bluuebunny/crossref_metadata_2025 huggingface dataset:
PBP1a/LpoA but Not PBP1b/LpoB Are Involved in Regulation of the Major β-Lactamase GeneblaAin Shewanella oneidensis
Authors: Yin Jianhua , Sun Yiyang , Mao Yinting , Jin Miao , Gao Haichun
Published: April 2015

β-Lactamase production is one of the most important strategies for Gram-negative bacteria to combat β-lactam antibiotics. Studies of the regulation of β-lactamase expression have largely been focused on the class C β-lactamase AmpC, whose induction by β-lactams requires LysR-type regulator AmpR and permease AmpG-dependent peptidoglycan recycling intermediates. InShewanella, which is ubiquitous in aquatic environments and is a reservoir for antibiotic resistance, production of the class D β-lactamase BlaA confers bacteria with natural resistance to many β-lactams. Expression of theblaAgene in the genus representativeShewanella oneidensisis distinct from the AmpC paradigm because of the lack of an AmpR homologue and the presence of an additional AmpG-independent regulatory pathway. In this study, using transposon mutagenesis, we identify proteins that are involved inblaAregulation. Inactivation ofmrcAandlpoA, which encode penicillin binding protein 1a (PBP1a) and its lipoprotein cofactor, LpoA, respectively, drastically enhancesblaAexpression in the absence of β-lactams. Although PBP1b and its cognate, LpoB, also exist inS. oneidensis, their roles inblaAinduction are dispensable. We further show that themrcA-mediatedblaAexpression is independent of AmpG.
Activity of Clarithromycin or Rifampin Alone or in Combination against Experimental Rhodococcus equi Infection in Mice
Authors: Burton Alexandra J. , Giguère Steeve , Berghaus Londa J. , Hondalus Mary K.
Published: April 2015

Treatment of mice with the combination of clarithromycin with rifampin resulted in a significantly lower number of
Rhodococcus equi
CFU in the organs of mice than treatment with either drug alone or placebo. There was no significant difference in the number of
R. equi
CFU between mice treated with clarithromycin monotherapy, rifampin monotherapy, or placebo. The combination of clarithromycin with rifampin conferred a clear advantage over either drug as monotherapy in this model of chronic
R. equi
infection.
In VitroAntibacterial Activity of AZD0914 against Human Mycoplasmas and Ureaplasmas
Authors: Waites Ken B. , Crabb Donna M. , Duffy Lynn B. , Huband Michael D.
Published: April 2015

In this study, susceptibilities were determined for AZD0914, a spiropyrimidinetrione DNA gyrase inhibitor, azithromycin, doxycycline, and levofloxacin againstMycoplasmaandUreaplasmaspecies. The activity of AZD0914 was comparable to that of levofloxacin and doxycycline againstMycoplasma genitaliumandMycoplasma pneumoniae. The AZD0914 MIC90againstMycoplasma hominiswas 8-fold greater than that for levofloxacin. The AZD0914 MIC90againstUreaplasmaspecies was 4-fold less than that for azithromycin and 8-fold less than that for levofloxacin and doxycycline.
The CpAL Quorum Sensing System Regulates Production of Hemolysins CPA and PFO To Build Clostridium perfringens Biofilms
Authors: Vidal Jorge E. , Shak Joshua R. , Canizalez-Roman Adrian
Published: April 2015

Clostridium perfringensstrains produce severe diseases, including myonecrosis and enteritis necroticans, in humans and animals. Diseases are mediated by the production of potent toxins that often damage the site of infection, e.g., skin epithelium during myonecrosis. In planktonic cultures, the regulation of important toxins, such as CPA, CPB, and PFO, is controlled by theC. perfringensAgr-like (CpAL) quorum sensing (QS) system. Strains also encode a functional LuxS/AI-2 system. AlthoughC. perfringensstrains form biofilm-like structures, the regulation of biofilm formation is poorly understood. Therefore, our studies investigated the role of CpAL and LuxS/AI-2 QS systems and of QS-regulated factors in controlling the formation of biofilms. We first demonstrate that biofilm production by reference strains differs depending on the culture medium. Increased biomass correlated with the presence of extracellular DNA in the supernatant, which was released by lysis of a fraction of the biofilm population and planktonic cells. Whereas ΔagrBmutant strains were not able to produce biofilms, a ΔluxSmutant produced wild-type levels. The transcript levels of CpAL-regulatedcpaandpfoAgenes, but notcpb, were upregulated in biofilms compared to planktonic cultures. Accordingly, Δcpaand ΔpfoAmutants, in type A (S13) or type C (CN3685) backgrounds, were unable to produce biofilms, whereas CN3685Δcpbmade wild-type levels. Biofilm formation was restored in complemented Δcpa/cpaand ΔpfoA/pfoAstrains. Confocal microscopy studies further detected CPA partially colocalizing with eDNA on the biofilm structure. Thus, CpAL regulates biofilm formation inC. perfringensby increasing levels of certain toxins required to build biofilms.
The Human P-Glycoprotein Transporter Enhances the Type I Interferon Response to Listeria monocytogenes Infection
Authors: Sigal Nadejda , Kaplan Zeevi Millie , Weinstein Shiri , Peer Dan , Herskovits Anat A.
Published: April 2015

