bluuebunny/crossref_metadata_2025_split huggingface dataset:
Molecular Epidemiology of Mutations in Antimicrobial Resistance Loci of Pseudomonas aeruginosa Isolates from Airways of Cystic Fibrosis Patients
Authors: Greipel Leonie , Fischer Sebastian , Klockgether Jens , Dorda Marie , Mielke Samira , Wiehlmann Lutz , Cramer Nina , Tümmler Burkhard 
Published: August 2016

ABSTRACT

The chronic airway infections with
Pseudomonas aeruginosa
in people with cystic fibrosis (CF) are treated with aerosolized antibiotics, oral fluoroquinolones, and/or intravenous combination therapy with aminoglycosides and β-lactam antibiotics. An international strain collection of 361
P. aeruginosa
isolates from 258 CF patients seen at 30 CF clinics was examined for mutations in 17 antimicrobial susceptibility and resistance loci that had been identified as hot spots of mutation by genome sequencing of serial isolates from a single CF clinic. Combinatorial amplicon sequencing of pooled PCR products identified 1,112 sequence variants that were not present in the genomes of representative strains of the 20 most common clones of the global
P. aeruginosa
population. A high frequency of singular coding variants was seen in
spuE
,
mexA
,
gyrA
,
rpoB
,
fusA1
,
mexZ
,
mexY
,
oprD
,
ampD
,
parR
,
parS
, and
envZ
(
amgS
), reflecting the pressure upon
P. aeruginosa
in lungs of CF patients to generate novel protein variants. The proportion of nonneutral amino acid exchanges was high. Of the 17 loci,
mexA
,
mexZ
, and
pagL
were most frequently affected by independent stop mutations. Private and
de novo
mutations seem to play a pivotal role in the response of
P. aeruginosa
populations to the antimicrobial load and the individual CF host.
Antimicrobial Susceptibility of Pseudomonas aeruginosa Isolated from Cystic Fibrosis Patients in Northern Europe
Authors: Mustafa Muhammad-Hariri , Chalhoub Hussein , Denis Olivier , Deplano Ariane , Vergison Anne , Rodriguez-Villalobos Hector , Tunney Michael M. , Elborn J. Stuart , Kahl Barbara C. , Traore Hamidou , Vanderbist Francis , Tulkens Paul M. , Van Bambeke Françoise 
Published: August 2016

ABSTRACT

Pseudomonas aeruginosa
is a major cause of morbidity and mortality in cystic fibrosis patients. This study compared the antimicrobial susceptibilities of 153
P. aeruginosa
isolates from the United Kingdom (UK) (
n =
58), Belgium (
n =
44), and Germany (
n =
51) collected from 118 patients during routine visits over the period from 2006 to 2012. MICs were measured by broth microdilution. Genes encoding extended-spectrum β-lactamases (ESBL), metallo-β-lactamases, and carbapenemases were detected by PCR. Pulsed-field gel electrophoresis and multilocus sequence typing were performed on isolates resistant to ≥3 antibiotic classes among the penicillins/cephalosporins, carbapenems, fluoroquinolones, aminoglycosides, and polymyxins. Based on EUCAST/CLSI breakpoints, susceptibility rates were ≤30%/≤40% (penicillins, ceftazidime, amikacin, and ciprofloxacin), 44 to 48%/48 to 63% (carbapenems), 72%/72% (tobramycin), and 92%/78% (colistin) independent of patient age. Sixty percent of strains were multidrug resistant (MDR; European Centre for Disease Prevention and Control criteria). Genes encoding the most prevalent ESBL (BEL, PER, GES, VEB, CTX-M, TEM, SHV, and OXA), metallo-β-lactamases (VIM, IMP, and NDM), or carbapenemases (OXA-48 and KPC) were not detected. The Liverpool epidemic strain (LES) was prevalent in UK isolates only (75% of MDR isolates). Four MDR sequence type 958 (ST958) isolates were found to be spread over the three countries. The other MDR clones were evidenced in ≤3 isolates and localized in a single country. A new sequence type (ST2254) was discovered in one MDR isolate in Germany. Clonal and nonclonal isolates with different susceptibility profiles were found in 20 patients. Thus, resistance and MDR are highly prevalent in routine isolates from 3 countries, with meropenem, tobramycin, and colistin remaining the most active drugs.
Bayesian Estimation of Tobramycin Exposure in Patients with Cystic Fibrosis
Authors: Barras Michael A. , Serisier David , Hennig Stefanie , Jess Katrina , Norris Ross L. G. 
Published: August 2016

