bluuebunny/crossref_metadata_2025 huggingface dataset:
In Situ Analysis of Nitrifying Biofilms as Determined by In Situ Hybridization and the Use of Microelectrodes
Authors: Okabe Satoshi , Satoh Hisashi , Watanabe Yoshimasa
Published: December 2019

We investigated the in situ spatial organization of ammonia-oxidizing and nitrite-oxidizing bacteria in domestic wastewater biofilms and autotrophic nitrifying biofilms by using microsensors and fluorescent in situ hybridization (FISH) performed with 16S rRNA-targeted oligonucleotide probes. The combination of these techniques made it possible to relate in situ microbial activity directly to the occurrence of nitrifying bacterial populations. In situ hybridization revealed that bacteria belonging to the genus
Nitrosomonas
were the numerically dominant ammonia-oxidizing bacteria in both types of biofilms. Bacteria belonging to the genus
Nitrobacter
were not detected; instead,
Nitrospira
-like bacteria were the main nitrite-oxidizing bacteria in both types of biofilms.
Nitrospira
-like cells formed irregularly shaped aggregates consisting of small microcolonies, which clustered around the clusters of ammonia oxidizers. Whereas most of the ammonia-oxidizing bacteria were present throughout the biofilms, the nitrite-oxidizing bacteria were restricted to the active nitrite-oxidizing zones, which were in the inner parts of the biofilms. Microelectrode measurements showed that the active ammonia-oxidizing zone was located in the outer part of a biofilm, whereas the active nitrite-oxidizing zone was located just below the ammonia-oxidizing zone and overlapped the location of nitrite-oxidizing bacteria, as determined by FISH.
Phylogeny of the Defined Murine Microbiota: Altered Schaedler Flora
Authors: Dewhirst Floyd E. , Chien Chih-Ching , Paster Bruce J. , Ericson Rebecca L. , Orcutt Roger P. , Schauer David B. , Fox James G.
Published: December 2019

The “altered Schaedler flora” (ASF) was developed for colonizing germfree rodents with a standardized microbiota. The purpose of this study was to identify each of the eight ASF strains by 16S rRNA sequence analysis. Three strains were previously identified as
Lactobacillus acidophilus
(strain ASF 360),
Lactobacillus salivarius
(strain ASF 361), and
Bacteroides distasonis
(strain ASF 519) based on phenotypic criteria. 16S rRNA analysis indicated that each of the strains differed from its presumptive identity. The 16S rRNA sequence of strain ASF 361 is essentially identical to the 16S rRNA sequences of the type strains of
Lactobacillus murinis
and
Lactobacillus animalis
(both isolated from mice), and all of these strains probably belong to a single species. Strain ASF 360 is a novel lactobacillus that clusters with
L. acidophilus
and
Lactobacillus lactis
. Strain ASF 519 falls into an unnamed genus containing [
Bacteroides
]
distasonis
, [
Bacteroides
]
merdae
, [
Bacteroides
]
forsythus
, and CDC group DF-3. This unnamed genus is in the
Cytophaga-Flavobacterium-Bacteroides
phylum and is most closely related to the genus
Porphyromonas
. The spiral-shaped strain, strain ASF 457, is in the
Flexistipes
phylum and exhibits sequence identity with rodent isolates of Robertson. The remaining four ASF strains, which are extremely oxygen-sensitive fusiform bacteria, group phylogenetically with the low-G+C-content gram-positive bacteria (
Firmicutes
,
Bacillus-Clostridium
group). ASF 356, ASF 492, and ASF 502 fall into
Clostridium
cluster XIV of Collins et al. Morphologically, ASF 492 resembles members of this cluster,
Roseburia cecicola
, and
Eubacterium plexicaudatum
. The 16S rRNA sequence of ASF 492 is identical to that of
E. plexicaudatum
. Since the type strain and other viable original isolates of
E. plexicaudatum
have been lost, strain ASF 492 is a candidate for a neotype strain. Strain ASF 500 branches deeply in the low-G+C-content gram-positive phylogenetic tree but is not closely related to any organisms whose 16S rRNA sequences are currently in the GenBank database. The 16S rRNA sequence information determined in the present study should allow rapid identification of ASF strains and should permit detailed analysis of the interactions of ASF organisms during development of intestinal disease in mice that are coinfected with a variety of pathogenic microorganisms.
Degradation of 3-Phenoxybenzoic Acid in Soil by
Pseudomonas pseudoalcaligenes
POB310(pPOB) and Two Modified
Pseudomonas
Strains
Authors: Halden Rolf U. , Tepp Sandra M. , Halden Barbara G. , Dwyer Daryl F.
Published: December 2019

Pseudomonas pseudoalcaligenes
POB310(pPOB) and
Pseudomonas
sp. strains B13-D5(pD30.9) and B13-ST1(pPOB) were introduced into soil microcosms containing 3-phenoxybenzoic acid (3-POB) in order to evaluate and compare bacterial survival, degradation of 3-POB, and transfer of plasmids to a recipient bacterium. Strain POB310 was isolated for its ability to use 3-POB as a growth substrate; degradation is initiated by POB-dioxygenase, an enzyme encoded on pPOB. Strain B13-D5 contains pD30.9, a cloning vector harboring the genes encoding POB-dioxygenase; strain B13-ST1 contains pPOB. Degradation of 3-POB in soil by strain POB310 was incomplete, and bacterial densities decreased even under the most favorable conditions (100 ppm of 3-POB, supplementation with P and N, and soil water-holding capacity of 90%). Strains B13-D5 and B13-ST1 degraded 3-POB (10 to 100 ppm) to concentrations of <50 ppb with concomitant increases in density from 10
6
to 10
8
CFU/g (dry weight) of soil. Thus, in contrast to strain POB310, the modified strains had the following two features that are important for in situ bioremediation: survival in soil and growth concurrent with removal of an environmental contaminant. Strains B13-D5 and B13-ST1 also completely degraded 3-POB when the inoculum was only 30 CFU/g (dry weight) of soil. This suggests that in situ bioremediation may be effected, in some cases, with low densities of introduced bacteria. In pure culture, transfer of pPOB from strains POB310 and B13-ST1 to
Pseudomonas
sp. strain B13 occurred at frequencies of 5 × 10
−7
and 10
−1
transconjugant per donor, respectively. Transfer of pPOB from strain B13-ST1 to strain B13 was observed in autoclaved soil but not in nonautoclaved soil; formation of transconjugant bacteria was more rapid in soil containing clay and organic matter than in sandy soil. Transfer of pPOB from strain POB310 to strain B13 in soil was never observed.
Phenotypic and Genotypic Characterization of Non-Starter Lactic Acid Bacteria in Mature Cheddar Cheese
Authors: Fitzsimons N. A. , Cogan T. M. , Condon S. , Beresford T.
Published: December 2019

