bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mutational Events in Cefotaximase Extended-Spectrum β-Lactamases of the CTX-M-1 Cluster Involved in Ceftazidime Resistance
Authors: Novais Ângela , Cantón Rafael , Coque Teresa M. , Moya Andrés , Baquero Fernando , Galán Juan Carlos 
Published: April 2008

ABSTRACT

CTX-M β-lactamases, which show a high cefotaxime hydrolytic activity, constitute the most prevalent extended-spectrum β-lactamase (ESBL) type found among clinical isolates. The recent explosive diversification of CTX-M enzymes seems to have taken place due to the appearance of more efficient enzymes which are capable of hydrolyzing both cefotaxime and ceftazidime, especially among the CTX-M-1 cluster. A combined strategy of in vitro stepwise evolution experiments using
bla
CTX-M-1
,
bla
CTX-M-3
, and
bla
CTX-M-10
genes and site-directed mutagenesis has been used to evaluate the role of ceftazidime and other β-lactam antibiotics in triggering the diversity found among enzymes belonging to this cluster. Two types of mutants, P167S and D240G, displaying high ceftazidime MICs but reduced resistance to cefotaxime and/or cefepime, respectively, were identified. Such an antagonistic pleiotropic effect was particularly evident with P167S/T mutations. The incompatibility between P167S and D240G changes was demonstrated, since double mutants reduced susceptibility to both ceftazidime and cefotaxime-cefepime; this may explain the absence of strains containing both mutations in the clinical environment. The role of A77V and N106S mutations, which are frequently associated with P167S/T and/or D240G, respectively, in natural strains, was investigated. The presence of A77V and N106S contributes to restore a high-level cefotaxime resistance phenotype, but only when associated with mutations P167S and D240G, respectively. However, A77V mutation increases resistance to both cefotaxime and ceftazidime when associated with CTX-M-10. This suggests that in this context this mutation might be considered a primary site involved in resistance to broad-spectrum cephalosporins.
Tools for Functional Postgenomic Analysis ofListeria monocytogenes
Authors: Monk Ian R. , Gahan Cormac G. M. , Hill Colin 
Published: April 2008

ABSTRACTWe describe the development of genetic tools for regulated gene expression, the introduction of chromosomal mutations, and improved plasmid transfer by electroporation in the food-borne pathogenListeria monocytogenes. pIMK, a kanamycin-resistant, site-specific, integrative listeriophage vector was constructed and then modified for overexpression (pIMK2) or for isopropyl-β-d-thiogalactopyranoside (IPTG)-regulated expression (pIMK3 and pIMK4). The dynamic range of promoters was assessed by determining luciferase activity, P60 secretion, and internalin A-mediated invasion. These analyses demonstrated that pIMK4 and pIMK3 have a stringently controlled dynamic range of 540-fold. Stable gene overexpression was achieved with pIMK2, giving a range of expression for the three vectors of 1,350-fold. The lactococcal pORI280 system was optimized for the generation of chromosomal mutations and used to create five newprfAstar mutants. The combination of pIMK4 and pORI280 allowed streamlined creation of “IPTG-dependent” mutants. This was exemplified by creation of a clean deletion mutant with deletion of the universally essentialsecAgene, and this mutant exhibited a rapid loss of viability upon withdrawal of IPTG. We also improved plasmid transfer by electroporation into three commonly used laboratory strains ofL. monocytogenes. A 125-fold increase in transformation efficiency for EGDe compared with the widely used protocol of Park and Stewart (S. F. Park and G. S. Stewart, Gene 94:129-132, 1990) was observed. Maximal transformation efficiencies of 5.7 × 106and 6.7 × 106CFU per μg were achieved for EGDe and 10403S, respectively, with a replicating plasmid. An efficiency of 2 × 107CFU per μg is the highest efficiency reported thus far forL. monocytogenesF2365.
Molecular Analysis of the
Enterobacter sakazakii
O-Antigen Gene Locus
Authors: Mullane N. , O'Gaora P. , Nally J. E. , Iversen C. , Whyte P. , Wall P. G. , Fanning S. 
Published: April 2008

