICs of other beta-lactam antimicrobials for
Streptococcus pneumoniae
.
Skin Disease Presenting as an Outbreak of Pseudobacteremia in a Laboratory Worker
Authors: Simhon A. , Rahav G. , Shapiro M. , Block C. 
Published: July 2002

ABSTRACT

An outbreak of pseudobacteremia due to
Streptococcus pyogenes
(group A streptococci [GAS]) and methicillin-susceptible
Staphylococcus aureus
(MSSA) was traced to the venting procedure for aerobic bottles prior to their loading into the incubator of the BacT/Alert analyzer (Organon Teknika). Bacteria shed by a laboratory worker suffering from impetigo and cellulitis contaminated the aerobic bottles of 10 patients. All blood culture isolates, in addition to the isolates from the laboratory worker, were of the same GAS M and T types. All MSSA isolates from blood cultures and the index case's hands had the same lytic phage profile. Procedural breakdowns were identified in the laboratory. Bottles were vented outside the biological safety cabinet, gloves were not worn, and unprotected needles were used for the venting procedure. The source of the aspirated bacteria that contaminated the bottles was identified and the index case was treated promptly.
Influence of Variations in Test Methods on Susceptibility of
Haemophilus influenzae
to Ampicillin, Azithromycin, Clarithromycin, and Telithromycin
Authors: Fuchs Peter C. , Barry Arthur L. , Brown Steven D. 
Published: July 2002

ABSTRACT

The National Committee for Clinical Laboratory Standards standard broth microdilution method for testing the susceptibility of
Haemophilus influenzae
to ampicillin, azithromycin, clarithromycin, and telithromycin was evaluated by altering one variable at a time. Variables that were tested included age of colony for inoculum preparation, inoculum density, test medium, incubation atmosphere, and incubation time. For the macrolide, azalide, and ketolide agents, incubation in 5 to 7% CO
2
most significantly affected the MICs, producing nearly twofold increases for clarithromycin and telithromycin and a greater than threefold increase for azithromycin. For ampicillin, a 10-fold increase in inoculum density increased the geometric mean MICs for β-lactamase-negative strains from 1.50 to 2.45 μg/ml. In addition, 206
H. influenzae
strains were tested for their susceptibilities to the same drugs by the broth microdilution tests in two media, as well as by agar dilution tests, disk diffusion tests, and Etests, on six different agar media. The three standard methods with
Haemophilus
test medium (HTM) compared favorably with each other except for a high minor discrepancy rate (27%) by the disk diffusion test with ampicillin and clarithromycin. Agar dilution test MICs on the five comparative media were generally higher than those on HTM agar but were only rarely more than one twofold concentration higher. Etest MICs of azithromycin and telithromycin were more than twofold higher than agar dilution and broth microdilution MICs on HTM; ampicillin Etest MICs were nearly twofold lower. The use of media other than HTM agar appears to have a minimal effect on susceptibility test results for the ketolide, azalide, or macrolide drugs that we tested against
H. influenzae
.
Comparison of the Vitek Gram-Positive Susceptibility 106 Card, the MRSA-Screen Latex Agglutination Test, and
mecA
Analysis for Detecting Oxacillin Resistance in a Geographically Diverse Collection of Clinical Isolates of Coagulase-Negative Staphylococci
Authors: Yamazumi T. , Furuta I. , Diekema D. J. , Pfaller M. A. , Jones R. N. 
Published: July 2002

ABSTRACT

The Vitek automated susceptibility testing system with a modified gram-positive susceptibility (GPS) 106 card (bioMerieux Vitek, Inc., Hazelwood. Mo.) and a rapid slide latex agglutination test (MRSA-Screen test; Denka Seiken Co., Ltd., Tokyo, Japan) were evaluated for their abilities to detect oxacillin resistance in coagulase-negative staphylococci (CoNS). The reference broth microdilution method and the detection of the
mecA
gene by PCR (“gold standard” reference result) were used to compare the results obtained with the commercial products. A total of 123 clinical isolates consisting of eight species were selected from U.S. surveillance collections. Among the
mecA
-positive isolates (95 strains), 30 isolates were initially negative on the MRSA-Screen test read at 3 min. When the agglutination reaction was extended for 10 min, 26 of the 30 isolates became positive. For a different four isolates, the oxacillin MIC was ≤0.25 μg/ml on the Vitek GPS 106 card. Among the
mecA
-negative isolates (28 strains), for two
Staphylococcus warneri
, two
S. lugdunensis
, and two
S. saprophyticus
strains MICs were ≥0.5 μg/ml by the reference broth microdilution method. Four of these isolates were also categorized as resistant with the Vitek GPS 106 card and two isolates were positive by the MRSA-Screen test. Overall, the MRSA-Screen test, GPS 106 card, and reference broth microdilution method had sensitivities of 95.7 (result at 10 min), 95.7, and 100%, respectively, and specificities of 92.8, 85.7, and 78.5%, respectively. Although the MRSA-Screen test required a slight procedural modification, both commercial methods achieved a sensitivity and specificity at detecting oxacillin resistance in CoNS at a level that was acceptable for clinical laboratory use.
Identification of
Staphylococcus
spp. by PCR-Restriction Fragment Length Polymorphism of
gap
Gene
Authors: Yugueros Javier , Temprano Alejandro , Sánchez Marı́a , Luengo José Marı́a , Naharro Germán 
Published: July 2002

