bluuebunny/crossref_metadata_2025_split huggingface dataset:
Construction of a Gene Knockdown System Based on Catalytically Inactive (“Dead”) Cas9 (dCas9) in Staphylococcus aureus
Authors: Zhao Changlong , Shu Xueqin , Sun Baolin 
Published: April 2017

ABSTRACT

There has been an absence of an efficient method of gene knockdown in the important human pathogen
Staphylococcus aureus
like RNA interference in eukaryotes. The previously developed antisense RNA technology is mainly applied for forward genetic screening but is rather limited in specific gene knockdown because of the lack of rational antisense RNA design strategies. Here we report an efficient and specific system for gene knockdown in
S. aureus
based on the type II clustered regularly interspaced short palindromic repeat (CRISPR) system from
Streptococcus pyogenes
. We can achieve gene silencing with the coexpression of dCas9, an RNA-guided DNA binding protein, and a small guide RNA complementary to the target gene. With this system, we have successfully silenced diverse sets of genes varying in size and expression level in different
S. aureus
strains. This system exhibited high-efficiency knockdown of both essential and nonessential genes, and its effect is inducible and reversible. In addition, the system can repress the expression of multiple genes simultaneously and silence an entire operon or part of it. This RNA-guided DNA targeting system thus provides a simple, rapid, and affordable method for selective gene knockdown in
S. aureus
.

IMPORTANCE
Staphylococcus aureus
is an important human and animal pathogen that can cause a diversity of infectious diseases. Molecular genetic study of
S. aureus
has provided an avenue for the understanding of its virulence, pathogenesis, and drug resistance, leading to the discovery of new therapies for the treatment of staphylococcal infections. However, methodologies developed for genetic manipulation of
S. aureus
usually involve either low efficiency or laborious procedures. Here we report an RNA-guided system for gene knockdown in
S. aureus
and show its high efficiency and simplicity for selective gene silencing in different strains of
S. aureus
. This simple, rapid, and affordable system may serve as a promising tool for functional gene study in
S. aureus
, especially for the study of essential genes, thus facilitating the understanding of this pathogen and its interaction with its hosts.
An Extracellular Cell-Attached Pullulanase Confers Branched α-Glucan Utilization in Human Gut Lactobacillus acidophilus
Authors: Møller Marie S. , Goh Yong Jun , Rasmussen Kasper Bøwig , Cypryk Wojciech , Celebioglu Hasan Ufuk , Klaenhammer Todd R. , Svensson Birte , Abou Hachem Maher 
Published: April 2017

ABSTRACT

Of the few predicted extracellular glycan-active enzymes, glycoside hydrolase family 13 subfamily 14 (GH13_14) pullulanases are the most common in human gut lactobacilli. These enzymes share a unique modular organization, not observed in other bacteria, featuring a catalytic module, two starch binding modules, a domain of unknown function, and a C-terminal surface layer association protein (SLAP) domain. Here, we explore the specificity of a representative of this group of pullulanases,
Lactobacillus acidophilus
Pul13_14 (
La
Pul13_14), and its role in branched α-glucan metabolism in the well-characterized
Lactobacillus acidophilus
NCFM, which is widely used as a probiotic. Growth experiments with
L. acidophilus
NCFM on starch-derived branched substrates revealed a preference for α-glucans with short branches of about two to three glucosyl moieties over amylopectin with longer branches. Cell-attached debranching activity was measurable in the presence of α-glucans but was repressed by glucose. The debranching activity is conferred exclusively by
La
Pul13_14 and is abolished in a mutant strain lacking a functional
La
Pul13_14 gene. Hydrolysis kinetics of recombinant
La
Pul13_14 confirmed the preference for short-branched α-glucan oligomers consistent with the growth data. Curiously, this enzyme displayed the highest catalytic efficiency and the lowest

K
m

reported for a pullulanase. Inhibition kinetics revealed mixed inhibition by β-cyclodextrin, suggesting the presence of additional glucan binding sites besides the active site of the enzyme, which may contribute to the unprecedented substrate affinity. The enzyme also displays high thermostability and higher activity in the acidic pH range, reflecting adaptation to the physiologically challenging conditions in the human gut.