ABSTRACT

Nucleotide polymorphism associated with the O-antigen-encoding locus,
rfb
, in
Enterobacter sakazakii
was determined by PCR-restriction fragment length polymorphism analysis. Based on the analysis of these DNA profiles, 12 unique banding patterns were detected among a collection of 62 strains from diverse origins. Two common profiles were identified and were designated serotypes O:1 and O:2. DNA sequencing of the 12,500-bp region flanked by
galF
and
gnd
identified 11 open reading frames, all with the same transcriptional direction. Analysis of the proximal region of both sequences demonstrated remarkable heterogeneity. A PCR assay targeting genes specific for the two prominent serotypes was developed and applied for the identification of these strains recovered from food, environmental, and clinical samples.
Coexistence of Antibiotic-Producing and Antibiotic-Sensitive Bacteria in Biofilms Is Mediated by Resistant Bacteria
Authors: Narisawa Naoki , Haruta Shin , Arai Hiroyuki , Ishii Masaharu , Igarashi Yasuo 
Published: April 2008

ABSTRACT

Antibiotic-sensitive bacteria have been found to coexist with antibiotic-producing bacteria in biofilms, but little is known about how the former develop in such an environment. Here we isolated pyocyanin-sensitive bacteria belonging to the genus
Brevibacillus
from a biofilm derived from soil extract and based on the preestablished biofilm of a pyocyanin producer,
Pseudomonas aeruginosa
strain P1. In addition, pyocyanin-resistant strains belonging to the genus
Raoultella
were isolated from the same biofilm. Microbial relationships within biofilms were examined by using three strains, strain P1,
Brevibacillus
strain S1, and
Raoultella
strain R1, each of which individually formed a biofilm within 2 days in a flow cell. Strain S1 did not fully develop on the preestablished biofilm of strain P1 during 4 days of cultivation, whereas a mutant of strain P1 which was deficient in pyocyanin production allowed strain S1 to cocolonize within a biofilm. On the other hand, strain R1 developed on the biofilm of strain P1 regardless of pyocyanin production. When mixed 1:1 inocula of strains S1 and R1 were introduced into the strain P1 biofilm, all three species were found in the 4-day biofilm. In the mixed biofilm, strain S1 was surrounded by the layer of strain R1 and seemed to be separated from strain P1 and the outflow solution. However, strain S1 did not survive in a three-species mixed culture under planktonic conditions. These results indicate that the survival of sensitive bacteria in biofilm with a pyocyanin producer is achieved by covering them with a layer of resistant bacteria. We also evaluated the influence of antibiotic production on the producer.
Effect of Extraction and Assay Media on Analysis of Airborne Endotoxin
Authors: Spaan Suzanne , Doekes Gert , Heederik Dick , Thorne Peter S. , Wouters Inge M. 
Published: April 2008

ABSTRACT

The measurement of airborne endotoxins is thus far not standardized. Earlier studies reported higher endotoxin yields when Tween 20 was added to the media used for filter extraction and in the
Limulus
amebocyte lysate (LAL) assay. This study compared four common media and assessed the effects of Tween during extraction and analysis separately. Parallel airborne dust samples from five work environments (
n
= 250) were used to compare the four media (pyrogen-free water [PFW], PFW-Tween 20, PFW-Tris, and PFW-triethylamine-phosphate [TAP]) and an extraction time of 10 or 60 min. A subset of the extracts in PFW or PFW-Tween (
n
= 40) were analyzed in parallel LAL assays with PFW or PFW-Tween as the assay medium. The results produced by a shorter extraction time or the presence of Tris were similar to the results for the reference procedure (PFW and 60 min of shaking). The use of PFW-TAP showed overall lower yields and a deviant calibration curve. The presence of Tween in the extraction medium resulted in significantly (
P
< 0.05) higher endotoxin yields from all dust types, independent of the effect of Tween in the assay. Tween in the LAL assay, however, also strongly inhibited the reactivity of the lipopolysaccharide (LPS) standard, thus shifting the calibration curve to higher values. The inhibition of LPS in test samples was less pronounced and varied between dust sources, resulting in enhanced calculated concentrations. This assay effect could be circumvented by diluting extracts at least 50-fold before the LAL assay. In conclusion, of the media tested, only Tween enhances the efficiency of endotoxin extraction from airborne dust samples in a consistent manner. We recommend extraction in PFW-Tween combined with dilution and LAL analysis in PFW.
Loss of Virulence Genes in
Escherichia coli
Populations during Manure Storage on a Commercial Swine Farm
Authors: Duriez Patrick , Zhang Yun , Lu Zexun , Scott Andrew , Topp Edward 
Published: April 2008