Human multidrug efflux transporters are known for their ability to extrude antibiotics and toxic compounds out of cells, yet accumulating data indicate they have additional functions in diverse physiological processes not related to drug efflux. Here, we show that the human multidrug transporter P-glycoprotein (P-gp) (also named MDR1 and ABCB1) is transcriptionally induced in the monocytic cell line THP-1 upon infection with the human intracellular bacterial pathogen
Listeria monocytogenes
. Notably, we found that P-gp is important for full activation of the type I interferon response elicited against
L. monocytogenes
bacteria. Both inhibition of P-gp function by verapamil and inhibition of its transcription using mRNA silencing led to a reduction in the magnitude of the type I response in infected cells. This function of P-gp was specific to type I interferon cytokines elicited against cytosolic replicating bacteria and was not observed in response to cyclic di-AMP (c-di-AMP), a molecule that was shown to be secreted by
L. monocytogenes
during infection and to trigger type I interferons. Moreover, P-gp was not involved in activation of other proinflammatory cytokines, such as those triggered by vacuolar-restricted
L. monocytogenes
or lipopolysaccharide (LPS). Taken together, these findings demonstrate a role for P-gp in proper development of an innate immune response against intracellular pathogens, highlighting the complexity in employing therapeutic strategies that involve inhibition of multidrug resistance (MDR) efflux pumps.
The Cpx System Regulates Virulence Gene Expression in Vibrio cholerae
Authors: Acosta Nicole , Pukatzki Stefan , Raivio Tracy L.
Published: April 2015

Bacteria possess signal transduction pathways capable of sensing and responding to a wide variety of signals. The Cpx envelope stress response, composed of the sensor histidine kinase CpxA and the response regulator CpxR, senses and mediates adaptation to insults to the bacterial envelope. The Cpx response has been implicated in the regulation of a number of envelope-localized virulence determinants across bacterial species. Here, we show that activation of the Cpx pathway in
Vibrio cholerae
El Tor strain C6706 leads to a decrease in expression of the major virulence factors in this organism, cholera toxin (CT) and the toxin-coregulated pilus (TCP). Our results indicate that this occurs through the repression of production of the ToxT regulator and an additional upstream transcription factor, TcpP. The effect of the Cpx response on CT and TCP expression is mostly abrogated in a cyclic AMP receptor protein (CRP) mutant, although expression of the
crp
gene is unaltered. Since TcpP production is controlled by CRP, our data suggest a model whereby the Cpx response affects CRP function, which leads to diminished TcpP, ToxT, CT, and TCP production.
Characterization of Clostridium perfringens TpeL Toxin Gene Carriage, Production, Cytotoxic Contributions, and Trypsin Sensitivity
Authors: Chen Jianming , McClane Bruce A.
Published: April 2015

Large clostridial toxins (LCTs) are produced by at least four pathogenic clostridial species, and several LCTs are proven pivotal virulence factors for both human and veterinary diseases. TpeL is a recently identified LCT produced by
Clostridium perfringens
that has received relatively limited study. In response, the current study surveyed carriage of the
tpeL
gene among different
C. perfringens
strains, detecting this toxin gene in some type A, B, and C strains but not in any type D or E strains. This study also determined that all tested strains maximally produce, and extracellularly release, TpeL at the late-log or early-stationary growth stage during
in vitro
culture, which is different from the maximal late-stationary-phase production reported previously for other LCTs and for TpeL production by
C. perfringens
strain JIR12688. In addition, the present study found that TpeL levels in culture supernatants can be repressed by either glucose or sucrose. It was also shown that, at natural production levels, TpeL is a significant contributor to the cytotoxic activity of supernatants from cultures of
tpeL
-positive strain CN3685. Lastly, this study identified TpeL, which presumably is produced in the intestines during diseases caused by TpeL-positive type B and C strains, as a toxin whose cytotoxicity decreases after treatment with trypsin; this finding may have pathophysiologic relevance by suggesting that, like beta toxin, TpeL contributes to type B and C infections in hosts with decreased trypsin levels due to disease, diet, or age.
A Homology Model Reveals Novel Structural Features and an Immunodominant Surface Loop/Opsonic Target in the Treponema pallidum BamA Ortholog TP_0326
Authors: Luthra Amit , Anand Arvind , Hawley Kelly L. , LeDoyt Morgan , La Vake Carson J. , Caimano Melissa J. , Cruz Adriana R. , Salazar Juan C. , Radolf Justin D.
Published: April 2015

We recently demonstrated that TP_0326 is a bona fide rare outer membrane protein (OMP) inTreponema pallidumand that it possesses characteristic BamA bipartite topology. Herein, we used immunofluorescence analysis (IFA) to show that only the β-barrel domain of TP_0326 contains surface-exposed epitopes in intactT. pallidum. Using the solved structure ofNeisseria gonorrhoeaeBamA, we generated a homology model of full-length TP_0326. Although the model predicts a typical BamA fold, the β-barrel harbors features not described in other BamAs. Structural modeling predicted that a dome comprised of three large extracellular loops, loop 4 (L4), L6, and L7, covers the barrel's extracellular opening. L4, the dome's major surface-accessible loop, contains mainly charged residues, while L7 is largely neutral and contains a polyserine tract in a two-tiered conformation. L6 projects into the β-barrel but lacks the VRGF/Y motif that anchors L6 within other BamAs. IFA and opsonophagocytosis assay revealed that L4 is surface exposed and an opsonic target. Consistent with B cell epitope predictions, immunoblotting and enzyme-linked immunosorbent assay (ELISA) confirmed that L4 is an immunodominant loop inT. pallidum-infected rabbits and humans with secondary syphilis. Antibody capture experiments usingEscherichia coliexpressing OM-localized TP_0326 as aT. pallidumsurrogate further established the surface accessibility of L4. Lastly, we found that a naturally occurring substitution (Leu593→ Gln593) in the L4 sequences ofT. pallidumstrains affects antibody binding in sera from syphilitic patients. Ours is the first study to employ a “structure-to-pathogenesis” approach to map the surface topology of aT. pallidumOMP within the context of syphilitic infection.