IMPORTANCE
Starch is one of the most abundant glycans in the human diet. Branched α-1,6-glucans in dietary starch and glycogen are nondegradable by human enzymes and constitute a metabolic resource for the gut microbiota. The role of health-beneficial lactobacilli prevalent in the human small intestine in starch metabolism remains unexplored in contrast to colonic bacterial residents. This study highlights the pivotal role of debranching enzymes in the breakdown of starchy branched α-glucan oligomers (α-limit dextrins) by human gut lactobacilli exemplified by
Lactobacillus acidophilus
NCFM, which is one of the best-characterized strains used as probiotics. Our data bring novel insight into the metabolic preference of
L. acidophilus
for α-glucans with short α-1,6-branches. The unprecedented affinity of the debranching enzyme that confers growth on these substrates reflects its adaptation to the nutrient-competitive gut ecological niche and constitutes a potential advantage in cross-feeding from human and bacterial dietary starch metabolism.
The Current State of Macrolide Resistance in Campylobacter spp.: Trends and Impacts of Resistance Mechanisms
Authors: Bolinger Hannah , Kathariou Sophia 
Published: April 2017

ABSTRACT

Campylobacter
spp., especially
Campylobacter jejuni
and
C. coli
, are leading bacterial foodborne pathogens worldwide. In the United States, an estimated 0.8 million cases of campylobacteriosis occur annually, mostly involving
C. jejuni
. Campylobacteriosis is generally self-limiting, but in severe cases, treatment with antibiotics may be mandated. The increasing incidence of fluoroquinolone resistance in
Campylobacter
has rendered macrolides such as erythromycin and azithromycin the drugs of choice for human campylobacteriosis. The prevalence of macrolide resistance in
C. jejuni
remains low, but macrolide resistance can be common in
C. coli
. Substitutions in the 23S rRNA gene, specifically A2075G, and less frequently A2074C/G, remain the most common mechanism for high-level resistance to macrolides. In
C. jejuni
, resistance mediated by such substitutions is accompanied by a reduced ability to colonize chickens and other fitness costs, potentially contributing to the low incidence of macrolide resistance. Interestingly, similar fitness impacts have not been noted in
C. coli
. Also noteworthy is a novel mechanism first reported in 2014 for a
C. coli
isolate from China and mediated by
erm
(B) harbored on multidrug resistance genomic islands. The incidence of
erm
(B) appears to reflect clonal expansion of certain strains, and whole-genome sequencing has been critical to the elucidation of
erm
(B)-associated macrolide resistance in
Campylobacter
spp. With the exception of one report from Spain,
erm
(B)-mediated macrolide resistance has been restricted to
Campylobacter
spp., mostly
C. coli
, of animal and human origin from China. If
erm
(B)-mediated macrolide resistance does not confer fitness costs in
C. jejuni
, the range of this gene may expand in
C. jejuni
, threatening to compromise treatment effectiveness for severe campylobacteriosis cases.
Fate and Persistence of a Pathogenic NDM-1-Positive Escherichia coli Strain in Anaerobic and Aerobic Sludge Microcosms
Authors: Mantilla-Calderon David , Hong Pei-Ying 
Published: April 2017