ABSTRACT

Confined livestock production farms typically store their wastes prior to land application. Here, we employed three complementary approaches to evaluate changes in the population structure and stability of virulence genes in
Escherichia coli
during manure storage on a commercial farm that housed healthy swine. Isolates were genotyped by repetitive extragenic palindromic PCR using the BOXA1R primer and evaluated for the presence of selected virulence genes by PCR. Isolates obtained from the manure holding tank (
n
= 392) carried
estB
,
fedA
,
stx
2e
,
astA
,
paa
,
aida-I
, and
sepA
at lower frequencies than isolates obtained from fresh feces (
n
= 412). Fresh fecal material from the barn was added into diffusion chambers and immersed in the manure holding tank for 7 weeks. The fecal
E. coli
population was initially dominated by a single genotype, all isolates of which carried
fedA
and
aida-I
. After 7 weeks, a genotype that did not carry any virulence genes dominated the surviving population. In a second experiment, 48 fecal isolates of
E. coli
that varied in their genotypes and virulence gene complement were incubated in diffusion chambers in the manure holding tank for 3 weeks. Over 95% of the inoculum population carried at least one virulence gene, whereas after 3 weeks 90% of the recovered isolates carried no virulence genes. Taken together, these results indicate that during commercial manure storage, there was a significant reduction in the carriage of these virulence genes by
E. coli
. We propose that loss of virulence genes from enteric pathogens in the farm and in natural environments may, if generalized, contribute to the attenuation of a public health risk from contamination with agricultural wastes.
Mycelium Differentiation and Antibiotic Production in Submerged Cultures ofStreptomyces coelicolor
Authors: Manteca Angel , Alvarez Ruben , Salazar Nuria , Yagüe Paula , Sanchez Jesus 
Published: April 2008

ABSTRACTDespite the fact that most industrial processes for secondary metabolite production are performed with submerged cultures, a reliable developmental model forStreptomycesunder these culture conditions is lacking. With the exception of a few species which sporulate under these conditions, it is assumed that no morphological differentiation processes take place. In this work, we describe new developmental features ofStreptomyces coelicolorA3(2) grown in liquid cultures and integrate them into a developmental model analogous to the one previously described for surface cultures. Spores germinate as a compartmentalized mycelium (first mycelium). These young compartmentalized hyphae start to form pellets which grow in a radial pattern. Death processes take place in the center of the pellets, followed by growth arrest. A new multinucleated mycelium with sporadic septa (second mycelium) develops inside the pellets and along the periphery, giving rise to a second growth phase. Undecylprodigiosin and actinorhodin antibiotics are produced by this second mycelium but not by the first one. Cell density dictates how the culture will behave in terms of differentiation processes and antibiotic production. When diluted inocula are used, the growth arrest phase, emergence of a second mycelium, and antibiotic production are delayed. Moreover, pellets are less abundant and have larger diameters than in dense cultures. This work is the first to report on the relationship between differentiation processes and secondary metabolite production in submergedStreptomycescultures.
Defects in Conidiophore Development and Conidium-Macrophage Interactions in a Dioxygenase Mutant of
Aspergillus fumigatus
Authors: Dagenais Taylor R. T. , Chung DaWoon , Giles Steven S. , Hull Christina M. , Andes David , Keller Nancy P. 
Published: April 2008

ABSTRACT

Oxygenated fatty acids, or oxylipins, play an essential role in physiological signaling and developmental processes in animals, plants, and fungi. Previous characterization of three
Aspergillus fumigatus
dioxygenases (PpoA, PpoB, and PpoC), similar in sequence to mammalian cyclooxygenases, showed that PpoA is responsible for the production of the oxylipins 8
R
-hydroperoxyoctadecadienoic acid and 5
S
,8
R
-dihydroxy-9
Z
,12
Z
-octadecadienoic acid and that PpoC is responsible for 10
R
-hydroxy-8
E
,12
Z
-hydroperoxyoctadecadienoic acid. Here, Δ
ppo
mutants were characterized to elucidate the role of fungal dioxygenases in
A. fumigatus
development and host interactions. The Δ
ppoC
strain displayed distinct phenotypes compared to those of other Δ
ppo
mutants and the wild type, including altered conidium size, germination, and tolerance to oxidative stress as well as increased uptake and killing by primary alveolar macrophages. These experiments implicate oxylipins in pathogen development and suggest that Δ
ppoC
represents a useful model for studying the
A. fumigatus
-host interaction.
A Cyanobacterial Lipopolysaccharide Antagonist Inhibits Cytokine Production Induced byNeisseria meningitidisin a Human Whole-Blood Model of Septicemia
Authors: Jemmett Kim , Macagno Annalisa , Molteni Monica , Heckels John E. , Rossetti Carlo , Christodoulides Myron 
Published: April 2008

