bluuebunny/crossref_metadata_2025_split huggingface dataset:
Quorum Sensing Is Not Required for Twitching Motility in
Pseudomonas aeruginosa
Authors: Beatson Scott A. , Whitchurch Cynthia B. , Semmler Annalese B. T. , Mattick John S. 
Published: July 2002

ABSTRACT

It has been reported that mutations in the quorum-sensing genes
lasI
and
rhlI
in
Pseudomonas aeruginosa
result in, among many other things, loss of twitching motility (A. Glessner, R. S. Smith, B. H. Iglewski, and J. B. Robinson, J. Bacteriol. 181:1623-1629, 1999). We constructed knockouts of
lasI
and
rhlI
and the corresponding regulatory genes
lasR
and
rhlR
and found no effect on twitching motility. However, twitching-defective variants accumulated during culturing of
lasI
and
rhlI
mutants. Further analysis showed that the stable twitching-defective variants of
lasI
and
rhlI
mutants had arisen as a consequence of secondary mutations in
vfr
and
algR
, respectively, both of which encode key regulators affecting a variety of phenotypes, including twitching motility. In addition, when grown in shaking broth culture,
lasI
and
rhlI
mutants, but not the wild-type parent, also accumulated unstable variants that lacked both twitching motility and swimming motility and appeared to be identical in phenotype to the S1 and S2 variants that were recently reported to occur at high frequencies in
P. aeruginosa
strains grown as a biofilm or in static broth culture (E. Deziel, Y. Comeau, and R. Villemur, J. Bacteriol. 183:1195-1204, 2001). These results indicate that mutations in one regulatory system may create distortions that select during subsequent culturing for compensatory mutations in other regulatory genes within the cellular network. This problem may have compromised some past studies of regulatory hierarchies controlled by quorum sensing and of bacterial regulatory systems in general.
Point Mutations in HpuB Enable Gonococcal HpuA Deletion Mutants To Grow on Hemoglobin
Authors: Chen Ching-Ju , Mclean Dalton , Thomas Christopher E. , Anderson James E. , Sparling P. Frederick 
Published: July 2002

ABSTRACT

Neisseria gonorrhoeae
ordinarily requires both HpuA and HpuB to use hemoglobin (Hb) as a source of iron for growth. Deletion of HpuA resulted in reduced Hb binding and failure of growth on Hb. We identified rare Hb-utilizing colonies (Hb
+
) from an
hpuA
deletion mutant of FA1090, which fell into two phenotypic classes. One class of the Hb
+
revertants required expression of both TonB and HpuB for growth on Hb, while the other class required neither TonB nor HpuB. All TonB/HpuB-dependent mutants had single amino acid alterations in HpuB, which occurred in clusters, particularly near the C terminus. The point mutations in HpuB did not restore normal Hb binding. Human serum albumin inhibited Hb-dependent growth of HpuB point mutants lacking HpuA but did not inhibit growth when expression of HpuA was restored. Thus, HpuB point mutants internalized heme in the absence of HpuA despite reduced binding of Hb. HpuA facilitated Hb binding and was important in allowing use of heme from Hb for growth.
Lateral Flagella and Swarming Motility in
Aeromonas
Species
Authors: Kirov Sylvia M. , Tassell Bronwen C. , Semmler Annalese B. T. , O’Donovan Lisa A. , Rabaan Ali A. , Shaw Jonathan G. 
Published: July 2002

ABSTRACT

Swarming motility, a flagellum-dependent behavior that allows bacteria to move over solid surfaces, has been implicated in biofilm formation and bacterial virulence. In this study, light and electron microscopic analyses and genetic and functional investigations have shown that at least 50% of
Aeromonas
isolates from the species most commonly associated with diarrheal illness produce lateral flagella which mediate swarming motility.
Aeromonas
lateral flagella were optimally produced when bacteria were grown on solid medium for ≈8 h. Transmission and thin-section electron microscopy confirmed that these flagella do not possess a sheath structure. Southern analysis of
Aeromonas
reference strains and strains of mesophilic species (
n
= 84, varied sources and geographic regions) with a probe designed to detect lateral flagellin genes (
lafA1
and
lafA2
) showed there was no marked species association of
laf
distribution. Approximately 50% of these strains hybridized strongly with the probe, in good agreement with the expression studies. We established a reproducible swarming assay (0.5% Eiken agar in Difco broth, 30°C) for
Aeromonas
spp. The
laf
-positive strains exhibited vigorous swarming motility, whereas
laf
-negative strains grew but showed no movement from the inoculation site. Light and scanning electron microscopic investigations revealed that lateral flagella formed bacterium-bacterium linkages on the agar surface. Strains of an
Aeromonas caviae
isolate in which lateral flagellum expression was abrogated by specific mutations in flagellar genes did not swarm, proving conclusively that lateral flagella are required for the surface movement. Whether lateral flagella and swarming motility contribute to
Aeromonas
intestinal colonization and virulence remains to be determined.
Identification and Functional Characterization of
flgM
, a Gene Encoding the Anti-Sigma 28 Factor in
Pseudomonas aeruginosa
Authors: Frisk A. , Jyot J. , Arora S. K. , Ramphal R. 
Published: July 2002