ABSTRACT

Fixed tobramycin (mg/kg) dosing is often inappropriate in patients with cystic fibrosis (CF), as pharmacokinetics are highly variable. The area under the concentration-time curve (AUC) is an exposure metric suited to monitoring in this population. Bayesian strategies to estimate AUC have been available for over 20 years but are not standard practice in the clinical setting. To assess their suitability for use in clinical practice, three AUC estimation methods using limited sampling were compared to measured true exposure by using intensive sampling tobramycin data. Adults prescribed once daily intravenous tobramycin had eight concentrations taken over 24 h. An estimate of true exposure within one dosing interval was calculated using the trapezoidal method and compared to three alternate estimates determined using (i) a two-sample log-linear regression (LLR) method (local hospital practice); (ii) a Bayesian estimate using one concentration (AUC
1
); and (iii) a Bayesian estimate using two concentrations (AUC
2
). Each method was evaluated against the true measured exposure by a Bland-Altman analysis. Twelve patients with a median (range) age and weight of 25 (18 to 36) years and 66.5 (51 to 76) kg, respectively, were recruited. There was good agreement between the true exposure and the three alternate estimates of AUC, with a mean AUC bias of <10 mg/liter · h in each case, i.e., −8.2 (LLR), 3.8 (AUC
1
), and 1.0 (AUC
2
). Bayesian analysis-based and LLR estimation methods of tobramycin AUC are equivalent to true exposure estimation. All three methods may be suitable for use in the clinical setting; however, a one-sample Bayesian method may be most useful in ambulatory patients for which coordinating blood samples is difficult. Suitably powered, randomized clinical trials are required to assess patient outcomes.
Effect of Antifungal Treatment in a Diet-Based Murine Model of Disseminated Candidiasis Acquired via the Gastrointestinal Tract
Authors: Kadosh David , Najvar Laura K. , Bocanegra Rosie , Olivo Marcos , Kirkpatrick William R. , Wiederhold Nathan P. , Patterson Thomas F. 
Published: August 2016

ABSTRACT

Candida albicans
, normally found as a commensal in the gut, is a major human fungal pathogen responsible for both mucosal and systemic infections in a wide variety of immunocompromised individuals, including cancer patients and organ transplant recipients. The gastrointestinal tract represents a major portal of entry for the establishment of disseminated candidiasis in many of these individuals. Here we report the development of a diet-based mouse model for disseminated candidiasis acquired via the gastrointestinal tract. Using this model, as well as an appropriate immunosuppression regimen, we demonstrate that dissemination of
C. albicans
from the gastrointestinal tract can result in mortality within 30 days postinfection. We also show a significant increase in fungal burden in systemic organs, but not gastrointestinal tract organs, upon immunosuppression. Importantly, we demonstrate that the administration of two widely used antifungals, fluconazole and caspofungin, either pre- or postimmunosuppression, significantly reduces fungal burdens. This model should prove to be of significant value for testing the ability of both established and experimental therapeutics to inhibit
C. albicans
dissemination from the gastrointestinal tract in an immunocompromised host as well as the subsequent mortality that can result from disseminated candidiasis.
Characterization of a Novel IncHI2 Plasmid Carrying Tandem Copies of
bla
CTX-M-2
in a
fosA6
-Harboring Escherichia coli Sequence Type 410 Strain
Authors: Guo Qinglan , Ding Baixing , Jové Thomas , Stoesser Nicole , Cooper Vaughn S. , Wang Minggui , Doi Yohei 
Published: August 2016

ABSTRACT

The extended-spectrum β-lactamase gene
bla
CTX-M-2
is mainly associated with IS
CR1
embedded in complex
sul1
-type integrons, but information on the genetic context of plasmids harboring the IS
CR1-bla
CTX-M-2
module remains limited. In this study, a
bla
CTX-M-2
-harboring plasmid (pYD786-1) belonging to the sequence type 2 (ST2)-IncHI2 plasmid type and isolated from an
Escherichia coli
ST410 clinical strain was sequenced and analyzed. pYD786-1 belongs to the APEC-O1-R-type IncHI2 plasmids, which are widely distributed in human, poultry, and livestock strains. It contains a multidrug resistance mosaic region (MRR) consisting of a Tn
21
::In2 transposon backbone augmented by acquisition of duplicate IS
CR1-bla
CTX-M-2
modules. Tn
2411
, a Tn
21
::In2 precursor, likely played a role in the generation of the MRR in pN13-01290_23, the putative progenitor plasmid of pYD786-1, found in a foodborne
Salmonella
strain. Tn
21/
Tn
2411
::In::IS
CR1-bla
CTX-M-2
derivatives, including pYD786-1, have been identified in strains from Europe, South America, and the United States, suggesting potential global dissemination of the
bla
CTX-M-2
modules mediated by this vehicle.
Bioluminescent Reporters for Rapid Mechanism of Action Assessment in Tuberculosis Drug Discovery
Authors: Naran Krupa , Moosa Atica , Barry Clifton E. , Boshoff Helena I. M. , Mizrahi Valerie , Warner Digby F. 
Published: August 2016