ABSTRACTSepticemia caused byNeisseria meningitidisis characterized by increasing levels of meningococcal lipopolysaccharide (Nm-LPS) and cytokine production in the blood. We have used an in vitro human whole-blood model of meningococcal septicemia to investigate the potential of CyP, a selective Toll-like receptor 4 (TLR4)-MD-2 antagonist derived from the cyanobacteriumOscillatoria planktothrixFP1, for reducing LPS-mediated cytokine production. CyP (≥1 μg/ml) inhibited the secretion of the proinflammatory cytokines tumor necrosis factor alpha, interleukin-1β (IL-1β), and IL-6 (by >90%) and chemokines IL-8 and monocyte chemoattractant protein 1 (by ∼50%) induced by the treatment of blood with pure Nm-LPS, by isolated outer membranes, and after infection with live meningococci of different serogroups. In vitro studies with human dendritic cells and TLR4-transfected Jurkat cells demonstrated that CyP competitively inhibited Nm-LPS interactions with TLR4 and subsequent NF-κB activation. These data demonstrate that CyP is a potent antagonist of meningococcal LPS and could be considered a new adjunctive therapy for treating septicemia.
New Adhesin of Enteroaggregative
Escherichia coli
Related to the Afa/Dr/AAF Family
Authors: Boisen Nadia , Struve Carsten , Scheutz Flemming , Krogfelt Karen A. , Nataro James P. 
Published: April 2008

ABSTRACT

Enteroaggregative
Escherichia coli
(EAEC) is an important cause of diarrhea worldwide. We analyzed 17 Danish EAEC strains, isolated in the course of a case control study, for phenotypic and genotypic properties. The strains belonged to at least 14 different serotypes. Using PCR to investigate the prevalence of various putative virulence genes, we found that all but two strains were typical EAEC, as they harbored all or part of the previously described AggR regulon. The majority of the strains harbored genes encoding aggregative adherence fimbriae (AAF). The most common was AAF/I, found in nine strains; eight strains carried no known AAF-related genes. We utilized Tn
phoA
mutagenesis to localize the aggregative adherence (AA) adhesin from one typical EAEC strain, C1010-00, which lacked a known AAF. We identified a Tn
phoA
insertion in a hypothetical Dr-related pilin deposited in GenBank as HdaA. Four additional Danish strains harbored HdaA, and all but one displayed AA to HEp-2 cells. By using PCR primers derived from the pilins and ushers from the three AAF and Hda, we found that 16 of 17 strains exhibited evidence of one of these factors; importantly, the one negative strain also lacked the
aggR
gene. Cloning of the complete Hda gene cluster and expression in
E. coli
DH5α resulted in AA and complementation of the C1010-00 nonadherent mutant. Four related adhesins have now been found to confer AA in typical EAEC strains; our data suggest that, together, these variants may account for AA in the large majority of strains.
Effect of Multiple Genetic Polymorphisms on Antigen Presentation and Susceptibility toMycobacterium tuberculosisInfection
Authors: Chang Stewart T. , Linderman Jennifer J. , Kirschner Denise E. 
Published: April 2008

ABSTRACTSeveral molecules related to antigen presentation, including gamma interferon (IFN-γ) and the major histocompatibility complex (MHC), are encoded by polymorphic genes. Some polymorphisms were found to affect susceptibility to tuberculosis (TB) when they were considered singly in epidemiological studies, but how multiple polymorphisms interact to determine susceptibility to TB in an individual remains an open question. We hypothesized that polymorphisms in some genes may counteract or intensify the effects of polymorphisms in other genes. For example, an increase in IFN-γ expression may counteract the weak binding that a particular MHC variant displays for a peptide fromMycobacterium tuberculosisto establish the same T-cell response as another, more strongly binding MHC variant. To test this hypothesis, we developed a mathematical model of antigen presentation based on experimental data for the known effects of genetic polymorphisms and simulated time courses when multiple polymorphisms were present. We found that polymorphisms in different genes could affect antigen presentation to the same extent and therefore compensate for each other. Furthermore, we defined the conditions under which such relationships could exist. For example, increased IFN-γ expression compensated for decreased peptide-MHC affinity in the model only above a certain threshold of expression. Below this threshold, changes in IFN-γ expression were ineffectual compared to changes in peptide-MHC affinity. The finding that polymorphisms exhibit such relationships could explain discrepancies in the epidemiological literature, where some polymorphisms have been inconsistently associated with susceptibility to TB. Furthermore, the model allows polymorphisms to be ranked by effect, providing a new tool for designing association studies.
The Hybrid Sensor Kinase RscS Integrates Positive and Negative Signals To Modulate Biofilm Formation in
Vibrio fischeri
Authors: Geszvain Kati , Visick Karen L. 
Published: April 2008

