bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterizing the Performance of the Conway‐Maxwell Poisson Generalized Linear Model
Authors: Francis Royce A. , Geedipally Srinivas Reddy , Guikema Seth D. , Dhavala Soma Sekhar , Lord Dominique , LaRocca Sarah
Published: July 2011

Count data are pervasive in many areas of risk analysis; deaths, adverse health outcomes, infrastructure system failures, and traffic accidents are all recorded as count events, for example. Risk analysts often wish to estimate the probability distribution for the number of discrete events as part of doing a risk assessment. Traditional count data regression models of the type often used in risk assessment for this problem suffer from limitations due to the assumed variance structure. A more flexible model based on the Conway‐Maxwell Poisson (COM‐Poisson) distribution was recently proposed, a model that has the potential to overcome the limitations of the traditional model. However, the statistical performance of this new model has not yet been fully characterized. This article assesses the performance of a maximum likelihood estimation method for fitting the COM‐Poisson generalized linear model (GLM). The objectives of this article are to (1) characterize the parameter estimation accuracy of the MLE implementation of the COM‐Poisson GLM, and (2) estimate the prediction accuracy of the COM‐Poisson GLM using simulated data sets. The results of the study indicate that the COM‐Poisson GLM is flexible enough to model under‐, equi‐, and overdispersed data sets with different sample mean values. The results also show that the COM‐Poisson GLM yields accurate parameter estimates. The COM‐Poisson GLM provides a promising and flexible approach for performing count data regression.
Current and Future Particulate‐Matter‐Related Mortality Risks in the United States from Aviation Emissions During Landing and Takeoff
Authors: Levy Jonathan I. , Woody Matthew , Baek Bok Haeng , Shankar Uma , Arunachalam Saravanan
Published: July 2011

Demand for air travel is projected to increase in the upcoming years, with a corresponding influence on emissions, air quality, and public health. The trajectory of health impacts would be influenced by not just emissions growth, but also changes in nonaviation ambient concentrations that influence secondary fine particulate matter (PM2.5) formation, population growth and aging, and potential shifts in PM2.5 concentration‐response functions (CRFs). However, studies to date have not systematically evaluated the individual and joint contributions of these factors to health risk trajectories. In this study, we simulated emissions during landing and takeoff from aircraft at 99 airports across the United States for 2005 and for a 2025 flight activity projection scenario. We applied the Community Multiscale Air Quality (CMAQ) model with the Speciated Modeled Attainment Test (SMAT) to determine the contributions of these emissions to ambient concentrations, including scenarios with 2025 aircraft emissions and 2005 nonaviation air quality. We combined CMAQ outputs with PM2.5 mortality CRFs and population projections, and evaluated the influence of changing emissions, nonaviation concentrations, and population factors. Given these scenarios, aviation‐related health impacts would increase by a factor of 6.1 from 2005 to 2025, with a factor of 2.1 attributable to emissions, a factor of 1.3 attributable to population factors, and a factor of 2.3 attributable to changing nonaviation concentrations which enhance secondary PM2.5 formation. Our study emphasizes that the public health burden of aviation emissions would be significantly influenced by the joint effects of flight activity increases, nonaviation concentration changes, and population growth and aging.
Little impact of hatchery supplementation that uses native broodstock on the genetic structure and diversity of steelhead trout revealed by a large‐scale spatio‐temporal microsatellite survey
Authors: Gow Jennifer L. , Tamkee Patrick , Heggenes Jan , Wilson Greg A. , Taylor Eric B.
Published: July 2011

Artificial breeding programs initiated to enhance the size of animal populations are often motivated by the desire to increase harvest opportunities. The introduction of non‐native genotypes, however, can have negative evolutionary impacts. These may be direct, such as introgressive hybridization, or indirect via competition. Less is known about the effects of stocking with native genotypes. We assayed variation at nine microsatellite loci in 902 steelhead trout (Oncorhynchus mykiss) from five rivers in British Columbia, Canada. These samples were collected over 58 years, a time period that spanned the initiation of native steelhead trout broodstock hatchery supplementation in these rivers. We detected no changes in estimates of effective population size, genetic variation or temporal genetic structure within any population, nor of altered genetic structure among them. Genetic interactions with nonmigratoryO. mykiss, the use of substantial numbers of primarily native broodstock with an approximate 1:1 male‐to‐female ratio, and/or poor survival and reproductive success of hatchery fish may have minimized potential genetic changes. Although no genetic changes were detected, ecological effects of hatchery programs still may influence wild population productivity and abundance. Their effects await the design and implementation of a more comprehensive evaluation program.
Induction of Attachment-Independent Biofilm Formation and Repression ofhfqExpression by Low-Fluid-Shear Culture of Staphylococcus aureus
Authors: Castro Sarah L. , Nelman-Gonzalez Mayra , Nickerson Cheryl A. , Ott C. Mark
Published: July 2011