Non-starter lactic acid bacteria were isolated from 14 premium-quality and 3 sensorially defective mature Irish Cheddar cheeses, obtained from six manufacturers. From countable plates of
Lactobacillus
-selective agar, 20 single isolated colonies were randomly picked per cheese. All 331 viable isolates were biochemically characterized as mesophilic (i.e., group II)
Lactobacillus
spp. Phenotypically, the isolates comprised 96.4%
L. paracasei
, 2.1%
L. plantarum
, 0.3%
L. curvatus
, 0.3%
L. brevis
, and 0.9% unidentified species. Randomly amplified polymorphic DNA (RAPD) analysis was used to rapidly identify the dominant strain groups in nine cheeses from three of the factories, and through clustering by the unweighted pair group method with arithmetic averages, an average of seven strains were found per cheese. In general, strains isolated from cheese produced at the same factory clustered together. The majority of isolates associated with premium-quality cheese grouped together and apart from clusters of strains from defective-quality cheese. No correlation was found between the isomer of lactate produced and RAPD profiles, although isolates which did not ferment ribose clustered together. The phenotypic and genotypic methods employed were validated with a selection of 31 type and reference strains of mesophilic
Lactobacillus
spp. commonly found in Cheddar cheese. RAPD analysis was found to be a useful and rapid method for identifying isolates to the species level. The low homology exhibited between RAPD banding profiles for cheese isolates and collection strains demonstrated the heterogeneity of the
L. paracasei
complex.
Detection of Infectious
Cryptosporidium parvum
Oocysts in Surface and Filter Backwash Water Samples by Immunomagnetic Separation and Integrated Cell Culture-PCR
Authors: Di Giovanni George D. , Hashemi F. Helen , Shaw Nancy J. , Abrams Felicia A. , LeChevallier Mark W. , Abbaszadegan Morteza
Published: December 2019

A new strategy for the detection of infectious
Cryptosporidium parvum
oocysts in water samples, which combines immunomagnetic separation (IMS) for recovery of oocysts with in vitro cell culturing and PCR (CC-PCR), was field tested with a total of 122 raw source water samples and 121 filter backwash water grab samples obtained from 25 sites in the United States. In addition, samples were processed by Percoll-sucrose flotation and oocysts were detected by an immunofluorescence assay (IFA) as a baseline method. Samples of different water quality were seeded with viable
C. parvum
to evaluate oocyst recovery efficiencies and the performance of the CC-PCR protocol. Mean method oocyst recoveries, including concentration of seeded 10-liter samples, from raw water were 26.1% for IMS and 16.6% for flotation, while recoveries from seeded filter backwash water were 9.1 and 5.8%, respectively. There was full agreement between IFA oocyst counts of IMS-purified seeded samples and CC-PCR results. In natural samples, CC-PCR detected infectious
C. parvum
in 4.9% (6) of the raw water samples and 7.4% (9) of the filter backwash water samples, while IFA detected oocysts in 13.1% (16) of the raw water samples and 5.8% (7) of the filter backwash water samples. All CC-PCR products were confirmed by cloning and DNA sequence analysis and were greater than 98% homologous to the
C. parvum
KSU-1
hsp70
gene product. DNA sequence analysis also revealed reproducible nucleotide substitutions among the
hsp70
fragments, suggesting that several different strains of infectious
C. parvum
were detected.
Development of a System for Genetic Manipulation of
Bartonella bacilliformis
Authors: Battisti James M. , Minnick Michael F.
Published: December 2019

Lack of a system for site-specific genetic manipulation has severely hindered studies on the molecular biology of all
Bartonella
species. We report the first site-specific mutagenesis and complementation for a
Bartonella
species. A highly transformable strain of
B. bacilliformis
, termed JB584, was isolated and found to exhibit a significant increase in transformation efficiency with the broad-host-range plasmid pBBR1MCS-2, relative to wild-type strains. Restriction analyses of genomic preparations with the methylation-sensitive restriction enzymes
Cla
I and
Stu
I suggest that strain JB584 possesses a
dcm
methylase mutation that contributes to its enhanced transformability. A suicide plasmid, pUB1, which contains a polylinker, a pMB1 replicon, and a
nptI
kanamycin resistance cassette, was constructed. An internal 508-bp fragment of the
B. bacilliformis
flagellin gene (
fla
) was cloned into pUB1 to generate pUB508, a
fla
-targeting suicide vector. Introduction of pUB508 into JB584 by electroporation generated eight Kan
r
clones of
B. bacilliformis
. Characterization of one of these strains, termed JB585, indicated that allelic exchange between pUB508 and
fla
had occurred. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and electron microscopy showed that synthesis of flagellin encoded by
fla
and secretion/assembly of flagella were abolished. Complementation of
fla
in
trans
was accomplished with a pBBR1MCS recombinant containing the entire wild-type
fla
gene (pBBRFLAG). These data conclusively show that inactivation of
fla
results in a bald, nonmotile phenotype and that pMB1 and REP replicons make suitable
B. bacilliformis
suicide and shuttle vectors, respectively. When used in conjunction with the highly transformable strain JB584, this system for site-specific genetic manipulation and complementation provides a new venue for studying the molecular biology of
B. bacilliformis
.
A Predictive Model That Describes the Effect of Prolonged Heating at 70 to 90°C and Subsequent Incubation at Refrigeration Temperatures on Growth from Spores and Toxigenesis by Nonproteolytic
Clostridium botulinum
in the Presence of Lysozyme
Authors: Fernández Pablo S. , Peck Michael W.
Published: December 2019