ABSTRACT

The tuberculosis (TB) drug discovery pipeline is fueled by compounds identified in whole-cell screens against the causative agent,
Mycobacterium tuberculosis
. Phenotypic screening enables the selection of molecules that inhibit essential cellular functions in live, intact bacilli grown under a chosen
in vitro
condition. However, deducing the mechanism of action (MOA), which is important to avoid promiscuous targets, often requires significant biological resources in a lengthy process that risks decoupling medicinal chemistry and biology efforts. Therefore, there is a need to develop methods enabling rapid MOA assessment of putative “actives” for triage decisions. Here, we describe a modified version of a bioluminescence reporter assay that allows nondestructive detection of compounds targeting either of two macromolecular processes in
M. tuberculosis
: cell wall biosynthesis or maintenance of DNA integrity. Coupling the
luxCDABE
operon from
Photorhabdus luminescens
to mycobacterial promoters driving expression of the
iniBAC
operon (P
iniB
-LUX) or the DNA damage-inducible genes,
recA
(P
recA
-LUX) or
radA
(P
radA
-LUX), provided quantitative detection in real time of compounds triggering expression of any of these promoters over an extended 10- to 12-day incubation. Testing against known anti-TB agents confirmed the specificity of each reporter in registering the MOA of the applied antibiotic in
M. tuberculosis
, independent of bactericidal or bacteriostatic activity. Moreover, profiles obtained for experimental compounds indicated the potential to infer complex MOAs in which multiple cellular processes are disrupted. These results demonstrate the utility of the reporters for early triage of compounds based on the provisional MOA and suggest their application to investigate polypharmacology in known and experimental anti-TB agents.
Cefazolin and Ertapenem, a Synergistic Combination Used To Clear Persistent Staphylococcus aureus Bacteremia
Authors: Sakoulas George , Olson Joshua , Yim Juwon , Singh Niedita B. , Kumaraswamy Monika , Quach Diana T. , Rybak Michael J. , Pogliano Joseph , Nizet Victor 
Published: August 2016

ABSTRACT

Ertapenem and cefazolin were used in combination to successfully clear refractory methicillin-susceptible
Staphylococcus aureus
(MSSA) bacteremia. In addition, recent work has demonstrated activity of combination therapy with beta-lactams from different classes against methicillin-resistant
S. aureus
(MRSA). The ertapenem-plus-cefazolin combination was evaluated for synergy
in vitro
and
in vivo
in a murine skin infection model using an index MSSA bloodstream isolate from a patient in whom persistent bacteremia was cleared with this combination and against a cadre of well-described research strains and clinical strains of MSSA and MRSA. Against the index MSSA bloodstream isolate, ertapenem and cefazolin showed synergy using both checkerboard (fractional inhibitory concentration [FIC] index = 0.375) and time-kill assays. Using a disk diffusion ertapenem potentiation assay, the MSSA isolate showed a cefazolin disk zone increased from 34 to 40 mm.
In vitro
pharmacokinetic/pharmacodynamic modeling at clinically relevant drug concentrations demonstrated bactericidal activity (>3 log
10
-CFU/ml reduction) of the combination but bacteriostatic activity of ether drug alone at 48 h. A disk diffusion potentiation assay showed that ertapenem increased the cefazolin zone of inhibition by >3 mm for 34/35 (97%) MSSA and 10/15 (67%) MRSA strains. A murine skin infection model of MSSA showed enhanced activity of cefazolin plus ertapenem compared to monotherapy with these agents. After successful use in clearance of MSSA bacteremia, the combination of ertapenem and cefazolin showed synergy against MSSA
in vitro
and
in vivo
. This combination may warrant consideration for future clinical study in MSSA bacteremia.
Ehrlichia chaffeensis TRP32 Is a Nucleomodulin That Directly Regulates Expression of Host Genes Governing Differentiation and Proliferation
Authors: Farris Tierra R. , Dunphy Paige S. , Zhu Bing , Kibler Clayton E. , McBride Jere W. 
Published: August 2016