ABSTRACT

Oligonucleotide primers specific for the
Staphylococcus aureus gap
gene were previously designed to identify 12
Staphylococcus
spp. by PCR. In the present study,
Alu
I digestion of PCR-generated products rendered distinctive restriction fragment length polymorphism patterns that allowed 24
Staphylococcus
spp. to be identified with high specificity.
Contamination of the Clinical Microbiology Laboratory with Vancomycin-Resistant Enterococci and Multidrug- Resistant
Enterobacteriaceae
: Implications for Hospital and Laboratory Workers
Authors: Collins Susan M. , Hacek Donna M. , Degen Lisa A. , Wright Marc O. , Noskin Gary A. , Peterson Lance R. 
Published: July 2002

ABSTRACT

We surveyed environmental surfaces in our clinical microbiology laboratory to determine the prevalence of vancomycin-resistant enterococci (VRE) and multidrug-resistant
Enterobacteriaceae
(MDRE) during a routine working day. From a total of 193 surfaces, VRE were present on 20 (10%) and MDRE were present on 4 (2%) of the surfaces tested. In a subsequent survey after routine cleaning, all of the 24 prior positive surfaces were found to be negative. Thus, those in the laboratory should recognize that many surfaces may be contaminated by resistant organisms during routine processing of patient specimens.
PCR Analyses of tRNA Intergenic Spacer, 16S-23S Internal Transcribed Spacer, and Randomly Amplified Polymorphic DNA Reveal Inter- and Intraspecific Relationships of
Enterobacter cloacae
Strains
Authors: Clementino Maysa M. , de Filippis Ivano , Nascimento Carlos R. , Branquinho Regina , Rocha Carmem L. , Martins Orlando B. 
Published: July 2002

ABSTRACT

PCR analysis of tRNA intergenic spacer (tDNA-PCR) and of the 16S-23S internal transcribed spacer (ITS-PCR) and random amplified polymorphic DNA (RAPD) analysis were evaluated for their usefulness in characterization of
Enterobacter cloacae
strains isolated from both clinical origins and vaccine microbial contamination. tDNA-PCR presented specific and reproducible patterns for
Enterobacter sakazakii
ATCC 29004,
Enterobacter aerogenes
ATCC 13048, and
Enterobacter cloacae
ATCC 13047 and 23355 that presented the same profile for all 16
E. cloacae
isolates, offering an alternative tool for species-level identification. ITS-PCR and RAPD analysis yielded completely different banding patterns for the 20 strains studied, except for
E. cloacae
strains isolated from different batches of vaccine that exhibited a unique pattern, suggesting contamination by the same strain. The combined use of tDNA-PCR and ITS-PCR in a one-step protocol allows accurate identification and typing of
E. cloacae
strains a few hours after the colony has been isolated.
Origin of
Cryptococcus neoformans
var.
neoformans
Diploid Strains
Authors: Cogliati Massimo , Esposto Maria C. , Clarke David L. , Wickes Brian L. , Viviani Maria A. 
Published: July 2002