Refrigerated processed foods of extended durability such as cook-chill and sous-vide foods rely on a minimal heat treatment at 70 to 95°C and then storage at a refrigeration temperature for safety and preservation. These foods are not sterile and are intended to have an extended shelf life, often up to 42 days. The principal microbiological hazard in foods of this type is growth of and toxin production by nonproteolytic
Clostridium botulinum
. Lysozyme has been shown to increase the measured heat resistance of nonproteolytic
C. botulinum
spores. However, the heat treatment guidelines for prevention of risk of botulism in these products have not taken into consideration the effect of lysozyme, which can be present in many foods. In order to assess the botulism hazard, the effect of heat treatments at 70, 75, 80, 85, and 90°C combined with refrigerated storage for up to 90 days on growth from 10
6
spores of nonproteolytic
C. botulinum
(types B, E, and F) in an anaerobic meat medium containing 2,400 U of lysozyme per ml (50 μg per ml) was studied. Provided that the storage temperature was no higher than 8°C, the following heat treatments each prevented growth and toxin production during 90 days; 70°C for ≥2,545 min, 75°C for ≥463 min, 80°C for ≥230 min, 85°C for ≥84 min, and 90°C for ≥33.5 min. A factorial experimental design allowed development of a predictive model that described the incubation time required before the first sample showed growth, as a function of heating temperature (70 to 90°C), period of heat treatment (up to 2,545 min), and incubation temperature (5 to 25°C). Predictions from the model provided a valid description of the data used to generate the model and agreed with observations made previously.
IS
1631
Occurrence in
Bradyrhizobium japonicum
Highly Reiterated Sequence-Possessing Strains with High Copy Numbers of Repeated Sequences RSα and RSβ
Authors: Isawa Tsuyoshi , Sameshima Reiko , Mitsui Hisayuki , Minamisawa Kiwamu
Published: December 2019

From
Bradyrhizobium japonicum
highly reiterated sequence-possessing (HRS) strains indigenous to Niigata and Tokachi in Japan with high copy numbers of the repeated sequences RSα and RSβ (K. Minamisawa, T. Isawa, Y. Nakatsuka, and N. Ichikawa, Appl. Environ. Microbiol. 64:1845–1851, 1998), several insertion sequence (IS)-like elements were isolated by using the formation of DNA duplexes by denaturation and renaturation of total DNA, followed by treatment with S1 nuclease. Most of these sequences showed structural features of bacterial IS elements, terminal inverted repeats, and homology with known IS elements and transposase genes. HRS and non-HRS strains of
B. japonicum
differed markedly in the profiles obtained after hybridization with all the elements tested. In particular, HRS strains of
B. japonicum
contained many copies of IS
1631
, whereas non-HRS strains completely lacked this element. This association remained true even when many field isolates of
B. japonicum
were examined. Consequently, IS
1631
occurrence was well correlated with
B. japonicum
HRS strains possessing high copy numbers of the repeated sequence RSα or RSβ. DNA sequence analysis indicated that IS
1631
is 2,712 bp long. In addition, IS
1631
belongs to the IS
21
family, as evidenced by its two open reading frames, which encode putative proteins homologous to IstA and IstB of IS
21
, and its terminal inverted repeat sequences with multiple short repeats.
Gene Sequence and Properties of an
s
-Triazine Ring-Cleavage Enzyme from
Pseudomonas
sp. Strain NRRLB-12227
Authors: Karns Jeffrey S.
Published: December 2019

Pesticides based on the
s
-triazine ring structure are widely used in cultivation of food crops. Cleavage of the
s
-triazine ring is an important step in the mineralization of
s
-triazine compounds and hence in their complete removal from the environment. Cyanuric acid amidohydrolase cleaves cyanuric acid (2,4,6-trihydroxy-
s
-triazine), which yields carbon dioxide and biuret; the biuret is subject to further metabolism, which yields CO
2
and ammonia. The
trzD
gene encoding cyanuric acid amidohydrolase was cloned into pMMB277 from
Pseudomonas
sp. strain NRRLB-12227, a strain that is capable of utilizing
s
-triazines as nitrogen sources. Hydrolysis of cyanuric acid was detected in crude extracts of
Escherichia coli
containing the cloned gene by monitoring the disappearance of cyanuric acid and the appearance of biuret by high-performance liquid chromatography (HPLC). DEAE and hydrophobic interaction HPLC were used to purify cyanuric acid amidohydrolase to homogeneity, and a spectrophotometric assay for the purified enzyme was developed. The purified enzyme had an apparent

K
m

of 0.05 mM for cyanuric acid at pH 8.0. The enzyme did not cleave any other
s
-triazine or hydroxypyrimidine compound, although barbituric acid (2,4,6-trihydroxypyrimidine) was found to be a strong competitive inhibitor. Neither the nucleotide sequence of
trzD
nor the amino acid sequence of the gene product exhibited a significant level of similarity to any known gene or protein.
The Viable-but-Nonculturable Condition Is Induced by Copper in
Agrobacterium tumefaciens
and
Rhizobium leguminosarum
Authors: Alexander Emily , Pham Dat , Steck Todd R.
Published: December 2019