IMPORTANCEPreviously, we reported that TP_0326 is a bona fide rare outer membrane protein (OMP) inTreponema pallidumand that it possesses the bipartite topology characteristic of a BamA ortholog. Using a homology model as a guide, we found that TP_0326 displays unique features which presumably relate to its function(s) in the biogenesis ofT. pallidum's unorthodox OM. The model also enabled us to identify an immunodominant epitope in a large extracellular loop that is both an opsonic target and subject to immune pressure in a human population. Ours is the first study to follow a structure-to-pathogenesis approach to map the surface topology of aT. pallidumrare OMP within the context of syphilitic infection.
Genome Modification in Enterococcus faecalis OG1RF Assessed by Bisulfite Sequencing and Single-Molecule Real-Time Sequencing
Authors: Huo Wenwen , Adams Hannah M. , Zhang Michael Q. , Palmer Kelli L.
Published: April 2015

Enterococcus faecalis
is a Gram-positive bacterium that natively colonizes the human gastrointestinal tract and opportunistically causes life-threatening infections. Multidrug-resistant (MDR)
E. faecalis
strains have emerged, reducing treatment options for these infections. MDR
E. faecalis
strains have large genomes containing mobile genetic elements (MGEs) that harbor genes for antibiotic resistance and virulence determinants. Bacteria commonly possess genome defense mechanisms to block MGE acquisition, and we hypothesize that these mechanisms have been compromised in MDR
E. faecalis
. In restriction-modification (R-M) defense, the bacterial genome is methylated at cytosine (C) or adenine (A) residues by a methyltransferase (MTase), such that nonself DNA can be distinguished from self DNA. A cognate restriction endonuclease digests improperly modified nonself DNA. Little is known about R-M in
E. faecalis
. Here, we use genome resequencing to identify DNA modifications occurring in the oral isolate OG1RF. OG1RF has one of the smallest
E. faecalis
genomes sequenced to date and possesses few MGEs. Single-molecule real-time (SMRT) and bisulfite sequencing revealed that OG1RF has global 5-methylcytosine (m5C) methylation at 5′-GCWGC-3′ motifs. A type II R-M system confers the m5C modification, and disruption of this system impacts OG1RF electrotransformability and conjugative transfer of an antibiotic resistance plasmid. A second DNA MTase was poorly expressed under laboratory conditions but conferred global
N
4
-methylcytosine (m4C) methylation at 5′-CCGG-3′ motifs when expressed in
Escherichia coli
. Based on our results, we conclude that R-M can act as a barrier to MGE acquisition and likely influences antibiotic resistance gene dissemination in the
E. faecalis
species.

IMPORTANCE
The horizontal transfer of antibiotic resistance genes among bacteria is a critical public health concern.
Enterococcus faecalis
is an opportunistic pathogen that causes life-threatening infections in humans. Multidrug resistance acquired by horizontal gene transfer limits treatment options for these infections. In this study, we used innovative DNA sequencing methodologies to investigate how a model strain of
E. faecalis
discriminates its own DNA from foreign DNA, i.e., self versus nonself discrimination. We also assess the role of an
E. faecalis
genome modification system in modulating conjugative transfer of an antibiotic resistance plasmid. These results are significant because they demonstrate that differential genome modification impacts horizontal gene transfer frequencies in
E. faecalis
.
Role of Bacillus subtilis DNA Glycosylase MutM in Counteracting Oxidatively Induced DNA Damage and in Stationary-Phase-Associated Mutagenesis
Authors: Gómez-Marroquín Martha , Vidales Luz E. , Debora Bernardo N. , Santos-Escobar Fernando , Obregón-Herrera Armando , Robleto Eduardo A. , Pedraza-Reyes Mario
Published: April 2015

Reactive oxygen species (ROS) promote the synthesis of the DNA lesion 8-oxo-G, whose mutagenic effects are counteracted in distinct organisms by the DNA glycosylase MutM. We report here that in
Bacillus subtilis
,
mutM
is expressed during the exponential and stationary phases of growth. In agreement with this expression pattern, results of a Western blot analysis confirmed the presence of MutM in both stages of growth. In comparison with cells of a wild-type strain, cells of
B. subtilis
lacking MutM increased their spontaneous mutation frequency to Rif
r
and were more sensitive to the ROS promoter agents hydrogen peroxide and 1,1′-dimethyl-4,4′-bipyridinium dichloride (Paraquat). However, despite MutM's proven participation in preventing ROS-induced-DNA damage, the expression of
mutM
was not induced by hydrogen peroxide, mitomycin C, or NaCl, suggesting that transcription of this gene is not under the control of the RecA, PerR, or σ
B
regulons. Finally, the role of MutM in stationary-phase-associated mutagenesis (SPM) was investigated in the strain
B. subtilis
YB955 (
hisC952 metB5 leuC427
). Results revealed that under limiting growth conditions, a
mutM
knockout strain significantly increased the amount of stationary-phase-associated
his
,
met
, and
leu
revertants produced. In summary, our results support the notion that the absence of MutM promotes mutagenesis that allows nutritionally stressed
B. subtilis
cells to escape from growth-limiting conditions.