ABSTRACTThe presence of emerging biological pollutants in treated wastewater effluents has gained attention due to increased interest in water reuse. To evaluate the effectiveness of the removal of such contaminants by the conventional wastewater treatment process, the fate and decay kinetics of NDM-1-positiveEscherichia colistrain PI7 and its plasmid-encoded antibiotic resistance genes (ARGs) were assessed in microcosms of anaerobic and aerobic sludge. Results showed thatE. coliPI7 decayed at a significantly lower rate under anaerobic conditions. Approximate half-lives were 32.4 ± 1.4 h and 5.9 ± 0.9 h in the anaerobic and aerobic microcosms, respectively. In the aerobic microcosms, after 72 h of operation,E. coliPI7 remained detectable, but no further decay was observed. Instead, 1 in every 10,000E. colicells was identified to be recalcitrant to decay and persist indefinitely in the sludge. ARGs associated with theE. coliPI7 strain were detected to have transferred to other native microorganisms in the sludge or were released to the liquid fraction upon host decay. Extracellular DNA quickly degraded in the liquid fraction of the aerobic sludge. In contrast, no DNA decay was detected in the anaerobic sludge water matrix throughout the 24-h sampling period. This study suggests an increased likelihood of environmental dispersion of ARGs associated with anaerobically treated wastewater effluents and highlights the potential importance of persister cells in the dissemination ofE. coliin the environment during reuse events of treated wastewater.IMPORTANCEThis study examines the decay kinetics of a pathogenic and antibiotic resistant strain ofEscherichia coliin microcosms simulating biological treatment units of aerobic and anaerobic sludge. The results of this study point at a significantly prolonged persistence of theE. coliand the associated antibiotic resistance gene in the anaerobic sludge. However, horizontal transfer of the plasmid encoding the antibiotic resistance gene was detected in the aerobic sludge by a cultivation method. A subpopulation of persisterE. colicells was also detected in the aerobic sludge. The findings of this study suggest potential areas of concern arising from pathogenic and antibiotic-resistantE. coliduring both anaerobic and aerobic sludge treatment processes.
Transfer Potential of Plasmids Conferring Extended-Spectrum-Cephalosporin Resistance in Escherichia coli from Poultry
Authors: Mo Solveig Sølverød , Sunde Marianne , Ilag Hanna Karin , Langsrud Solveig , Heir Even 
Published: April 2017

ABSTRACT

Escherichia coli
strains resistant to extended-spectrum cephalosporins (ESC) are widely distributed in Norwegian broiler production, and the majority harbor transferable IncK or IncI1 plasmids carrying
bla
CMY-2
. Persistent occurrence in broiler farms may occur through the survival of ESC-resistant
E. coli
strains in the farm environment, or by transfer and maintenance of resistance plasmids within a population of environmental bacteria with high survival abilities. The aim of this study was to determine the transferability of two successful
bla
CMY-2
-carrying plasmids belonging to the incompatibility groups IncK and IncI1 into
E. coli
and
Serratia
species recipients. Initially, conjugative plasmid transfer from two
E. coli
donors to potential recipients was tested in an agar assay. Conjugation was further investigated for selected mating pairs in surface and planktonic assays at temperatures from 12°C to 37°C. Transfer of plasmids was observed on agar, in broth, and in biofilm at temperatures down to 25°C. The IncK plasmid was able to transfer into
Serratia marcescens
, and transconjugants were able to act as secondary plasmid donors to different
E. coli
and
Serratia
species recipients. All transconjugants displayed an AmpC phenotype corresponding to the acquisition of
bla
CMY-2
. In summary, the results indicate that the IncK plasmid may transfer between
E. coli
and
Serratia
spp. under conditions relevant for broiler production.

IMPORTANCE
Certain
bla
CMY-2
-carrying plasmids are successful and disseminated in European broiler production. Traditionally, plasmid transferability has been studied under conditions that are optimal for bacterial growth. Plasmid transfer has previously been reported between
E. coli
bacteria in biofilms at 37°C and in broth at temperatures ranging from 8 to 37°C. However, intergenus transfer of
bla
CMY-2
-carrying plasmids from
E. coli
to environmental bacteria in the food-processing chain has not been previously studied. We demonstrate that
bla
CMY-2
-carrying plasmids are capable of conjugative transfer between different poultry-associated bacterial genera under conditions relevant for broiler production. Transfer to
Serratia
spp. and to hosts with good biofilm-forming abilities and with the potential to act as secondary plasmid donors to new hosts might contribute to the persistence of these resistance plasmids. These results contribute to increased knowledge of factors affecting the persistence of ESC resistance in broiler production and can provide a basis for improvement of routines and preventive measures.
Day-to-Day Dynamics of Commensal Escherichia coli in Zimbabwean Cows Evidence Temporal Fluctuations within a Host-Specific Population Structure
Authors: Massot Méril , Couffignal Camille , Clermont Olivier , D'Humières Camille , Chatel Jérémie , Plault Nicolas , Andremont Antoine , Caron Alexandre , Mentré France , Denamur Erick 
Published: April 2017