Many bacteria respond to changes in environmental conditions by entering the viable-but-nonculturable state. We have determined that copper can induce nutrient-starved
Agrobacterium tumefaciens
and
Rhizobium leguminosarum
cells to become viable but nonculturable. This is the first report of a chemical inducer of this condition.
Role of
N
γ-Acetyldiaminobutyrate as an Enzyme Stabilizer and an Intermediate in the Biosynthesis of Hydroxyectoine
Authors: Cánovas David , Borges Nuno , Vargas Carmen , Ventosa Antonio , Nieto Joaquín J. , Santos Helena
Published: December 2019

Strain CHR63 is a salt-sensitive mutant of the moderately halophilic wild-type strain
Halomonas elongata
DSM 3043 that is affected in the ectoine synthase gene (
ectC
). This strain accumulates large amounts of
N
γ-acetyldiaminobutyrate (NADA), the precursor of ectoine (D. Cánovas, C. Vargas, F. Iglesias-Guerra, L. N. Csonka, D. Rhodes, A. Ventosa, and J. J. Nieto, J. Biol. Chem. 272:25794–25801, 1997). Hydroxyectoine, ectoine, and glucosylglycerate were also identified by nuclear magnetic resonance (NMR) as cytoplasmic organic solutes in this mutant. Accumulation of NADA, hydroxyectoine, and ectoine was osmoregulated, whereas the levels of glucosylglycerate decreased at higher salinities. The effect of the growth stage on the accumulation of solutes was also investigated. NADA was purified from strain CHR63 and was shown to protect the thermolabile enzyme rabbit muscle lactate dehydrogenase against thermal inactivation. The stabilizing effect of NADA was greater than the stabilizing effect of ectoine or potassium diaminobutyrate. A
1
H NMR analysis of the solutes accumulated by the wild-type strain and mutants CHR62 (
ectA
::Tn
1732
) and CHR63 (
ectC
::Tn
1732
) indicated that
H. elongata
can synthesize hydroxyectoine by two different pathways—directly from ectoine or via an alternative pathway that converts NADA into hydroxyectoine without the involvement of ectoine.
Amplified Fragment Length Polymorphism Fingerprinting of
Pseudomonas
Strains from a Poultry Processing Plant
Authors: Geornaras Ifigenia , Kunene Nokuthula F. , von Holy Alexander , Hastings John W.
Published: December 2019

Molecular typing has been used previously to identify and trace dissemination of pathogenic and spoilage bacteria associated with food processing. Amplified fragment length polymorphism (AFLP) is a novel DNA fingerprinting technique which is considered highly reproducible and has high discriminatory power. This technique was used to fingerprint 88
Pseudomonas fluorescens
and
Pseudomonas putida
strains that were previously isolated from plate counts of carcasses at six processing stages and various equipment surfaces and environmental sources of a poultry abattoir. Clustering of the AFLP patterns revealed a high level of diversity among the strains. Six clusters (clusters I through VI) were delineated at an arbitrary Dice coefficient level of 0.65; clusters III (31 strains) and IV (28 strains) were the largest clusters. More than one-half (52.3%) of the strains obtained from carcass samples, which may have represented the resident carcass population, grouped together in cluster III. By contrast, 43.2% of the strains from most of the equipment surfaces and environmental sources grouped together in cluster IV. In most cases, the clusters in which carcass strains from processing stages grouped corresponded to the clusters in which strains from the associated equipment surfaces and/or environmental sources were found. This provided evidence that there was cross-contamination between carcasses and the abattoir environment at the DNA level. The AFLP data also showed that strains were being disseminated from the beginning to the end of the poultry processing operation, since many strains associated with carcasses at the packaging stage were members of the same clusters as strains obtained from carcasses after the defeathering stage.
Development and Use of a Reverse Transcription-PCR Assay To Study Expression of
Tri5
by
Fusarium
Species In Vitro and In Planta
Authors: Doohan F. M. , Weston G. , Rezanoor H. N. , Parry D. W. , Nicholson P.
Published: December 2019

The
Tri5
gene encodes trichodiene synthase, which catalyzes the first reaction in the trichothecene biosynthetic pathway. In vitro, a direct relationship was observed between
Tri5
expression and the increase in deoxynivalenol production over time. We developed a reverse transcription (RT)-PCR assay to quantify
Tri5
gene expression in trichothecene-producing strains of
Fusarium
species. We observed an increase in
Tri5
expression following treatment of
Fusarium culmorum
with fungicides, and we also observed an inverse relationship between
Tri5
expression and biomass, as measured by β-
d
-glucuronidase activity, during colonization of wheat (cv. Avalon) seedlings by
F. culmorum
. RT-PCR analysis also showed that for ears of wheat cv. Avalon inoculated with
F. culmorum
, there were different levels of
Tri5
expression in grain and chaff at later growth stages. We used the
Tri5
-specific primers to develop a PCR assay to detect trichothecene-producing
Fusarium
species in infected plant material.
Cloning of the Gene Encoding a Novel Thermostable α-Galactosidase from
Thermus brockianus
ITI360
Authors: Fridjonsson Olafur , Watzlawick Hildegard , Gehweiler Axel , Rohrhirsch Thilo , Mattes Ralf
Published: December 2019

An α-galactosidase gene from
Thermus brockianus
ITI360 was cloned, sequenced, and expressed in
Escherichia coli
, and the recombinant protein was purified. The gene, designated
agaT
, codes for a 476-residue polypeptide with a calculated molecular mass of 53,810 Da. The native structure of the recombinant enzyme (AgaT) was estimated to be a tetramer. AgaT displays amino acid sequence similarity to the α-galactosidases of
Thermotoga neapolitana
and
Thermotoga maritima
and a low-level sequence similarity to α-galactosidases of family 36 in the classification of glycosyl hydrolases. The enzyme is thermostable, with a temperature optimum of activity at 93°C with
para
-nitrophenyl-α-galactopyranoside as a substrate. Half-lives of inactivation at 92 and 80°C are 100 min and 17 h, respectively. The pH optimum is between 5.5 and 6.5. The enzyme displayed high affinity for oligomeric substrates. The