IMPORTANCE
The present study describes the role played by a DNA repair protein (MutM) in protecting the soil bacterium
Bacillus subtilis
from the genotoxic effects induced by reactive oxygen species (ROS) promoter agents. Moreover, it reveals that the genetic inactivation of
mutM
allows nutritionally stressed bacteria to escape from growth-limiting conditions, putatively by a mechanism that involves the accumulation and error-prone processing of oxidized DNA bases.
Regulation by the Modulation of Gene Expression Variability
Authors: Dubnau David
Published: April 2015

Classically, transcription is regulated so that the average expression per cell changes, often with a distribution that extends across the population. Roggiani and Goulian (
M. Roggiani and M. Goulian
, J. Bacteriol. 197:1976–1987, 2015, doi:
http://dx.doi.org/10.1128/JB.00074-15
) have shown that this is what happens when the
torCAD
operon of
Escherichia coli
is induced anaerobically by the addition of trimethylamine-
N
-oxide (TMAO). However, when the same inducer is added to aerobically growing cells, only a subset of the cells respond, although the mean expression per cell is similar to that obtained anaerobically. Thus, in the presence of oxygen, the variance but not the expression mean is altered. The regulation of gene expression variance appears to be due to noise in the phosphorelay that governs
torCAD
transcription.
Basal Body Structures Differentially Affect Transcription of RpoN- and FliA-Dependent Flagellar Genes in Helicobacter pylori
Authors: Tsang Jennifer , Hoover Timothy R.
Published: April 2015

Flagellar biogenesis in
Helicobacter pylori
is regulated by a transcriptional hierarchy governed by three sigma factors, RpoD (σ
80
), RpoN (σ
54
), and FliA (σ
28
), that temporally coordinates gene expression with the assembly of the flagellum. Previous studies showed that loss of flagellar protein export apparatus components inhibits transcription of flagellar genes. The FlgS/FlgR two-component system activates transcription of RpoN-dependent genes though an unknown mechanism. To understand better the extent to which flagellar gene regulation is coupled to flagellar assembly, we disrupted flagellar biogenesis at various points and determined how these mutations affected transcription of RpoN-dependent (
flaB
and
flgE
) and FliA-dependent (
flaA
) genes. The MS ring (encoded by
fliF
) is one of the earliest flagellar structures assembled. Deletion of
fliF
resulted in the elimination of RpoN-dependent transcripts and an ∼4-fold decrease in
flaA
transcript levels. FliH is a cytoplasmic protein that functions with the C ring protein FliN to shuttle substrates to the export apparatus. Deletions of
fliH
and genes encoding C ring components (
fliM
and
fliY
) decreased transcript levels of
flaB
and
flgE
but had little or no effect on transcript levels of
flaA
. Transcript levels of
flaB
and
flgE
were elevated in mutants where genes encoding rod proteins (
fliE
and
flgBC
) were deleted, while transcript levels of
flaA
was reduced ∼2-fold in both mutants. We propose that FlgS responds to an assembly checkpoint associated with the export apparatus and that FliH and one or more C ring component assist FlgS in engaging this flagellar structure.

IMPORTANCE
The mechanisms used by bacteria to couple transcription of flagellar genes with assembly of the flagellum are poorly understood. The results from this study identified components of the
H. pylori
flagellar basal body that either positively or negatively affect expression of RpoN-dependent flagellar genes. Some of these basal body proteins may interact directly with regulatory proteins that control transcription of the
H. pylori
RpoN regulon, a hypothesis that can be tested by examining protein-protein interactions
in vitro
.
Recoding of the Vesicular Stomatitis Virus L Gene by Computer-Aided Design Provides a Live, Attenuated Vaccine Candidate
Authors: Wang Bingyin , Yang Chen , Tekes Gergely , Mueller Steffen , Paul Aniko , Whelan Sean P. J. , Wimmer Eckard
Published: April 2015

Codon pair bias (CPB), which has been observed in all organisms, is a neglected genomic phenomenon that affects gene expression. CPB results from synonymous codons that are paired more or less frequently in ORFeomes regardless of codon bias. The effect of an individual codon pair change is usually small, but when it is amplified by large-scale genome recoding, strikingly altered biological phenotypes are observed. The utility of codon pair bias in the development of live attenuated vaccines was recently demonstrated by recodings of poliovirus (a positive-strand RNA virus) and influenza virus (a negative-strand segmented RNA virus). Here, the L gene of vesicular stomatitis virus (VSV), a nonsegmented negative-sense RNA virus, was partially recoded based on codon pair bias. Totals of 858 and 623 silent mutations were introduced into a 5′-terminal segment of the viral L gene (designated L1) to create sequences containing either overrepresented or underrepresented codon pairs, designated L1
sdmax
and L1
min
, respectively. Analysis revealed that recombinant VSV containing the L1
min
sequence could not be recovered, whereas the virus with the sdmax sequence showed a modest level of attenuation in cell culture. More strikingly, in mice the L1
sdmax
virus was almost as immunogenic as the parental strain but highly attenuated. Taken together, these results open a new road to attain a balance between VSV virulence and immunogenicity, which could serve as an example for the attenuation of other negative-strand, nonsegmented RNA viruses.