ABSTRACT

We describe here the functional characterization of the putative
flgM
gene of
Pseudomonas aeruginosa
. FlgM of
P. aeruginosa
is most similar to FlgM of
Vibrio parahaemolyticus
. A conserved region is present in the C-terminal half of the FlgM of
P. aeruginosa
and in FlgM homologues of other organisms that includes the σ
28
binding domain. A role for the
flgM
gene of
P. aeruginosa
in motility was demonstrated by its inactivation. The β-galactosidase activity of a transcriptional fusion of the
fliC
promoter to
lacZ
was upregulated in the
flgM
mutant, suggesting that the activity of FliA, the sigma factor that regulates
fliC
, was increased. Consistent with these results, an increased amount of flagellin was demonstrated in the
flgM
mutant of
P. aeruginosa
strain PAK by Western blot, suggesting that FlgM negatively regulates transcription of
fliC
by inhibiting the activity of FliA. Direct interaction of the
P. aeruginosa
FlgM with the alternative sigma factor σ
28
was demonstrated by utilizing the yeast two-hybrid system. Three putative consensus σ
54
recognition sites and one σ
28
site were found in the
flgM
upstream region. However, analysis of the transcriptional fusion of the
flgM
promoter to
lacZ
in different mutant backgrounds showed that the
flgM
promoter was not entirely dependent on either σ
28
or σ
54
. A transcript was detected by primer extension that was 8 bp downstream of the consensus σ
28
-binding site. Thus, a system for the control of flagellin synthesis by FlgM exists in
P. aeruginosa
that is different from that in the enteric bacteria and seems to be most similar to that of
V. cholerae
where both σ
28
-dependent and -independent mechanisms of transcription exist.
Frequency of
rpoB
Mutations Inside and Outside the Cluster I Region in Rifampin-Resistant Clinical
Mycobacterium tuberculosis
Isolates
Authors: Heep Markus , Brandstätter Barbara , Rieger Ulrich , Lehn Norbert , Richter Elvira , Rüsch-Gerdes Sabine , Niemann Stefan 
Published: July 2002

ABSTRACT

The prevalence of recently described mutation V176F, located in the beginning of the
rpoB
gene and associated with rifampin resistance and the wild-type cluster I sequence, was determined by analyzing the distribution of
rpoB
mutations among 80 rifampin (RIF)-resistant
Mycobacterium tuberculosis
strains isolated in Germany during 1997. The most frequent
rpoB
mutations were changes in codon 456 (52 isolates, 65%), followed by changes in codon 441 (13 isolates, 16%) and codon 451 (11 isolates, 14%). The V176F mutation was detected in one isolate of the study population and in 5 of 18 RIF-resistant strains with no cluster I mutation from six previously published studies. In three isolates, a mixture of resistant and susceptible subpopulations (heteroresistance) prohibited the detection of
rpoB
mutations in the initial analysis; however, in these isolates, cluster I mutations could be verified after a passage on RIF-containing medium. IS
6110
DNA fingerprinting of 76 strains revealed eight clusters comprising 27 strains with identical restriction fragment length polymorphism patterns that mainly also show identical
rpoB
mutations and identical or similar drug resistance patterns. In conclusion, our results indicate that the V176F mutation should be included in molecular tests for prediction of RIF resistance in
M. tuberculosis
. We further demonstrated that heteroresistance caused by a mixture of mycobacterial subpopulations with different susceptibilities to RIF may influence the sensitivity of molecular tests for detection of resistance.
Evaluation of Etest Method for Determining Posaconazole MICs for 314 Clinical Isolates of
Candida
Species
Authors: Pfaller M. A. , Messer S. A. , Mills K. , Bolmström A. , Jones R. N. 
Published: July 2002