K
m

s for melibiose and raffinose at 80°C were determined as 4.1 and 11.0 mM, respectively. The α-galactosidase gene in
T. brockianus
ITI360 was inactivated by integrational mutagenesis. Consequently, no α-galactosidase activity was detectable in crude extracts of the mutant strain, and it was unable to use melibiose or raffinose as a single carbohydrate source.
Cold-Adapted Alanine Dehydrogenases from Two Antarctic Bacterial Strains: Gene Cloning, Protein Characterization, and Comparison with Mesophilic and Thermophilic Counterparts
Authors: Galkin Andrey , Kulakova Ljudmila , Ashida Hiroyuki , Sawa Yoshihiro , Esaki Nobuyoshi
Published: December 2019

The genes encoding NAD
+
-dependent alanine dehydrogenases (AlaDHs) (EC
1.4.1.1
) from the Antarctic bacterial organisms
Shewanella
sp. strain Ac10 (SheAlaDH) and
Carnobacterium
sp. strain St2 (CarAlaDH) were cloned and expressed in
Escherichia coli
. Of all of the AlaDHs that have been sequenced, SheAlaDH exhibited the highest level of sequence similarity to the AlaDH from the gram-negative bacterium
Vibrio proteolyticus
(VprAlaDH). CarAlaDH was most similar to AlaDHs from mesophilic and thermophilic
Bacillus
strains. SheAlaDH and CarAlaDH had features typical of cold-adapted enzymes; both the optimal temperature for catalytic activity and the temperature limit for retaining thermostability were lower than the values obtained for the mesophilic counterparts. The
k
cat
/

K
m

value for the SheAlaDH reaction was about three times higher than the
k
cat
/

K
m

value for VprAlaDH, but it was much lower than the
k
cat
/

K
m

value for the AlaDH from
Bacillus subtilis
. Homology-based structural models of various AlaDHs, including the two psychrotrophic AlaDHs, were constructed. The thermal instability of SheAlaDH and CarAlaDH may result from relatively low numbers of salt bridges in these proteins.
Distribution of Bacterial Growth Activity in Flow-Chamber Biofilms
Authors: Sternberg Claus , Christensen Bjarke B. , Johansen Tove , Toftgaard Nielsen Alex , Andersen Jens Bo , Givskov Michael , Molin Søren
Published: December 2019

In microbial communities such as those found in biofilms, individual organisms most often display heterogeneous behavior with respect to their metabolic activity, growth status, gene expression pattern, etc. In that context, a novel reporter system for monitoring of cellular growth activity has been designed. It comprises a transposon cassette carrying fusions between the growth rate-regulated
Escherichia coli rrnB
P1 promoter and different variant
gfp
genes. It is shown that the P1 promoter is regulated in the same way in
E. coli
and
Pseudomonas putida
, making it useful for monitoring of growth activity in organisms outside the group of enteric bacteria. Construction of fusions to genes encoding unstable Gfp proteins opened up the possibility of the monitoring of rates of rRNA synthesis and, in this way, allowing on-line determination of the distribution of growth activity in a complex community. With the use of these reporter tools, it is demonstrated that individual cells of a toluene-degrading
P. putida
strain growing in a benzyl alcohol-supplemented biofilm have different levels of growth activity which develop as the biofilm gets older. Cells that eventually grow very slowly or not at all may be stimulated to restart growth if provided with a more easily metabolizable carbon source. Thus, the dynamics of biofilm growth activity has been tracked to the level of individual cells, cell clusters, and microcolonies.
Isolation of Nitrogen-Fixing Bacteria Containing Molybdenum-Independent Nitrogenases from Natural Environments
Authors: Loveless Telisa M. , Saah J. Royden , Bishop Paul E.
Published: December 2019

Seven diazotrophs that grow well under Mo-deficient, N
2
-fixing conditions were isolated from a variety of environments. These isolates fall in the γ subdivision of the class
Proteobacteria
and have genes that encode the Mo nitrogenase (nitrogenase 1) and the V nitrogenase (nitrogenase 2). Four of the isolates also harbor genes that encode the iron-only nitrogenase (nitrogenase 3).
Bacterial Spores Survive Treatment with Commercial Sterilants and Disinfectants
Authors: Sagripanti Jose-Luis , Bonifacino Aylin
Published: December 2019

This study compared the activity of commercial liquid sterilants and disinfectants on
Bacillus subtilis
spores deposited on three types of devices made of noncorrodible, corrodible, or polymeric material. Products like Renalin, Exspor, Wavicide-01, Cidexplus, and cupric ascorbate were tested under conditions specified for liquid sterilization. These products, at the shorter times indicated for disinfection, and popular disinfectants, like Clorox, Cavicide, and Lysol were also studied. Data obtained with a sensitive and quantitative test suggest that commercial liquid sterilants and disinfectants are less effective on contaminated surfaces than generally acknowledged.
ADAR2 Is Involved in Self and Nonself Recognition of Borna Disease Virus Genomic RNA in the Nucleus
Authors: Yanai Mako , Kojima Shohei , Sakai Madoka , Komorizono Ryo , Tomonaga Keizo , Makino Akiko
Published: December 2019