IMPORTANCE
Vesicular stomatitis virus (VSV) is the prototypic rhabdovirus in the order
Mononegavirales
. A wide range of human pathogens belong to this family. Using a unique computer algorithm and large-scale genome synthesis, we attempted to develop a live attenuated vaccine strain for VSV, which could be used as an antigen delivery platform for humans. Recombinant VSVs with distinct codon pair biases were rationally designed, constructed, and analyzed in both cell culture and an animal model. One such recombinant virus, L1
sdmax
, contained extra overrepresented codon pairs in its L gene open reading frame (ORF) and showed promise as an effective vaccine candidate because of a favorable balance between virulence and immunogenicity. Our study not only contributes to the understanding of the underlying mechanism of codon pair bias but also may facilitate the development of live attenuated vaccines for other viruses in the order
Mononegavirales
.
Distinct Roles of the Repeat-Containing Regions and Effector Domains of the Vibrio vulnificus Multifunctional-Autoprocessing Repeats-in-Toxin (MARTX) Toxin
Authors: Kim Byoung Sik , Gavin Hannah E. , Satchell Karla J. F.
Published: April 2015

Vibrio vulnificus
is a seafood-borne pathogen that destroys the intestinal epithelium, leading to rapid bacterial dissemination and death. The most important virulence factor is the multifunctional-autoprocessing repeats-in-toxin (MARTX) toxin comprised of effector domains in the center region flanked by long repeat-containing regions which are well conserved among MARTX toxins and predicted to translocate effector domains. Here, we examined the role of the repeat-containing regions using a modified
V. vulnificus
MARTX (MARTX
Vv
) toxin generated by replacing all the internal effector domains with β-lactamase (Bla). Bla activity was detected in secretions from the bacterium and also in the cytosol of intoxicated epithelial cells. The modified MARTX
Vv
toxin without effector domains retained its necrotic activity but lost its cell-rounding activity. Further, deletion of the carboxyl-terminal repeat-containing region blocked toxin secretion from the bacterium. Deletion of the amino-terminal repeat-containing region had no effect on secretion but completely abolished translocation and necrosis. Neither secretion nor translocation was affected by enzymatically inactivating the cysteine protease domain of the toxin. These data demonstrate that the amino-terminal and carboxyl-terminal repeat-containing regions of the MARTX
Vv
toxin are necessary and sufficient for the delivery of effector domains and epithelial cell lysis
in vitro
but that effector domains are required for other cytopathic functions. Furthermore, Ca
2+
-dependent secretion of the modified MARTX
Vv
toxin suggests that nonclassical RTX-like repeats found in the carboxyl-terminal repeat-containing region are functionally similar to classical RTX repeats found in other RTX proteins.

IMPORTANCE
Up to 95% of deaths from seafood-borne infections in the United States are due solely to one pathogen,
V. vulnificus
. Among its various virulence factors, the MARTX
Vv
toxin has been characterized as a critical exotoxin for successful pathogenesis of
V. vulnificus
in mouse infection models. Similarly to MARTX toxins of other pathogens, MARTX
Vv
toxin is comprised of repeat-containing regions, central effector domains, and an autoprocessing cysteine protease domain. Yet how each of these regions contributes to essential activities of the toxins has not been fully identified for any of MARTX toxins. Using modified MARTX
Vv
toxin fused with β-lactamase as a reporter enzyme, the portion(s) responsible for toxin secretion from bacteria, effector domain translocation into host cells, rapid host cell rounding, and necrotic host cell death was identified. The results are relevant for understanding how MARTX
Vv
toxin serves as both a necrotic pore-forming toxin and an effector delivery platform.
The Glycerol-Dependent Metabolic Persistence of Pseudomonas putida KT2440 Reflects the Regulatory Logic of the GlpR Repressor
Authors: Nikel Pablo I. , Romero-Campero Francisco J. , Zeidman Joshua A. , Goñi-Moreno Ángel , de Lorenzo Víctor
Published: April 2015

The growth of the soil bacterium
Pseudomonas putida
KT2440 on glycerol as the sole carbon source is characterized by a prolonged lag phase, not observed with other carbon substrates. We examined the bacterial growth in glycerol cultures while monitoring the metabolic activity of individual cells. Fluorescence microscopy and flow cytometry, as well as the analysis of the temporal start of growth in single-cell cultures, revealed that adoption of a glycerol-metabolizing regime was not the result of a gradual change in the whole population but rather reflected a time-dependent bimodal switch between metabolically inactive (i.e., nongrowing) and fully active (i.e., growing) bacteria. A transcriptional Φ(
glpD-gfp
) fusion (a proxy of the glycerol-3-phosphate [G3P] dehydrogenase activity) linked the macroscopic phenotype to the expression of the
glp
genes. Either deleting
glpR
(encoding the G3P-responsive transcriptional repressor that controls the expression of the
glpFKRD
gene cluster) or altering G3P formation (by overexpressing
glpK
, encoding glycerol kinase) abolished the bimodal
glpD
expression. These manipulations eliminated the stochastic growth start by shortening the otherwise long lag phase. Provision of
glpR
in
trans
restored the phenotypes lost in the
ΔglpR
mutant. The prolonged nongrowth regime of
P. putida
on glycerol could thus be traced to the regulatory device controlling the transcription of the
glp
genes. Since the physiological agonist of GlpR is G3P, the arrangement of metabolic and regulatory components at this checkpoint merges a positive feedback loop with a nonlinear transcriptional response, a layout fostering the observed time-dependent shift between two alternative physiological states.