ABSTRACT

Overexpression of the
Vibrio fischeri
sensor kinase RscS induces expression of the
syp
(
sy
mbiosis
p
olysaccharide) gene cluster and promotes biofilm phenotypes such as wrinkled colony morphology, pellicle formation, and surface adherence. RscS is predicted to be a hybrid sensor kinase with a histidine kinase/ATPase (HATPase) domain, a receiver (Rec) domain, and a histidine phosphotransferase (Hpt) domain. Bioinformatic analysis also revealed the following three potential signal detection domains within RscS: two transmembrane helices forming a transmembrane region (TMR), a large periplasmic (PP) domain, and a cytoplasmic PAS domain. In this work, we genetically dissected the contributions of these domains to RscS function. Substitutions within the carboxy-terminal domain supported identification of RscS as a hybrid sensor kinase; disruption of both the HATPase and Rec domains eliminated induction of
syp
transcription, wrinkled colony morphology, pellicle formation, and surface adherence, while disruption of Hpt resulted in decreased activity. The PAS domain was also critical for RscS activity; substitutions in PAS resulted in a loss of activity. Generation of a cytoplasmic, N-terminal deletion derivative of RscS resulted in a partial loss of activity, suggesting a role for localization to the membrane and/or sequences within the TMR and PP domain. Finally, substitutions within the first transmembrane helix of the TMR and deletions within the PP domain both resulted in increased activity. Thus, RscS integrates both inhibitory and stimulatory signals from the environment to regulate biofilm formation by
V. fischeri
.
Spontaneous Amplification of the Actinorhodin Gene Cluster in
Streptomyces coelicolor
Involving Native Insertion Sequence IS
466
Authors: Widenbrant E. M. , Tsai Hsiu-Hui , Chen Carton W. , Kao C. M. 
Published: April 2008

ABSTRACT

We observed a spontaneous amplification of the
Streptomyces coelicolor
chromosome, including genes encoding biosynthetic enzymes of the antibiotic actinorhodin. A new junction of two tandem segments has, inserted within it, a third copy of a transposable element existing in two places elsewhere in the chromosome, suggesting its involvement in the amplification mechanism.
Response Regulator DegU ofListeria monocytogenesControls Temperature-Responsive Flagellar Gene Expression in Its Unphosphorylated State
Authors: Mauder Norman , Williams Tatjana , Fritsch Frederike , Kuhn Michael , Beier Dagmar 
Published: April 2008

ABSTRACTWe demonstrate that inListeria monocytogenes, temperature-responsive transcriptional control of flagellar genes does not rely on the phosphorylation of the conserved phosphorylation site (D55) in the receiver domain of response regulator DegU. Furthermore, proper control of DegU-regulated genes involved in ethanol tolerance and virulence is independent of receiver phosphorylation.
Salmonella enterica
Requires ApbC Function for Growth on Tricarballylate: Evidence of Functional Redundancy between ApbC and IscU
Authors: Boyd Jeffrey M. , Lewis Jeffrey A. , Escalante-Semerena Jorge C. , Downs Diana M. 
Published: April 2008

ABSTRACT

Mutants of
Salmonella enterica
lacking
apbC
have nutritional and biochemical properties indicative of defects in [Fe-S] cluster metabolism. Here we show that
apbC
is required for
S. enterica
to use tricarballylate as a carbon and energy source. Tricarballylate catabolism requires three gene products, TcuA, TcuB, and TcuC. Of relevance to this work is the TcuB protein, which has two [4Fe-4S] clusters required for function, making it a logical target for the
apbC
effect. TcuB activity was 100-fold lower in an
apbC
mutant than in the isogenic
apbC
+
strain. Genetic data show that derepression of the
iscRSUA-hscAB-fdx-orf3
operon or overexpression of
iscU
from a plasmid compensates for the lack of ApbC during growth on tricarballylate. The studies described herein provide evidence that the scaffold protein IscU has a functional overlap with ApbC and that ApbC function is involved in the synthesis of active TcuB.