ABSTRACT

The performance of the Etest for posaconazole (SCH 56592) susceptibility testing of 314 isolates of
Candida
spp. was assessed against the National Committee for Clinical Laboratory Standards (NCCLS) microdilution broth method. The NCCLS method employed RPMI 1640 broth medium, and MICs were read after incubation for 48 h at 35°C. MICs were determined by Etest for all 314 isolates with RPMI agar containing 2% glucose (RPG agar) and were read after incubation for 48 h at 35°C. The
Candida
isolates included
C. albicans
(
n
= 174),
C. glabrata
(
n
= 57),
C. tropicalis
(
n
= 31),
C. parapsilosis
(
n
= 39),
C. krusei
(
n
= 5),
C. guilliermondii
(
n
= 6), and
C. lusitaniae
(
n
= 2). The Etest results correlated well with reference MICs. Overall agreement was 95%, and agreements for individual species were as follows:
C. krusei
, 100%;
C. albicans
, 98%;
C. tropicalis
, 97%;
C. glabrata
, 93%;
C. parapsilosis
, 85%;
C. guilliermondii
, 83%; and
C.
lusitaniae
, 50%. The problem of trailing end points was minimized with RPG agar, and good agreement with broth dilution MICs was obtained when discernible growth within an established ellipse was ignored. The Etest method using RPG agar appears to be a useful method for determining posaconazole susceptibilities of
Candida
species.
Evaluation of the NucliSens Basic Kit for Detection of
Chlamydia trachomatis
and
Neisseria gonorrhoeae
in Genital Tract Specimens Using Nucleic Acid Sequence-Based Amplification of 16S rRNA
Authors: Mahony J. B. , Song X. , Chong S. , Faught M. , Salonga T. , Kapala J. 
Published: July 2002

ABSTRACT

We evaluated a new RNA amplification and detection kit, the NucliSens Basic Kit (Organon Teknika), for the detection of
Chlamydia trachomatis
and
Neisseria gonorrhoeae
in genitourinary specimens. The Basic Kit provides an open platform for RNA amplification and detection and contains isolation reagents for nucleic acid extraction, nucleic acid sequence-based amplification (NASBA) reagents (enzymes and buffers), and a generic ruthenium-labeled probe for electrochemiluminescent (ECL) detection of amplified product. Using freshly purified and titrated stocks of
C. trachomatis
and
N. gonorrhoeae
and in vitro-generated RNA transcripts for sensitivity determinations, the Basic Kit detected 1 inclusion-forming unit of
C. trachomatis
, 1 CFU of
N. gonorrhoeae
, and 100 RNA molecules of 16S rRNA for both bacteria. The clinical performance of the Basic Kit was evaluated by testing a total of 250 specimens for
N. gonorrhoeae
by culture and NASBA and a total of 96 specimens for
C. trachomatis
by PCR and NASBA. The Basic Kit detected 139 of 142
N. gonorrhoeae
culture-positive specimens and gave a negative result for 73 of 74 culture-negative specimens, for a sensitivity and specificity of 97.9 and 98.7%, respectively. For
C. trachomatis
, the Basic Kit detected 24 of 24 PCR-positive specimens and gave a negative result for 71 of 72 PCR-negative specimens, for a sensitivity and specificity of 100 and 98.6%, respectively. The Basic Kit also detected specimens containing both
N. gonorrhoeae
and
C. trachomatis
, using a multiplex NASBA assay using primers for both bacteria. The NucliSens Basic Kit offers a versatile platform for the development of sensitive RNA detection assays for sexually transmitted diseases.
Assignment of CDC Weak Oxidizer Group 2 (WO-2) to the Genus
Pandoraea
and Characterization of Three New
Pandoraea
Genomospecies
Authors: Daneshvar Maryam I. , Hollis Dannie G. , Steigerwalt Arnold G. , Whitney Anne M. , Spangler Laura , Douglas Michael P. , Jordan Jean G. , MacGregor John P. , Hill Bertha C. , Tenover Fred C. , Brenner Don J. , Weyant Robbin S. 
Published: July 2002