IMPORTANCE
Phenotypic variation is a widespread attribute of prokaryotes that leads,
inter alia
, to the emergence of persistent bacteria, i.e., live but nongrowing members within a genetically clonal population. Persistence allows a fraction of cells to avoid the killing caused by conditions or agents that destroy most growing bacteria (e.g., some antibiotics). Known molecular mechanisms underlying the phenomenon include genetic changes, epigenetic variations, and feedback-based multistability. We show that a prolonged nongrowing state of the bacterial population can be brought about by a distinct regulatory architecture of metabolic genes when cells face specific nutrients (e.g., glycerol).
Pseudomonas putida
may have adopted the resulting carbon source-dependent metabolic bet hedging as an advantageous trait for exploring new chemical and nutritional landscapes. Defeating such naturally occurring adaptive features of environmental bacteria is instrumental in improving the performance of these microorganisms as whole-cell catalysts in a bioreactor setup.
Rapid Genome Assembly and Comparison Decode Intrastrain Variation in Human Alphaherpesviruses
Authors: Parsons Lance R. , Tafuri Yolanda R. , Shreve Jacob T. , Bowen Christopher D. , Shipley Mackenzie M. , Enquist L. W. , Szpara Moriah L.
Published: April 2015

Herpes simplex virus (HSV) is a widespread pathogen that causes epithelial lesions with recurrent disease that manifests over a lifetime. The lifelong aspect of infection results from latent viral infection of neurons, a reservoir from which the virus reactivates periodically. Recent work has demonstrated the breadth of genetic variation in globally distributed HSV strains. However, the amount of variation or capacity for mutation within one strain has not been well studied. Here we developed and applied a streamlined new approach for assembly and comparison of large DNA viral genomes such as HSV-1. This viral genome assembly (VirGA) workflow incorporates a combination of
de novo
assembly, alignment, and annotation strategies to automate the generation of draft genomes for large viruses. We applied this approach to quantify the amount of variation between clonal derivatives of a common parental virus stock. In addition, we examined the genetic basis for syncytial plaque phenotypes displayed by a subset of these strains. In each of the syncytial strains, we found an identical DNA change, affecting one residue in the gB (UL27) fusion protein. Since these identical mutations could have appeared after extensive
in vitro
passaging, we applied the VirGA sequencing and comparison approach to two clinical HSV-1 strains isolated from the same patient. One of these strains was syncytial upon first culturing; its sequence revealed the same gB mutation. These data provide insight into the extent and origin of genome-wide intrastrain HSV-1 variation and present useful methods for expansion to
in vivo
patient infection studies.

IMPORTANCE
Herpes simplex virus (HSV) infects more than 70% of adults worldwide, causing epithelial lesions and recurrent disease that manifests over a lifetime. Prior work has demonstrated that HSV strains vary from country to country and between individuals. However, the amount of variation within one strain has not been well studied. To address this, we developed a new approach for viral genome assembly (VirGA) and analysis. We used this approach to quantify the amount of variation between sister clones of a common parental virus stock and to determine the basis of a unique fusion phenotype displayed by several variants. These data revealed that while sister clones of one HSV stock are more than 98% identical, these variants harbor enough genetic differences to change their observed characteristics. Comparative genomics approaches will allow us to explore the impacts of viral inter- and intrastrain diversity on drug and vaccine efficacy.
Neonatal seizures—part 1: Not everything that jerks, stiffens and shakes is a fit
Authors: Hart Anthony R , Pilling Elizabeth L , Alix James JP
Published: April 2015

The neonatal period is the most frequent time of life to have epileptic seizures. However, neonates can also exhibit unusual movements that are not epileptic seizures. Differentiating between epileptic and non-epileptic movements can be difficult. Many neonatal seizures exhibit few or no clinical features at all. This article is for the benefit of paediatric trainees and reviews the published evidence on which neonatal movements are likely to be epileptic seizures and which are not. We also discuss epileptic seizure classification.
Bilateral cystic encephalomalacia following multiple intrauterine transfusions for anti-Kell isoimmunisation
Authors: Elsayed Hesham , Ng Mansum , Rutherford Mary , Gupta Rajesh
Published: April 2015

Fetal and neonatal haemolytic diseases result from maternal allo-immunisation to fetal antigens. Maternal antibodies cross the placenta causing red cell haemolysis, resulting in fetal anaemia and, in severe cases, hydrops and perinatal death. Intravascular intrauterine blood transfusion (IUT) has markedly reduced perinatal mortality and is now a standard procedure. IUT is considered to be a safe procedure with fetal loss rate reported to be less than 5% and no reported increase in the rate of neurodevelopment impairment. In this report, we are presenting a case of bilateral cystic encephalomalacia following fetal anaemia secondary to anti-Kell iso-immunisation treated with multiple IUTs. Such a significant adverse outcome following IUT for anti-Kell iso-immunisation has not been reported in the literature. This case highlights the need for appropriate parental counselling and routine postnatal head ultrasound in all babies delivered following multiple IUTs.
Ophthalmic manifestations postlightning strike
Authors: Dhillon Permesh Singh , Gupta Mohit
Published: April 2015

Various ophthalmic complications affecting the anterior and posterior segments have been identified due to lightning strike. We report the first case of an indirect lightning-induced full thickness macular hole formation in the UK as evidenced by slit lamp examination and optical coherence tomography (OCT) scan in a 77-year-old woman presenting with sudden visual loss in her right eye and thermal skin injury affecting her scalp. Her best corrected visual acuities were LogMAR 0.46 and 0.12 in the right and left eyes, respectively. There were no other ocular manifestations observed in either eye. She was initially managed conservatively with non-steroidal anti-inflammatory drug eye drops but surgery was later advised due to minimal changes in the visual acuity and macular hole on follow-up. OCT scanning is important in diagnosing macular holes, which usually warrant surgical intervention.
Ruptured hepatic artery aneurysm: an unusual presentation of polyarteritis nodosa
Authors: Wicherts D A , Bruntink M M , Demirkiran A , van Santvoort H C , van Lienden K P , Ambarus C A , Besselink M G H , van Gulik T M
Published: April 2015