Cells use the editing activity of adenosine deaminase acting on RNA proteins (ADARs) to prevent autoimmune responses induced by self dsRNA, but viruses can exploit this process to their advantage. Borna disease virus (BoDV), a nuclear-replicating RNA virus, must escape nonself RNA sensing by the host to establish persistent infection in the nucleus. We evaluated whether BoDV utilizes ADARs to prevent innate immune induction. ADAR2 plays a key role throughout the BoDV life cycle. ADAR2 knockdown reduced A-to-I editing of BoDV genomic RNA, leading to the induction of a strong innate immune response. These data suggest that BoDV exploits ADAR2 to edit nonself genomic RNA to appear as self RNA for innate immune evasion and establishment of persistent infection.
Broad and Dynamic Diversification of Infectious Hepatitis C Virus in a Cell Culture Environment
Authors: Gallego Isabel , Soria María Eugenia , García-Crespo Carlos , Chen Qian , Martínez-Barragán Patricia , Khalfaoui Soumaya , Martínez-González Brenda , Sanchez-Martin Irene , Palacios-Blanco Inés , de Ávila Ana Isabel , García-Cehic Damir , Esteban Juan Ignacio , Gómez Jordi , Briones Carlos , Gregori Josep , Quer Josep , Perales Celia , Domingo Esteban
Published: December 2019

The study shows that extensive replication of an RNA virus in a constant biological environment does not limit exploration of sequence space and adaptive options. There was no convergence toward a restricted set of adapted genomes. Mutational waves and mutant spectrum broadening affected infectious genomes. Therefore, profound modifications of mutant spectrum composition and consensus sequence diversification are not exclusively dependent on environmental alterations or the intervention of population bottlenecks.
Does reducing the height of the tackle through law change in elite men’s rugby union (The Championship, England) reduce the incidence of concussion? A controlled study in 126 games
Authors: Stokes Keith A , Locke Duncan , Roberts Simon , Henderson Lewis , Tucker Ross , Ryan Dean , Kemp Simon
Published: December 2019

Objectives

Most concussions in rugby union occur during tackles. We investigated whether legislation to lower maximum tackle height would change tackle behaviour, and reduce concussion incidence rate.

Methods

In an observational evaluation using a prospective cohort design, 12 elite men’s teams played in two competitions during the 2018/2019 season. The Championship (90 games) retained standard Laws of Rugby for the tackle; the Championship Cup (36 games) used revised laws—the maximum tackle height was lowered from the line of the shoulders on the ball carrier to the line of the armpits. Videos of tackles were analysed for ball carrier and tackler behaviour. Injury data were collected using standardised methods.

Results

In the lowered tackle height setting, there was a significantly lower proportion of tackles; (1) in which ball carriers (rate ratio (RR) 0.83, 95% CI 0.79 to 0.86) and tacklers (RR 0.80, 95% CI 0.76 to 0.84) were upright, (2) in which the tackler’s initial contact was to the ball carrier’s head or neck (RR 0.70, 95% CI 0.58 to 0.84) and (3) in which initial contact was above the line of the ball carrier’s armpit (RR 0.84, 95% CI 0.80 to 0.88). Concussion incidence rate did not differ between conditions (RR 1.31, 95% CI 0.85 to 2.01). Unexpectedly, compared with the standard tackle height setting, tacklers in the lowered tackle height setting were themselves concussed at a higher rate as measured by; (1) incidence (RR 1.90, 95% CI 1.05 to 3.45) and (2) concussions per 1000 tackles (2.09, 95% CI 1.15 to 3.80).

Conclusions

Legislating to lower the height of the tackle meant that tacklers made contact with the ball carrier’s head and neck 30% less often. This did not influence concussion incidence rates. Tacklers in the lowered tackle height setting suffered more concussions than did tacklers in the standard tackle height setting.
Working 9 to 5, not the way to make an academic living: observational analysis of manuscript and peer review submissions over time
Authors: Barnett Adrian , Mewburn Inger , Schroter Sara
Published: December 2019

Objective

To determine whether researchers are submitting manuscripts and peer reviews to BMJ journals out of hours and whether this has changed over time.

Design

Observational study of research manuscripts and peer reviews submitted between 2012 and 2019 for which an author’s address could be geocoded.

Setting

Online BMJ submission systems for two large general medical journals.

Main outcome measures

Manuscript and peer review submissions on weekends, on national holidays, and by hour of day (to determine early mornings and late nights). Logistic regression was used to estimate the probability of manuscript and peer review submissions on weekends or holidays.

Results

The analyses included more than 49 000 manuscript submissions and 76 000 peer reviews. Little change over time was seen in the average probability of manuscript or peer review submissions occurring on weekends or holidays. The levels of out of hours work were high, with average probabilities of 0.14 to 0.18 for work on the weekends and 0.08 to 0.13 for work on holidays compared with days in the same week. Clear and consistent differences were seen between countries. Chinese researchers most often worked at weekends and at midnight, whereas researchers in Scandinavian countries were among the most likely to submit during the week and the middle of the day.

Conclusion

The differences between countries that are persistent over time show that a “culture of overwork” is a literal thing, not just a figure of speech.
Patterns of postmeal insulin secretion in individuals with sulfonylurea-treatedKCNJ11neonatal diabetes show predominance of non-KATP-channel pathways
Authors: Bowman Pamela , McDonald Timothy J , Knight Bridget A , Flanagan Sarah E , Leveridge Maria , Spaull Steve R , Shields Beverley M , Hammersley Suzanne , Shepherd Maggie H , Andrews Robert C , Patel Kashyap A , Hattersley Andrew T
Published: December 2019

Objective

Insulin secretion in sulfonylurea-treatedKCNJ11permanent neonatal diabetes mellitus (PNDM) is thought to be mediated predominantly through amplifying non-KATP-channel pathways such as incretins. Affected individuals report symptoms of postprandial hypoglycemia after eating protein/fat-rich foods. We aimed to assess the physiological response to carbohydrate and protein/fat in people with sulfonylurea-treatedKCNJ11PNDM.

Research design and methods

5 adults with sulfonylurea-treatedKCNJ11PNDM and five age, sex and body mass index-matched controls without diabetes had a high-carbohydrate and high-protein/fat meal on two separate mornings. Insulin(i) and glucose(g) were measured at baseline then regularly over 4 hours after the meal. Total area under the curve (tAUC) for insulin and glucose was calculated over 4 hours and compared between meals in controls andKCNJ11cases.