ABSTRACTEhrlichia chaffeensisis an obligately intracellular bacterium that reprograms the mononuclear phagocyte through diverse effector-host interactions to modulate numerous host cell processes, including transcription. In a previous study, we reported thatE. chaffeensisTRP32, a type 1 secreted effector, interacts with multiple host nucleus-associated proteins and also autoactivates reporter gene expression in yeast. In this study, we demonstrate that TRP32 is a nucleomodulin that binds host DNA and alters host gene transcription. TRP32 enters the host cell nucleus via a noncanonical translocation mechanism that involves phosphorylation of Y179 located in a C-terminal trityrosine motif. Both genistein and mutation of Y179 inhibited TRP32 nuclear entry. An electromobility shift assay (EMSA) demonstrated TRP32 host DNA binding via its tandem repeat domain. TRP32 DNA-binding and motif preference were further confirmed by supershift assays, as well as competition and mutant probe analyses. Using chromatin immunoprecipitation with next-generation sequencing (ChIP-seq), we determined that TRP32 binds a G-rich motif primarily within ±500 bp of the gene transcription start site. An ontology analysis identified genes involved in processes such as immune cell differentiation, chromatin remodeling, and RNA transcription and processing as primary TRP32 targets. TRP32-bound genes (n= 1,223) were distributed on all chromosomes and included several global regulators of proliferation and inflammation such as those encoding FOS, JUN, AKT3, and NRAS and noncoding RNA genes microRNA 21 (miRNA 21) and miRNA 142. TRP32 target genes were differentially regulated during infection, the majority of which were repressed, and direct repression/activation of these genes by TRP32 was confirmedin vitrowith a cellular luciferase reporter assay.
Requirements for Pseudomonas aeruginosa Type I-F CRISPR-Cas Adaptation Determined Using a Biofilm Enrichment Assay
Authors: Heussler Gary E. , Miller Jon L. , Price Courtney E. , Collins Alan J. , O'Toole George A. 
Published: August 2016

ABSTRACT

CRISPR (clustered regularly interspaced short palindromic repeat)-Cas (CRISPR-associated protein) systems are diverse and found in many archaea and bacteria. These systems have mainly been characterized as adaptive immune systems able to protect against invading mobile genetic elements, including viruses. The first step in this protection is acquisition of spacer sequences from the invader DNA and incorporation of those sequences into the CRISPR array, termed CRISPR adaptation. Progress in understanding the mechanisms and requirements of CRISPR adaptation has largely been accomplished using overexpression of
cas
genes or plasmid loss assays; little work has focused on endogenous CRISPR-acquired immunity from viral predation. Here, we developed a new biofilm-based assay system to enrich for
Pseudomonas aeruginosa
strains with new spacer acquisition. We used this assay to demonstrate that
P. aeruginosa
rapidly acquires spacers protective against DMS3
vir
, an engineered lytic variant of the Mu-like bacteriophage DMS3, through primed CRISPR adaptation from spacers present in the native CRISPR2 array. We found that for the
P. aeruginosa
type I-F system, the
cas1
gene is required for CRISPR adaptation,
recG
contributes to (but is not required for) primed CRISPR adaptation,
recD
is dispensable for primed CRISPR adaptation, and finally, the ability of a putative priming spacer to prime can vary considerably depending on the specific sequences of the spacer.

IMPORTANCE
Our understanding of CRISPR adaptation has expanded largely through experiments in type I CRISPR systems using plasmid loss assays, mutants of
Escherichia coli
, or
cas1-cas2
overexpression systems, but there has been little focus on studying the adaptation of endogenous systems protecting against a lytic bacteriophage. Here we describe a biofilm system that allows
P. aeruginosa
to rapidly gain spacers protective against a lytic bacteriophage. This approach has allowed us to probe the requirements for CRISPR adaptation in the endogenous type I-F system of
P. aeruginosa
. Our data suggest that CRISPR-acquired immunity in a biofilm may be one reason that many
P. aeruginosa
strains maintain a CRISPR-Cas system.
HubP, a Polar Landmark Protein, Regulates Flagellar Number by Assisting in the Proper Polar Localization of FlhG in Vibrio alginolyticus
Authors: Takekawa Norihiro , Kwon Soojin , Nishioka Noriko , Kojima Seiji , Homma Michio 
Published: August 2016