The opportunistic pathogenStaphylococcus aureusencounters a wide variety of fluid shear levels within the human host, and they may play a key role in dictating whether this organism adopts a commensal interaction with the host or transitions to cause disease. By using rotating-wall vessel bioreactors to create a physiologically relevant, low-fluid-shear environment,S. aureuswas evaluated for cellular responses that could impact its colonization and virulence.S. aureuscells grown in a low-fluid-shear environment initiated a novel attachment-independent biofilm phenotype and were completely encased in extracellular polymeric substances. Compared to controls, low-shear-cultured cells displayed slower growth and repressed virulence characteristics, including decreased carotenoid production, increased susceptibility to oxidative stress, and reduced survival in whole blood. Transcriptional whole-genome microarray profiling suggested alterations in metabolic pathways. Further genetic expression analysis revealed downregulation of the RNA chaperone Hfq, which parallels low-fluid-shear responses of certain Gram-negative organisms. This is the first study to report an Hfq association with fluid shear in a Gram-positive organism, suggesting an evolutionarily conserved response to fluid shear among structurally diverse prokaryotes. Collectively, our results suggestS. aureusresponds to a low-fluid-shear environment by initiating a biofilm/colonization phenotype with diminished virulence characteristics, which could lead to insight into key factors influencing the divergence between infection and colonization during the initial host-pathogen interaction.
Predominant Effect of Host Genetics on Levels of Lactobacillus johnsonii Bacteria in the Mouse Gut
Authors: Buhnik-Rosenblau Keren , Danin-Poleg Yael , Kashi Yechezkel
Published: July 2011

The gut microbiota is strongly associated with the well-being of the host. Its composition is affected by environmental factors, such as food and maternal inoculation, while the relative impact of the host's genetics have been recently uncovered. Here, we studied the effect of the host genetic background on the composition of intestinal bacteria in a murine model, focusing on lactic acid bacteria (LAB) as an important group that includes many probiotic strains. Based on 16S rRNA gene genotyping, variation was observed in fecal LAB populations of BALB/c and C57BL/6J mouse lines.
Lactobacillus johnsonii
, a potentially probiotic bacterium, appeared at significantly higher levels in C57BL/6J versus BALB/c mouse feces. In the BALB/c gut, the
L. johnsonii
level decreased rapidly after oral administration, suggesting that some selective force does not allow its persistence at higher levels. The genetic inheritance of
L. johnsonii
levels was further tested in reciprocal crosses between the two mouse lines. The resultant F1 offspring presented similar
L. johnsonii
levels, confirming that mouse genetics plays a major role in determining these levels compared to the smaller maternal effect. Our findings suggest that mouse genetics has a major effect on the composition of the LAB population in general and on the persistence of
L. johnsonii
in the gut in particular. Concentrating on a narrow spectrum of culturable LAB enables the isolation and characterization of such potentially probiotic bacterial strains, which might be specifically oriented to the genetic background of the host as part of a personalized-medicine approach.
Flavobacterium johnsoniae as a Model Organism for Characterizing Biopolymer Utilization in Oligotrophic Freshwater Environments
Authors: Sack Eveline L. W. , van der Wielen Paul W. J. J. , van der Kooij Dick
Published: July 2011

Biopolymers are important substrates for heterotrophic bacteria in oligotrophic freshwater environments, but information on bacterial growth kinetics with biopolymers is scarce. The objective of this study was to characterize bacterial biopolymer utilization in these environments by assessing the growth kinetics of
Flavobacterium johnsoniae
strain A3, which is specialized in utilizing biopolymers at μg liter
−1
levels. Growth of strain A3 with amylopectin, xyloglucan, gelatin, maltose, or fructose at 0 to 200 μg C liter
−1
in tap water followed Monod or Teissier kinetics, whereas growth with laminarin followed Teissier kinetics. Classification of the specific affinity of strain A3 for the tested substrates resulted in the following affinity order: laminarin (7.9 × 10
−2
liter·μg
−1
of C·h
−1
) ≫ maltose > amylopectin ≈ gelatin ≈ xyloglucan > fructose (0.69 × 10
−2
liter·μg
−1
of C·h
−1
). No specific affinity could be determined for proline, but it appeared to be high. Extracellular degradation controlled growth with amylopectin, xyloglucan, or gelatin but not with laminarin, which could explain the higher affinity for laminarin. The main degradation products were oligosaccharides or oligopeptides, because only some individual monosaccharides and amino acids promoted growth. A higher yield and a lower ATP cell
−1
level was achieved at ≤10 μg C liter
−1
than at >10 μg C liter
−1
with every substrate except gelatin. The high specific affinities of strain A3 for different biopolymers confirm that some representatives of the classes
Cytophagia
-
Flavobacteria
are highly adapted to growth with these compounds at μg liter
−1
levels and support the hypothesis that
Cytophagia-Flavobacteria
play an important role in biopolymer degradation in (ultra)oligotrophic freshwater environments.
The Pentose Moiety of Adenosine and Inosine Is an Important Energy Source for the Fermented-Meat Starter Culture Lactobacillus sakei CTC 494
Authors: Rimaux T. , Vrancken G. , Vuylsteke B. , De Vuyst L. , Leroy F.
Published: July 2011