ABSTRACT

CDC weak oxidizer group 2 (WO-2) consists of nine phenotypically similar human clinical isolates received by the Centers for Disease Control and Prevention between 1989 and 1998. Four of the isolates were from blood, three were from sputum, and one each was from bronchial fluid and maxillary sinus. All are aerobic nonfermentative, motile gram-negative rods with one to eight polar flagella per cell. All grew at 25 and 35°C and were positive for catalase, urease (usually delayed 3 to 7 days), citrate, alkalinization of litmus milk, oxidization of glycerol (weakly), and growth on MacConkey agar and in nutrient broth without NaCl. All except one strain were oxidase positive with the Kovács method, and all except one isolate weakly oxidized
d
-glucose. All were negative for oxidation of
d
-xylose,
d
-mannitol, lactose, sucrose, maltose, and 20 other carbohydrates, esculin hydrolysis, indole production, arginine dihydrolase, and lysine and ornithine decarboxylase. Only two of nine isolates reduced nitrate. Broth microdilution susceptibilities were determined for all strains against 13 antimicrobial agents. Most of the strains were resistant to ampicillin, extended-spectrum cephalosporins, and aminoglycosides, including gentamicin, tobramycin, and amikacin, but they varied in their susceptibility to fluoroquinolones. High-performance liquid chromatographic and mass spectrometric analyses of the WO-2 group identified ubiquinone-8 as the major quinone component. The percent G+C of the WO-2 strains ranged from 65.2 to 70.7% (thermal denaturation method). All shared a common cellular fatty acid (CFA) profile, which was characterized by relatively large amounts (7 to 22%) of 16:1ω7c, 16:0, 17:0cyc, 18:1ω7c, and 19:0cyc
11-12
; small amounts (1 to 3%) of 12:0 and 14:0; and eight hydroxy acids, 2-OH-12:0 (4%), 2-OH-14:0 (trace), 3-OH-14:0 (12%), 2-OH-16:1 (1%), 2-OH-16:0 (3%), 3-OH-16:0 (4%), 2-OH-18:1 (2%), and 2-OH-19:0cyc (3%). This profile is similar to the CFA profile of
Pandoraea
, a recently described genus associated with respiratory infections in cystic fibrosis patients (T. Coenye et al., Int. J. Syst. Evol. Microbiol., 50:887–899, 2000). Sequencing of the 16S rRNA gene (1,300 bp) for all nine strains indicated a high level (≥98.8%) of homogeneity with
Pandoraea
spp. type strains. DNA-DNA hybridization analysis (hydroxyapatite method; 70°C) confirmed the identity of WO-2 with the genus
Pandoraea
and assigned three strains to
Pandoraea apista
and three to
Pandoraea pnomenusa
, and identified three additional new genomospecies containing one strain each (ATCC BAA-108, ATCC BAA-109, ATCC BAA-110). This study also shows that
Pandoraea
isolates may be encountered in blood cultures from patients without cystic fibrosis.
Comparison of
Flavivirus
Universal Primer Pairs and Development of a Rapid, Highly Sensitive Heminested Reverse Transcription-PCR Assay for Detection of Flaviviruses Targeted to a Conserved Region of the NS5 Gene Sequences
Authors: Scaramozzino Natale , Crance Jean-Marc , Jouan Alain , DeBriel Dominique A. , Stoll Françoise , Garin Daniel 
Published: July 2002

ABSTRACT

Arthropod-transmitted flaviviruses are responsible for considerable morbidity and mortality, causing severe encephalitic, hemorrhagic, and febrile illnesses in humans. Because there are no specific clinical symptoms for infection by a determined virus and because different arboviruses could be present in the same area, a genus diagnosis by PCR would be a useful first-line diagnostic method. The six published
Flavivirus
genus primer pairs localized in the NS1, NS3, NS5, and 3′ NC regions were evaluated in terms of specificity and sensitivity with flaviviruses (including the main viruses pathogenic for humans) at a titer of 10
5
50% tissue culture infectious doses (TCID
50
s) ml
−1
with a common identification step by agarose gel electrophoresis. Only one NS5 primer pair allowed the detection of all tested flaviviruses with the sensitivity limit of 10
5
TCID
50
s ml
−1
. Using a heminested PCR with new primers designed in the same region after an alignment of 30 different flaviviruses, the sensitivity of reverse transcription-PCR was improved and allowed the detection of about 200 infectious doses ml
−1
with all of the tick- and mosquito-borne flaviviruses tested. It was confirmed that the sequenced amplified products in the NS5 region allowed predictability of flavivirus species by dendrogram, including the New York 99 West Nile strain. This technique was successfully performed with a cerebrospinal fluid sample from a patient hospitalized with West Nile virus encephalitis.
Phenotypic Switching and Genetic Diversity of
Cryptococcus neoformans
Authors: Sukroongreung Samaniya , Lim Shirlene , Tantimavanich Srisurang , Eampokalap Boonchaoy , Carter Dee , Nilakul Churairatana , Kulkeratiyut Suriyaphongse , Tansuphaswadikul Somsit 
Published: July 2002