ABSTRACT

The basidiomycetous yeast
Cryptococcus neoformans
is an important human fungal pathogen. Two varieties,
C. neoformans
var.
neoformans
and
C. neoformans
var.
gattii
, have been identified. Both are heterothallic with two mating types,
MATa
and
MATα.
Some rare isolates are self-fertile and are considered occasional diploid or aneuploid strains. In the present study, 133 isolates, mostly from Italian patients, were investigated to detect the presence of diploid strains in the Igiene UniversitàMilano culture collection. All of the diploid isolates were further investigated by different methods to elucidate their origins. Forty-nine diploid strains were identified by flow cytometry. PCR fingerprinting using the (GACA)
4
primer showed that the diploid state was associated with two specific genotypes identified as VN3 and VN4. Determination of mating type on V8 juice medium confirmed that the majority of the strains were sterile. PCR and dot blotting using the two pheromone genes (
MFa
and
MFα
) as probes identified 36 of the 49 diploid isolates as
MATa/α
. The results of pheromone gene sequencing showed that two allelic
MFα
genes exist and are distinct for serotypes A and D. In contrast, the
MFa
gene sequence was conserved in both serotype alleles. Amplification of serotype-specific
STE20
alleles demonstrated that the diploid strains contained one mating locus inherited from a serotype A parent and one inherited from a serotype D parent. The present results suggest that diploid isolates may be common among the
C. neoformans
population and that in Italy and other European countries serotype A and D populations are not genetically isolated but are able to recombine by sexual reproduction.
Diagnostic Examination of Human Intestinal Spirochetosis by Fluorescent In Situ Hybridization for
Brachyspira aalborgi
,
Brachyspira pilosicoli
, and Other Species of the Genus
Brachyspira
(
Serpulina
)
Authors: Jensen T. K. , Boye M. , Ahrens P. , Korsager B. , Teglbjærg P. S. , Lindboe C. F. , Møller K. 
Published: July 2002

ABSTRACT

Human intestinal spirochetosis, characterized by end-on attachment of densely packed spirochetes to the epithelial surface of the large intestines as a fringe has been associated with the weakly beta-hemolytic spirochetes
Brachyspira aalborgi
and
Brachyspira
(
Serpulina
)
pilosicoli
. In this study, fluorescent in situ hybridization with oligonucleotide probes targeting 16S or 23S rRNA of
B. aalborgi, B. pilosicoli
, and the genus
Brachyspira
was applied to 40 sections of formalin-fixed, paraffin-embedded intestinal biopsy specimens from 23 Danish and 15 Norwegian patients with histologic evidence of intestinal spirochetosis. Five biopsy specimens from patients without intestinal spirochetosis and three samples from pigs with experimental
B. pilosicoli
colitis were examined as well. In addition, the 16S ribosomal DNAs of two clinical isolates of
B. aalborgi
were sequenced, and a PCR procedure was developed for the identification of
B. aalborgi
in cultures. The genotypic characteristics of the two clinical isolates showed very high (99.5%) similarity with two existing isolates, the type strain of
B. aalborgi
and a Swedish isolate. Hybridization with the
Brachyspira
genus-specific probe revealed a brightly fluorescing fringe of spirochetes on the epithelia of 39 biopsy specimens, whereas 1 biopsy specimen was hybridization negative. The spirochetes in biopsy specimens from 13 Danish and 8 Norwegian patients (55.3%) were identified as
B. aalborgi
. The spirochetes in the biopsy specimens from the other 17 patients hybridized only with the
Brachyspira
probe, possibly demonstrating the involvement of as-yet-uncharacterized
Brachyspira
spirochetes in human intestinal spirochetosis.
Evaluation of a Latex Agglutination Test (MRSA-Screen) for Detection of Oxacillin Resistance in Coagulase-Negative Staphylococci
Authors: Louie L. , Majury A. , Goodfellow J. , Louie M. , Simor A. E. 
Published: July 2002

ABSTRACT

The MRSA-Screen (Denka-Seiken, Tokyo, Japan) latex agglutination test was evaluated for its ability to detect PBP 2a from 200 clinical isolates of coagulase-negative staphylococci (CoNS; 84
mecA
-positive strains and 116
mecA
-negative strains) consisting of 108
Staphylococcus epidermidis
, 37
S. saprophyticus
, 15
S. haemolyticus
, 11
S. hominis
, 10
S. capitis
, 10
S. warneri
, and 3
S. lugdunensis
species as well as 6 other species of CoNS. The assay was compared with susceptibility testing with an agar screen plate with oxacillin at 6 μg/ml (OXA6), by oxacillin disk diffusion (DD), by broth microdilution (BMDIL), by the E test, and with Vitek GPS-SV and Vitek GPS-107 susceptibility cards. PCR for the detection of the
mecA
gene was used as the “gold standard.” The sensitivities and specificities for the methods evaluated were as follows: MRSA-Screen, 100 and 100%, respectively; OXA6, 100 and 99%, respectively; DD, 98 and 62%, respectively; BMDIL, 100 and 60%, respectively; E test, 100 and 51%, respectively; Vitek GPS-SV susceptibility card, 98 and 87%, respectively; and Vitek GPS-107 susceptibility card, 100 and 61%, respectively. The MRSA-Screen test accurately and rapidly detected oxacillin resistance in CoNS.
Evaluation of a PCR/DNA Probe Colorimetric Membrane Assay for Identification of
Campylobacter
spp. in Human Stool Specimens
Authors: Collins Evelyn , Glennon Maura , Hanley Shirley , Murray Anne-Marie , Cormican Martin , Smith Terry , Maher Majella 
Published: July 2002