ABSTRACTTo get insights into the temporal pattern of commensalEscherichia colipopulations, we sampled the feces of four healthy cows from the same herd in the Hwange District of Zimbabwe daily over 25 days. The cows had not received antibiotic treatment during the previous 3 months. We performed viableE. colicounts and characterized the 326 isolates originating from the 98 stool samples at a clonal level, screened them forstxandeaegenes, and tested them for their antibiotic susceptibilities. We observed thatE. colicounts and dominant clones were different among cows, and very few clones were shared. No clone was shared by three or four cows. Clone richness and evenness were not different between cows. Within each host, the variability in theE. colicount was evidenced between days, and no clone was found to be dominant during the entire sampling period, suggesting the existence of clonal interference. Dominant clones tended to persist longer than subdominant ones and were mainly from phylogenetic groups A and B1. FiveE. coliclones were found to contain both thestx1andstx2genes, representing 6.3% of the studied isolates. All cows harbored at least one Shiga toxin-producingE. coli(STEC) strain. Resistance to tetracycline, penicillins, trimethoprim, and sulfonamides was rare and observed in three clones that were shed at low levels in two cows. This study highlights the fact that the commensalE. colipopulation, including the STEC population, is host specific, is highly dynamic over a short time frame, and rarely carries antibiotic resistance determinants in the absence of antibiotic treatment.IMPORTANCEThe literature about the dynamics of commensalEscherichia colipopulations is very scarce. Over 25 days, we followed the totalE. colicounts daily and characterized the sampled clones in the feces of four cows from the same herd living in the Hwange District of Zimbabwe. This study deals with the day-to-day dynamics of both quantitative and qualitative aspects ofE. colicommensal populations, with a focus on both Shiga toxin-producingE. coliand antibiotic-resistantE. colistrains. We show that the structure of these commensal populations was highly specific to the host, even though the cows ate and roamed together, and was highly dynamic between days. Such data are of importance to understand the ecological forces that drive the dynamics of the emergence ofE. coliclones of particular interest within the gastrointestinal tract and their transmission between hosts.
Global-Scale Structure of the Eelgrass Microbiome
Authors: Fahimipour Ashkaan K. , Kardish Melissa R. , Lang Jenna M. , Green Jessica L. , Eisen Jonathan A. , Stachowicz John J. 
Published: April 2017

ABSTRACT

Plant-associated microorganisms are essential for their hosts' survival and performance. Yet, most plant microbiome studies to date have focused on terrestrial species sampled across relatively small spatial scales. Here, we report the results of a global-scale analysis of microbial communities associated with leaf and root surfaces of the marine eelgrass
Zostera marina
throughout its range in the Northern Hemisphere. By contrasting host microbiomes with those of surrounding seawater and sediment, we uncovered the structure, composition, and variability of microbial communities associated with eelgrass. We also investigated hypotheses about the assembly of the eelgrass microbiome using a metabolic modeling approach. Our results reveal leaf communities displaying high variability and spatial turnover that mirror their adjacent coastal seawater microbiomes. By contrast, roots showed relatively low compositional turnover and were distinct from surrounding sediment communities, a result driven by the enrichment of predicted sulfur-oxidizing bacterial taxa on root surfaces. Predictions from metabolic modeling of enriched taxa were consistent with a habitat-filtering community assembly mechanism whereby similarity in resource use drives taxonomic cooccurrence patterns on belowground, but not aboveground, host tissues. Our work provides evidence for a core eelgrass root microbiome with putative functional roles and highlights potentially disparate processes influencing microbial community assembly on different plant compartments.