Results

In controls, glucose values after carbohydrate and protein/fat were similar (median glucose tAUC0-4h21.4 vs 19.7 mmol/L, p=0.08). InKCNJ11cases glucose levels were higher after carbohydrate than after protein/fat (median glucose tAUC0-4h58.1 vs 31.3 mmol/L, p=0.04). These different glycemic responses reflected different patterns of insulin secretion: in controls, insulin secretion was greatly increased after carbohydrate versus protein/fat (median insulin tAUC0-4h727 vs 335 pmol/L, p=0.04), but inKCNJ11cases insulin secretion was similar after carbohydrate and protein/fat (median insulin tAUC0-4h327 vs 378 pmol/L, p=0.50).

Conclusions

Individuals with sulfonylurea-treatedKCNJ11PNDM produce similar levels of insulin in response to both carbohydrate and protein/fat meals despite carbohydrate resulting in much higher glucose levels and protein/fat resulting in relatively low glucose levels. This suggests in an inability to modulate insulin secretion in response to glucose levels, consistent with a dependence on non-KATPpathways for insulin secretion.

Trial registration number

NCT02921906.
Clinical significance of obstructive sleep apnea in patients with acute coronary syndrome in relation to diabetes status
Authors: Wang Xiao , Fan Jingyao , Du Yunhui , Ma Changsheng , Ma Xinliang , Nie Shaoping , Wei Yongxiang
Published: December 2019

Objective

The prognostic significance of obstructive sleep apnea (OSA) in patients with acute coronary syndrome (ACS) according to diabetes mellitus (DM) status remains unclear. We aimed to elucidate the association of OSA with subsequent cardiovascular events in patients with ACS with or without DM.

Research design and methods

In this prospective cohort study, consecutive eligible patients with ACS underwent cardiorespiratory polygraphy between June 2015 and May 2017. OSA was defined as an Apnea Hypopnea Index ≥15 events/hour. The primary end point was major adverse cardiovascular and cerebrovascular events (MACCEs), including cardiovascular death, myocardial infarction, stroke, ischemia-driven revascularization, or hospitalization for unstable angina or heart failure.

Results

Among 804 patients, 248 (30.8%) had DM and 403 (50.1%) had OSA. OSA was associated with 2.5 times the risk of 1 year MACCE in patients with DM (22.3% vs 7.1% in the non-OSA group; adjusted HR (HR)=2.49, 95% CI 1.16 to 5.35, p=0.019), but not in patients without DM (8.5% vs 7.7% in the non-OSA group, adjusted HR=0.94, 95% CI 0.51 to 1.75, p=0.85). Patients with DM without OSA had a similar 1 year MACCE rate as patients without DM. The increased risk of events was predominately isolated to patients with OSA with baseline glucose or hemoglobin A1c levels above the median. Combined OSA and longer hypoxia duration (time with arterial oxygen saturation <90%>22 min) further increased the MACCE rate to 31.0% in patients with DM.

Conclusions

OSA was associated with increased risk of 1 year MACCE following ACS in patients with DM, but not in non-DM patients. Further trials exploring the efficacy of OSA treatment in high-risk patients with ACS and DM are warranted.
Cancer diagnosis and care among rural-to-urban migrants in China
Authors: Myerson Rebecca , Lu Tianyi , Yuan Yong , Liu Gordon Guo-En
Published: December 2019

Introduction

Cancer is a leading cause of death in China. Rural-to-urban migrants are a group of over 260 million people in China sometimes termed the ‘floating’ population. This study assessed the prevalence of cancer diagnosis and access to needed healthcare by residence and migration status in China.

Methods

We used data from the China Health and Retirement Longitudinal Survey, a nationally representative population-based random sample of adults age 45 years and older and their spouses in China. We used multivariable logistic regressions to compare outcomes among rural-to-urban migrants, local urban residents and local rural residents after adjusting for province of residence, socioeconomic status and demographic characteristics.

Results

The sample included 7335 urban residents, 9286 rural residents and 3255 rural-to-urban migrants. Prevalence of cancer diagnosis was 9.9 per 1000 population among rural-to-urban migrants (95% CI 6.5 to 15.1 per 1000 population). Rural-to-urban migrants had higher tobacco use (OR=2.01; 95% CI 1.59 to 2.56, p<0.001), lower use of a health check-up (OR=0.57; 95% CI 0.48 to 0.67, p<0.001) and lower prevalence of diagnosed cancer (OR=0.41; 95% CI 0.18 to 0.95, p=0.037) than urban residents. Among participants with diagnosed cancer, residence and migration status were not predictive of foregoing needed healthcare, but were predictive of diagnosis with a screen-detectable tumour (ie, breast, colon, prostate or cervical cancer) (OR=0.17; 95% CI 0.05 to 0.63, p=0.007 for rural residents; OR=0.34; 95% CI 0.09 to 1.22, p=0.098 for rural-to-urban migrants, compared with urban residents).

Conclusion

Rapid and large migration is still a driving force transitioning China. Due to some remaining dual policy settings in favour of local residents, rural migrants tend to use lower primary care and preventive health check-ups in general, and diagnosis of screen-detectable tumours in particular, leading to potentially higher risk of missing early diagnosis of cancers. Closing gaps in diagnosis of screen-detectable tumours could increase treatment and improve cancer outcomes.
Factors associated with antibiotic prescribing in patients with acute respiratory tract complaints in Malta: a 1-year repeated cross-sectional surveillance study
Authors: Saliba-Gustafsson Erika A , Dunberger Hampton Alexandra , Zarb Peter , Orsini Nicola , Borg Michael A , Stålsby Lundborg Cecilia
Published: December 2019

Objective

To identify factors that influence general practitioners’ (GPs’) oral antibiotic prescribing for acute respiratory tract complaints (aRTCs) in Malta.