ABSTRACT

DNA was extracted from 50 human stool specimens using the QIAamp DNA stool minikit. PCR amplification was followed by post-PCR hybridization to DNA probes specific for the
Campylobacter
genus,
Campylobacter jejuni
, and
Campylobacter coli
in a colorimetric membrane assay. Thirty-two of 38 culture-positive specimens were PCR/DNA probe positive for
C. jejuni
. The assay is rapid and simple and can be applied to stool specimens for the detection of
Campylobacter
.
Rapid PCR–Single-Strand Conformation Polymorphism Method To Differentiate and Estimate Relative Abundance of
Pneumocystis carinii
Special Forms Infecting Rats
Authors: Nahimana Aimable , Cushion Melanie T. , Blanc Dominique S. , Hauser Philippe M. 
Published: July 2002

ABSTRACT

A rapid method that uses PCR–single-strand conformation polymorphism analysis of the intron of the nuclear 26S rRNA gene was shown to differentiate the two
Pneumocystis carinii
special forms that infect rats,
P. carinii
f. sp.
carinii
and
P. carinii
f. sp.
ratti
. The method also provides a means for estimation of the relative abundance of the two special forms in the case of a coinfected rat. The results suggest that the method described will help to further standardize the immunosuppressed rat model of
P. carinii
infection and, thus, contribute to a better understanding of
P. carinii
infection in humans.
Detection of
Treponema denticola
in Atherosclerotic Lesions
Authors: Okuda Katsuji , Ishihara Kazuyuki , Nakagawa Taneaki , Hirayama Akihiko , Inayama Yoshiyuki , Okuda Kenji 
Published: July 2002

ABSTRACT

We examined 26 atherosclerotic lesions and 14 nondiseased aorta specimens to detect the periodontopathogenic part of the bacterial 16S rRNA locus by PCR.
Treponema denticola
sequence of the 16S rRNA locus was found in 6 out of 26 DNA samples (23.1%) from the formalin-fixed, paraffin-embeded atherosclerotic lesions obtained during surgery but not in any of the 14 nondiseased aorta samples from deceased persons. Utilizing immunofluorescence microscopy, we observed aggregated antigenic particles reacting with rabbit antiserum against
T. denticola
in thin sections of the PCR-positive samples, but we could not detect any reacting particles in the PCR-negative thin sections.
High-Throughput Detection of West Nile Virus RNA
Authors: Shi Pei-Yong , Kauffman Elizabeth B. , Ren Ping , Felton Andy , Tai Jennifer H. , Dupuis Alan P. , Jones Susan A. , Ngo Kiet A. , Nicholas David C. , Maffei Joseph , Ebel Gregory D. , Bernard Kristen A. , Kramer Laura D. 
Published: July 2002

ABSTRACT
The recent outbreaks of West Nile virus (WNV) in the northeastern United States and other regions of the world have made it essential to develop an efficient protocol for surveillance of WNV. In the present report, we describe a high-throughput procedure that combines automated RNA extraction, amplification, and detection of WNV RNA. The procedure analyzed 96 samples in approximately 4.5 h. A robotic system, the ABI Prism 6700 Automated Nucleic Acid workstation, extracted RNA and set up reactions for real-time reverse transcription (RT)-PCR in a 96-well format. The robot extracted RNA with a recovery as efficient as that of a commercial RNA extraction kit. A real-time RT-PCR assay was used to detect and quantitate WNV RNA. Using in vitro transcribed RNA, we estimated the detection limit of the real-time RT-PCR to be approximately 40 copies of RNA. A standard RT-PCR assay was optimized to a sensitivity similar to that of the real-time RT-PCR. The standard assay can be reliably used to test a small number of samples or to confirm previous test results. Using internal primers in a nested RT-PCR, we increased the sensitivity by approximately 10-fold compared to that of the standard RT-PCR. The results of the study demonstrated for the first time that the use of an automated system for the purpose of large-scale viral RNA surveillance dramatically increased the speed and efficiency of sample throughput for diagnosis.