A 52-year-old woman presented with severe acute right upper quadrant abdominal pain and signs of intra-abdominal haemorrhage. CT and selective angiography revealed a ruptured right hepatic artery aneurysm and diffuse aneurysmatic disease involving most intra-abdominal organs, suggestive of polyarteritis nodosa. Although treatment with high-dose steroids was initiated, the patient died of progressive bowel ischaemia.
Colovesical fistula presenting asListeria monocytogenesbacteraemia
Authors: Hobbs Mark
Published: April 2015

We present a case of colovesical fistula presenting with a clinical syndrome of urosepsis subsequently demonstrated to be due toListeria monocytogenesbacteraemia. The patient had a history of previous rectal cancer with a low anterior resection and a covering ileostomy that had been reversed 6 months prior to this presentation.L. monocytogeneswas also isolated among mixed enteric organisms on urine culture. There were no symptoms or signs of acute gastrointestinal listeriosis or meningoencephalitis. This unusual scenario prompted concern regarding the possibility of communication between bowel and bladder, which was subsequently confirmed with CT and a contrast enema. The patient recovered well with intravenous amoxicillin and to date has declined surgical management of his colovesical fistula. This case illustrates the importance of considering bowel pathology when enteric organisms such asListeriaare isolated from unusual sites.
Acute upper airway obstruction and emergency front of neck access in an achondroplastic patient
Authors: McCaffer Craig James , Douglas Catriona , Wickham Matthew H , Picozzi Gerard L
Published: April 2015

Dwarfism is defined as a failure to attain a height of 148 cm in adulthood. Achondroplasia is the most common form of short-limbed dwarfism. Although this condition is relatively rare, with an incidence of 0.5–1.5 per 10 000 live births, most medical professionals will come across the achondroplastic dwarf (AD) during their career. Faulty endochondral ossification produces the characteristic short stature phenotype, as well as severe craniofacial, central nervous system, spinal, respiratory and cardiac anomalies. These unusual characteristics may present airway management difficulties in elective as well as emergency situations. Within the literature there is very little information regarding the emergency insertion of a surgical airway in an adult AD. We present our experience of this situation in the form of a case report and a review of the relevant literature.
Which determinants predict tibiofemoral and patellofemoral osteoarthritis after anterior cruciate ligament injury? A systematic review
Authors: van Meer Belle L , Meuffels Duncan E , van Eijsden Wilbert A , Verhaar Jan A N , Bierma-Zeinstra Sita M A , Reijman Max
Published: April 2015

Background

Anterior cruciate ligament (ACL) injury is an important risk factor for development of knee osteoarthritis (OA). To identify those ACL injured patients at increased risk for knee OA, it is necessary to understand risk factors for OA.

Aim

To summarise the evidence for determinants of (1) tibiofemoral OA and (2) patellofemoral OA in ACL injured patients.

Methods

MEDLINE, EMBASE, Web of Science and CINAHL databases were searched up to 20 December 2013. Additionally, reference lists of eligible studies were manually and independently screened by two reviewers. 2348 studies were assessed for the following main inclusion criteria: ≥20 patients; ACL injured patients treated operatively or non-operatively; reporting OA as outcome; description of relationship between OA outcome and determinants; and a follow-up period ≥2 years. Two reviewers extracted the data, assessed the risk of bias and performed a best-evidence synthesis.

Results

Sixty-four publications were included and assessed for quality. Two studies were classified as low risk of bias. Medial meniscal injury/meniscectomy showed moderate evidence for influencing OA development (tibiofemoral OA and compartment unspecified). Lateral meniscal injury/meniscectomy showed moderate evidence for no relationship (compartment unspecified), as did time between injury and reconstruction (tibiofemoral and patellofemoral OA).

Conclusions

Medial meniscal injury/meniscectomy after ACL rupture increased the risk of OA development. In contrast, it seems that lateral meniscal injury/meniscectomy has no relationship with OA development. Our results suggest that time between injury and reconstruction does not influence patellofemoral and tibiofemoral OA development. Many determinants showed conflicting and limited evidence and no determinant showed strong evidence.
Practice guidelines for sedation and analgesia management of critically ill children: a pilot study evaluating guideline impact and feasibility in the PICU
Authors: Keogh Samantha J , Long Debbie A , Horn Desley V
Published: April 2015

Aims

The aim of this study was to develop and implement guidelines for sedation and analgesia management in the paediatric intensive care unit (PICU) and evaluate the impact, feasibility and acceptability of these as part of a programme of research in this area and as a prelude to future trial work.

Method

This pilot study used a pre–post design using a historical control.

Setting

Two PICUs at different hospitals in an Australian metropolitan city.

Participants

Patients admitted to the PICU and ventilated for ≥24 h, aged more than 1 month and not admitted for seizure management or terminal care.

Intervention

Guidelines for sedation and analgesia management for critically ill children including algorithm and assessment tools.

Outcome variables

In addition to key outcome variables (ventilation time, medication dose and duration, length of stay), feasibility outcomes data (recruitment, data collection, safety) were evaluated. Guideline adherence was assessed through chart audit and staff were surveyed about merit and the use of guidelines.