Design

Repeated, cross-sectional surveillance.

Setting

Maltese general practice; both public health centres and private GP clinics.

Participants

30 GPs registered on the Malta Medical Council’s Specialist Register and 3 GP trainees registered data of 4831 patients of all ages suffering from any aRTC. Data were collected monthly between May 2015 and April 2016 during predetermined 1-week periods.

Outcome measures

The outcome of interest was antibiotic prescription (yes/no), defined as an oral antibiotic prescription issued for an aRTC during an in-person consultation, irrespective of the number of antibiotics given. The association between GP, practice and consultation-level factors, patient sociodemographic factors and patient health status factors, and antibiotic prescription was investigated.

Results

The antibiotic prescription rate was 45.0%. Independent factors positively associated with antibiotic prescribing included female GP sex (OR 2.3, 95% CI 1.22 to 4.26), GP age with GPs ≥60 being the most likely (OR 34.7, 95% CI 14.14 to 84.98), patient age with patients ≥65 being the most likely (OR 2.3, 95% CI 1.71 to 3.18), number of signs and/or symptoms with patients having ≥4 being the most likely (OR 9.6, 95% CI 5.78 to 15.99), fever (OR 2.6, 95% CI 2.08 to 3.26), productive cough (OR 1.3, 95% CI 1.03 to 1.61), otalgia (OR 1.3, 95% CI 1.01 to 1.76), tender cervical nodes (OR 2.2, 95% CI 1.57 to 3.05), regular clients (OR 1.3, 95% CI 1.05 to 1.66), antibiotic requests (OR 4.8, 95% CI 2.52 to 8.99) and smoking (OR 1.4, 95% CI 1.13 to 1.71). Conversely, patients with non-productive cough (OR 0.3, 95% CI 0.26 to 0.41), sore throat (OR 0.6, 95% CI 0.53 to 0.78), rhinorrhoea (OR 0.3, 95% CI 0.23 to 0.36) or dyspnoea (OR 0.6, 95% CI 0.41 to 0.83) were less likely to receive an antibiotic prescription.

Conclusion

Antibiotic prescribing for aRTCs was high and influenced by a number of factors. Potentially inappropriate prescribing in primary care can be addressed through multifaceted interventions addressing modifiable factors associated with prescription.

Trial registration number

NCT03218930
Community-based rehabilitation programme for adolescents with mental health conditions in Israel: a qualitative study protocol
Authors: Tuaf Hila , Orkibi Hod
Published: December 2019

Introduction

In Israel, 12% of adolescents have mental health conditions. Approximately 600 adolescents with mental health conditions are hospitalised each year and about 40% of them return to the hospital and are thus cut-off from their daily lives and peers in the community. In contrast to adults, adolescents with mental health conditions in Israel are not eligible by law for rehabilitation services. Thus, the overarching goal of this qualitative study is to identify best practices for the implementation of community-based psychosocial rehabilitation programme for this population, by examining the first such programme in Israel. Amitim for Youth, which was established in 2018 by the Israel Association of Community Centers in cooperation with the Ministry of Health, the Ministry of Education and the Special Projects Fund of the National Insurance Institute.

Methods and analysis

Qualitative data will be collected through in depth semi-structured interviews and focus groups. To identify themes and patterns in the data, a six-stage reflexive thematic analysis approach will be used. A triangulation procedure will be conducted to strengthen the validity of the findings collected by different methods and from various stakeholders in the programme: the programme’s decision-makers, programme team members, the intended beneficiaries and referring mental health professionals. To insure the trustworthiness of the findings, three strategies will be employed: memo writing, reflexive journaling and member checking.

Ethics and dissemination

This study was approved by the Ethics Committee for Human Research in the Faculty of Social Welfare and Health Sciences at the University of Haifa (#455–18) and by the Chief Scientist in the Ministry of Education (#10566). All participants will sign an informed consent form and will be guaranteed confidentiality and anonymity. Data collection will be conducted in the next 2 years (2019 to 2020). After data analysis, the findings will be disseminated via publications.
Risk of bias and reporting completeness of randomised controlled trials in burn care: protocol for a systematic review
Authors: Young Amber , Reeves Barnaby C , Cheng Hung-Yuan , Wasiak Jason , Muir Duncan , Davies Anna , Blazeby Jane
Published: December 2019

Introduction

Burn care represents a healthcare and economic burden to patients internationally. Choice of the most clinically effective treatment strategies requires evidence which is best obtained through high-quality randomised controlled trials (RCT). The number of published RCTs of burn care is increasing. However, trial quality and reporting standards are unclear. This study will assess the risk of bias and adequacy of reporting in recent burn care RCTs using tools endorsed by the Cochrane Collaboration.

Methods and analysis

A systematic literature review will be undertaken, assessing parallel group RCTs evaluating therapeutic interventions for patients with cutaneous burns. Literature searches will use Ovid Medline, Ovid Embase, Web of Science and the Cochrane Library. Separate searches for each database will include medical subject heading and free text terms including ‘burn’, ‘scald’, ‘thermal injury’ and ‘RCT’. Two reviewers will independently assess each study for inclusion. Risk of bias (RoB) will be assessed with the revised tool (RoB 2) and reporting completeness with the CONsolidated Standards of Reporting Trials (CONSORT) 2010 guidelines. We will report a narrative synthesis of all studies, including domain specific, and overall risk of bias for the primary outcome of each trial. Inter-rater agreement for RoB 2 will be reported using Fleiss’s Kappa. For adherence to the CONSORT guidelines, we will generate a completeness of reporting index for the ﬁve domains.

Ethics and dissemination

No ethics approval is required because published documents will be used. Findings of the study will be disseminated in a peer-reviewed journal and presented at conferences.

PROSPERO registration number

CRD42018111020.