ABSTRACT

The marine bacterium
Vibrio alginolyticus
has a single polar flagellum, the number of which is regulated positively by FlhF and negatively by FlhG. FlhF is intrinsically localized at the cell pole, whereas FlhG is localized there through putative interactions with the polar landmark protein HubP. Here we focused on the role of HubP in the regulation of flagellar number in
V. alginolyticus
. Deletion of
hubP
increased the flagellar number and completely disrupted the polar localization of FlhG. It was thought that the flagellar number is determined primarily by the absolute amount of FlhF localized at the cell pole. Here we found that deletion of
hubP
increased the flagellar number although it did not increase the polar amount of FlhF. We also found that FlhG overproduction did not reduce the polar localization of FlhF. These results show that the absolute amount of FlhF is not always the determinant of flagellar number. We speculate that cytoplasmic FlhG works as a quantitative regulator, controlling the amount of FlhF localized at the pole, and HubP-anchored polar FlhG works as a qualitative regulator, directly inhibiting the activity of polar FlhF. This regulation by FlhF, FlhG, and HubP might contribute to achieving optimal flagellar biogenesis at the cell pole in
V. alginolyticus
.

IMPORTANCE
For regulation of the flagellar number in marine
Vibrio
, two proteins, FlhF and FlhG, work as positive and negative regulators, respectively. In this study, we found that the polar landmark protein HubP is involved in the regulation of flagellar biogenesis. Deletion of
hubP
increased the number of flagella without increasing the amount of pole-localizing FlhF, indicating that the number of flagella is not determined solely by the absolute amount of pole-localizing FlhF, which is inconsistent with the previous model. We propose that cytoplasmic FlhG and HubP-anchored polar FlhG negatively regulate flagellar formation through two independent schemes.
Effect of ischaemic brain injury on sexual function in adult mice
Authors: Tang Yaohui , Yuan Falei , Cai Beibei , Xia Weiliang , Wang Yongting , Yang Guo-Yuan 
Published: August 2016

ObjectivePriapism refers to a condition with persistent abnormal erection of the penis, which is usually caused by disease or injury in the brain or spinal cord, or obstruction to the outflow of blood through the dorsal vein at the root of the penis, without sexual desires. The effect of cerebral ischaemia on sexual function is unknown. The aim of this study is to explore whether priapism occurs in adult mice. Furthermore, we examined the relationship between priapism and the region of infarct in the brain.DesignAdult male CD-1 mice who underwent permanent middle cerebral artery occlusion (pMCAO) were closely examined from 2 hours to 14 days postoperation.ResultsWe found that priapism occurs in ∼80% of the mice with pMCAO, which could persist up to 14 days. Further study has demonstrated that the occurrence of priapism is related to the infarct region: priapism is found only in mice with ischaemic injury extending to the hypothalamus and the hippocampus regions.ConclusionOur result suggested priapism may be used as a deep brain injury marker for evaluating brain injury in mice after pMCAO.
ASEAN ECONOMIC INTEGRATION COMPARED: WHAT DO THE NUMBERS TELL US?
Authors: CHEN LURONG , CUYVERS LUDO , DE LOMBAERDE PHILIPPE 
Published: August 2016

In the comparative literature on regional (economic) integration processes, the ASEAN experience is often contrasted with the European (EU) integration process. More than any other integration process in the world, the ASEAN case is singled out as a counter-model for the EU. The ASEAN model is thereby associated with features such as: low levels of institutionalization, pragmatism, bottom-up or de facto regionalization, regional production platforms, and so on. The positive features of this ‘model’ are often emphasized; however, in recent years there have also been calls for deepening the institutionalization of ASEAN, including from the ADB.The purpose of our paper is double: On the one hand, we present a long-term meta-analysis of available indicators in order to compare more accurately the ASEAN experience with other integration experiences worldwide, and complement (and test) the mostly qualitative approaches in the comparative literature. This should allow to better identify convergences and divergences between ASEAN and other integration processes. On the other hand, by comparing ASEAN with benchmark cases, we shed new light on the potential for further deepening the economic integration process in Southeast Asia. The latter comparison is complemented with gravity-type model estimations to assess the potential for further developing intra-ASEAN trade.It is shown that ASEAN’s export space is expanding faster than the world average and that there is still space for ASEAN countries to further develop the role of its intra-regional trade. ASEAN can further improve the region’s competitiveness by reducing the non-tariff barriers and technical barriers, trade facilitation, strengthening regional value chains, facilitating factor mobility and enhancing regional cooperation.