Results

The guidelines were trialled for a total of 12 months on 63 patients and variables compared with the historical control group (n=75). Analysis revealed differences in median Morphine infusion duration between groups (pretest 3.63 days (87 h) vs post-test 2.83 days (68 h), p=0.05) and maximum doses (pretest 120 μg/kg/h vs post-test 97.5 μg/kg/h) with no apparent change to ventilation duration. Chart audit revealed varied use of tools, but staff were positive about the guidelines and their use in practice.

Conclusions

The sedation guidelines impacted on the duration and dosage of agents without any apparent impact on ventilation duration or length of stay. Furthermore, the guidelines appeared to be feasible and acceptable in clinical practice. The results of the study have laid the foundation for follow-up studies in withdrawal from sedation, point prevalence and longitudinal studies of sedation practices as well as drug trial work.
Educational interventions for improving control of blood pressure in patients with hypertension: a systematic review protocol
Authors: Ribeiro Cibele D , Resqueti Vanessa R , Lima Íllia , Dias Fernando A L , Glynn Liam , Fregonezi Guilherme A F
Published: April 2015

Introduction

The aim of this review is to evaluate the effectiveness of educational interventions on improving the control of blood pressure in patients with hypertension.

Methods

Randomised controlled trials including patients over 18 years of age, regardless of sex and ethnicity, with a diagnosis of hypertension (either treated or not treated with antihypertensive medications) will be assessed in our analysis. We will electronically search four databases: MEDLINE, CINAHL, PEDro and ScienceDirect. There will be no language restrictions in the search for studies. The data will be extracted independently by two authors using predefined criteria. Disagreements will be resolved between the authors. The risk of bias will be assessed using the Cochrane risk of bias tool. After searching and screening of the studies, we will run a meta-analysis of the included randomised controlled trials. We will summarise the results as risk ratio for dichotomous data and mean differences for continuous data.

Ethics and dissemination

The review will be published in a journal. The findings from the review will also be disseminated electronically and at conference presentations.

Trial registration number

PROSPERO CRD4201401071.
Levels, trends and reasons for unmet need for family planning among married women in Botswana: a cross-sectional study
Authors: Letamo Gobopamang , Navaneetham Kannan
Published: April 2015

Objectives

The objectives of this study are: (1) to estimate the prevalence of unmet need for family planning among married women using Botswana Family Health Survey 2007 data and (2) to identify risk factors for unmet need for family planning among married women.

Design

This study used secondary data from a cross-sectional survey that was conducted to provide a snapshot of health issues in Botswana.

Setting

Nationally representative population survey data.

Participants

2601 married or in union women aged 15–49 years who participated in the 2007 Botswana Family Health Survey were included in the analysis.

Primary outcome

Unmet need for family planning, which was defined as the percentage of all fecund married women who are not using a method of contraception even though they do not want to get pregnant.

Results

Married women who had unmet need for family planning were 9.6% in 2007. Most of the unmet need was for limiting (6.7%) compared to spacing (2.9%). Unmet need for family planning was more likely to be among women whose partners disapproved of family planning, non-Christians, had one partner and had never discussed family planning with their partner. Women of low parity, aged 25–34 years, and greater exposure to mass media, were less likely to have experienced unmet need. The patterns and magnitude of covariates differed between unmet need for limiting and for spacing.

Conclusions

The prevalence of unmet need for family planning was low in Botswana compared to other sub-Saharan African countries. The findings from this study reemphasise the importance of women's empowerment and men's involvement in women's sexual and reproductive healthcare needs and services. Different approaches are needed to satisfy the demand for family planning for spacing and limiting.
Impaired mental well-being and psychosocial risk: a cross-sectional study in female nursing home direct staff
Authors: Pélissier C , Fontana L , Fort E , Vohito M , Sellier B , Perrier C , Glerant V , Couprie F , Agard J P , Charbotel B
Published: April 2015

Objectives

The present study sought to quantify the impaired mental well-being and psychosocial stress experienced by nursing home staff and to determine the relationship between impaired mental well-being assessed on the 12-item General Health Questionnaire (GHQ-12) and exposure to psychosocial stress assessed on Siegrist's effort/reward and overcommitment model.

Methods

A transverse study was conducted in France on 2471 female employees in 105 nursing homes for the elderly. Personal and occupational data were collected by questionnaire for 668 housekeepers, 1454 nursing assistants and 349 nurses.

Results

36.8% of participants (n=896) showed impaired mental well-being, 42.7% (n=1039) overcommitment and 9% (n=224) effort/reward imbalance. Overcommitment (prevalence ratio (PR)=1.27; 95% CI (1.21 to 1.34)) and effort–reward imbalance (PR=1.19; 95% CI (1.12 to 1.27)) were significantly associated with presence of impaired mental well-being after adjustment for personal factors (age and private life events). Taking effort and reward levels into account, the frequency of impaired mental well-being was highest in case of exposure to great extrinsic effort and low rewards of any type: esteem, PR=3.53, 95% CI (3.06 to 4.08); earnings, PR=3.48, 95% CI (2.99 to 4.06); or job security, PR=3.30, 95% CI (2.88 to 3.78). Participants in situations of overcommitment and of effort/reward imbalance were at the highest risk of impaired mental well-being: PR=3.86, 95% CI (3.42 to 4.35).

Conclusions

Several changes in nursing home organisation can be suggested to reduce staff exposure to factors of psychosocial stress. Qualitative studies of the relation between impaired mental well-being and psychosocial stress in nursing home staff could guide prevention of impaired mental well-being at work.