IMPORTANCE
Plants depend critically on their associated microbiome, yet the structure of microbial communities found on marine plants remains poorly understood in comparison to that for terrestrial species. Seagrasses are the only flowering plants that live entirely in marine environments. The return of terrestrial seagrass ancestors to oceans is among the most extreme habitat shifts documented in plants, making them an ideal testbed for the study of microbial symbioses with plants that experience relatively harsh abiotic conditions. In this study, we report the results of a global sampling effort to extensively characterize the structure of microbial communities associated with the widespread seagrass species
Zostera marina
, or eelgrass, across its geographic range. Our results reveal major differences in the structure and composition of above- versus belowground microbial communities on eelgrass surfaces, as well as their relationships with the environment and host.
Effect of Antimicrobial and Physical Treatments on Growth of Multispecies Staphylococcal Biofilms
Authors: Stewart Elizabeth J. , Payne David E. , Ma Tianhui Maria , VanEpps J. Scott , Boles Blaise R. , Younger John G. , Solomon Michael J. 
Published: April 2017

ABSTRACT

The prevalence and structure of
Staphylococcus aureus
and
Staphylococcus epidermidis
within multispecies biofilms were found to depend sensitively on physical environment and antibiotic dosage. Although these species commonly infect similar sites, such as orthopedic implants, little is known about their behavior in multispecies communities, particularly in response to treatment. This research establishes that
S. aureus
is much more prevalent than
S. epidermidis
when simultaneously seeded and grown under unstressed conditions (pH 7, 37°C) in both laboratory and clinical strains. In multispecies communities,
S. epidermidis
is capable of growing a more confluent biofilm when the addition of
S. aureus
is delayed 4 to 6 h during 18 h of growth. Different vancomycin dosages generate various behaviors:
S. epidermidis
is more prevalent at a dose of 1.0 μg/ml vancomycin, but reduced growth of both species occurs at 1.9 μg/ml vancomycin. This variability is consistent with the different MICs of
S. aureus
and
S. epidermidis
. Growth at higher temperature (45°C) results in an environment where
S. aureus
forms porous biofilms. This porosity allows
S. epidermidis
to colonize more of the surface, resulting in detectable
S. epidermidis
biomass. Variations in pH result in increased prevalence of
S. epidermidis
at low pH (pH 5 and 6), while
S. aureus
remains dominant at high pH (pH 8 and 9). This work establishes the structural variability of multispecies staphylococcal biofilms as they undergo physical and antimicrobial treatments. It provides a basis for understanding the structure of these communities at infection sites and how treatments disrupt their multispecies behaviors.

IMPORTANCE
Staphylococcus aureus
and
Staphylococcus epidermidis
are two species of bacteria that are commonly responsible for biofilm infections on medical devices. Biofilms are structured communities of bacteria surrounded by polysaccharides, proteins, and DNA; bacteria are more resistant to antimicrobials as part of a biofilm than as individual cells. This work investigates the structure and prevalence of these two organisms when grown together in multispecies biofilms and shows shifts in the behavior of the polymicrobial community when grown in various concentrations of vancomycin (an antibiotic commonly used to treat staphylococcal infections), in a high-temperature environment (a condition previously shown to lead to cell disruption and death), and at low and high pH (a change that has been previously shown to soften the mechanical properties of staphylococcal biofilms). These shifts in community structure demonstrate the effect such treatments may have on multispecies staphylococcal infections.
Endoscopic advanced imaging of the respiratory tract: exploring probe-based confocal laser endomicroscopy in emphysema
Authors: Yserbyt J , Dooms C , Janssens W , Verleden G M 
Published: April 2017

Probe-based confocal laser endomicroscopy (pCLE) was performed in 15 patients with emphysema and 15 healthy subjects to visualise small airways in a direct and dynamic way. Morphometry shows that the median cross-sectional area of the alveolar openings at the level of the alveolar ducts is significantly larger in emphysema (7.2×104 µm2) as compared with healthy subjects (5.2×104 µm2) (p=0.0002). Normalised autofluorescence intensity histograms show a decrease in median autofluorescence intensity (mAFI) in emphysema (p=0.001). mAFI correlates well with Tiffeneau index (r=0.66, p=0.007, 95% CI 0.21 to 0.88). Autofluorescence intensity in emphysema correlates with corresponding data of CT-based quantification. pCLE-based morphometry and autofluorescence intensity analysis in emphysema is able to detect regional changes inside the ‘quiet zone’.Trial registration numberResults, NCT01204970.