ABSTRACT

Niger seed agar was used as a primary plating medium for the isolation of
Cryptococcus neoformans
from cerebrospinal fluid specimens from AIDS patients with untreated primary cryptococcosis. The medium was used as the primary means to detect variations in the colony morphology of the yeast. To search for phenotypic and genetic variations, nine patients individually harboring two or three types of colony morphology were studied. Intraindividual isolates from nine patients had minor variations in the API 20C profile, and the MICs of one or more antifungal agents (amphotericin B, fluconazole, and itraconazole) for isolates from three patients were significantly different. Intraindividual isolates from three patients had minor karyotype differences, and one showed a dramatic chromosomal length polymorphism. In addition, three serial isolates from a patient with two episodes of infection showed similar karyotypes, confirming persistent infection by the same strain. Random amplified polymorphic DNA products were identical for all isolates (including three isolates from a relapse case). Our results provided evidence suggesting that (i) in humans,
C. neoformans
may undergo phenotypic and genetic changes during early infection prior to antifungal agent administration; (ii) dramatic variations in electrophoretic karyotypes and in phenotypes, as demonstrated during the early infection of one patient, may be due to infection by different strains; and (iii) the use of niger seed agar as a primary plating medium is useful for studying antifungal susceptibility, phenotypic switching, genetic diversity, and multiple-strain infections.
Evaluation of Methods for Detection of Toxins in Specimens of Feces Submitted for Diagnosis of
Clostridium difficile
- Associated Diarrhea
Authors: O'Connor Don , Hynes Pearl , Cormican Martin , Collins Edward , Corbett-Feeney Geraldine , Cassidy Michael 
Published: July 2002

ABSTRACT

Clostridium difficile
is the principal pathogen associated with hospital-acquired acute diarrheal disease. We have evaluated the performances of six approaches for diagnosis of
C. difficile
-associated diarrhea (CDAD). Consecutive stool specimens (
n
= 200) from 133 patients were examined by cytotoxin assay, by culture of
C. difficile
on cycloserine-cefoxitin-fructose agar, and by toxin detection using four rapid immunoassay systems (Oxoid Toxin A test, ImmunoCard Toxin A test, TechLab Tox A/B II test, and Premier Toxins A&B test). A diagnosis of CDAD was established for 35 (27%) patients (representing 29% of specimens). The adjusted sensitivity and specificity of the methods were, respectively, 98 and 99% for the cytotoxin assay, 54 and 99% for ImmunoCard, 50 and 98% for Oxoid, 79 and 98% for TechLab, 80 and 98% for Premier, and 57 and 100% for culture. The TechLab and Premier assays are acceptable tests for diagnosis of CDAD but are not equivalent to the cytotoxin assay.
Burkholderia cepacia
Complex Infection in Italian Patients with Cystic Fibrosis: Prevalence, Epidemiology, and Genomovar Status
Authors: Agodi Antonella , Mahenthiralingam Eshwar , Barchitta Martina , Gianninò Viviana , Sciacca Agata , Stefani Stefania 
Published: July 2002

ABSTRACT

The prevalence, epidemiology, and genomovar status of
Burkholderia cepacia
complex strains recovered from Italian cystic fibrosis (CF) patients were investigated using genetic typing and species identification methods. Four CF treatment centers were examined: two in Sicily, one in central Italy, and one in northern Italy.
B. cepacia
complex bacteria were isolated from 59 out of 683 CF patients attending these centers (8.6%). For the two geographically related treatment centers in Sicily, there was a high incidence of infection caused by a single epidemic clone possessing the
cblA
gene and belonging to
B. cepacia
genomovar III,
recA
group III-A, closely related to the major North America-United Kingdom clone, ET12; instability of the
cblA
sequence was also demonstrated for clonal isolates. In summary, of all the strains of
B. cepacia
encountered in the Italian CF population, the genomovar III,
recA
group III-A strains were the most prevalent and transmissible. However, patient-to-patient spread was also observed with several other genomovars, including strains of novel taxonomic status within the
B. cepacia
complex. A combination of genetic identification and molecular typing analysis is recommended to fully define specific risks posed by the genomovar status of strains within the
B. cepacia
complex.