The genome sequence of
Lactobacillus sakei
23K has revealed that the species
L. sakei
harbors several genes involved in the catabolism of energy sources other than glucose in meat, such as glycerol, arginine, and nucleosides. In this study, a screening of 15
L. sakei
strains revealed that arginine, inosine, and adenosine could be used as energy sources by all strains. However, no glycerol catabolism occurred in any of the
L. sakei
strains tested. A detailed kinetic analysis of inosine and adenosine catabolism in the presence of arginine by
L. sakei
CTC 494, a fermented-meat starter culture, was performed. It showed that nucleoside catabolism occurred as a mixed-acid fermentation in a pH range (pH 5.0 to 6.5) relevant for sausage fermentation. This resulted in the production of a mixture of acetic acid, formic acid, and ethanol from ribose, while the nucleobase (hypoxanthine and adenine in the case of fermentations with inosine and adenosine, respectively) was excreted into the medium stoichiometrically. This indicates that adenosine deaminase activity did not take place. The ratios of the different fermentation end products did not vary with environmental pH, except for the fermentation with inosine at pH 5.0, where lactic acid was produced too. In all cases, no other carbon-containing metabolites were found; carbon dioxide was derived only from arginine catabolism. Arginine was cometabolized in all cases and resulted in the production of both citrulline and ornithine. Based on these results, a pathway for inosine and adenosine catabolism in
L. sakei
CTC 494 was presented, whereby both nucleosides are directly converted into their nucleobase and ribose, the latter entering the heterolactate pathway. The present study revealed that the pentose moiety (ribose) of the nucleosides inosine and adenosine is an effective fermentable substrate for
L. sakei
. Thus, the ability to use these energy sources offers a competitive advantage for this species in a meat environment.
Comparison of Real-Time PCR, Reverse Transcriptase Real-Time PCR, Loop-Mediated Isothermal Amplification, and the FDA Conventional Microbiological Method for the Detection of Salmonella spp. in Produce
Authors: Zhang Guodong , Brown Eric W. , González-Escalona Narjol
Published: July 2011

Contamination of foods, especially produce, with
Salmonella
spp. is a major concern for public health. Several methods are available for the detection of
Salmonella
in produce, but their relative efficiency for detecting
Salmonella
in commonly consumed vegetables, often associated with outbreaks of food poisoning, needs to be confirmed. In this study, the effectiveness of three molecular methods for detection of
Salmonella
in six produce matrices was evaluated and compared to the FDA microbiological detection method. Samples of cilantro (coriander leaves), lettuce, parsley, spinach, tomato, and jalapeno pepper were inoculated with
Salmonella
serovars at two different levels (10
5
and <10
1
CFU/25 g of produce). The inoculated produce was assayed by the FDA
Salmonella
culture method (
Bacteriological Analytical Manual
) and by three molecular methods: quantitative real-time PCR (qPCR), quantitative reverse transcriptase real-time PCR (RT-qPCR), and loop-mediated isothermal amplification (LAMP). Comparable results were obtained by these four methods, which all detected as little as 2 CFU of
Salmonella
cells/25 g of produce. All control samples (not inoculated) were negative by the four methods. RT-qPCR detects only live
Salmonella
cells, obviating the danger of false-positive results from nonviable cells. False negatives (inhibition of either qPCR or RT-qPCR) were avoided by the use of either a DNA or an RNA amplification internal control (IAC). Compared to the conventional culture method, the qPCR, RT-qPCR, and LAMP assays allowed faster and equally accurate detection of
Salmonella
spp. in six high-risk produce commodities.
Interesting Starter Culture Strains for Controlled Cocoa Bean Fermentation Revealed by Simulated Cocoa Pulp Fermentations of Cocoa-Specific Lactic Acid Bacteria
Authors: Lefeber Timothy , Janssens Maarten , Moens Frédéric , Gobert William , De Vuyst Luc
Published: July 2011

Among various lactic acid bacterial strains tested, cocoa-specific strains of
Lactobacillus fermentum
were best adapted to the cocoa pulp ecosystem. They fermented glucose to lactic acid and acetic acid, reduced fructose to mannitol, and converted citric acid into lactic acid and 2,3-butanediol.
Plasmids of Raw Milk Cheese Isolate Lactococcus lactis subsp. lactis Biovar diacetylactis DPC3901 Suggest a Plant-Based Origin for the Strain
Authors: Fallico Vincenzo , McAuliffe Olivia , Fitzgerald Gerald F. , Ross R. Paul
Published: July 2011

The four-plasmid complement of the raw milk cheese isolate
Lactococcus lactis
subsp.
lactis
biovar diacetylactis DPC3901 was sequenced, and some genetic features were functionally analyzed. The complete sequences of pVF18 (18,977 bp), pVF21 (21,739 bp), pVF22 (22,166 bp), and pVF50 (53,876 bp) were obtained. Each plasmid contained genes not previously described for
Lactococcus
, in addition to genes associated with plant-derived lactococcal strains. Most of the novel genes were found on pVF18 and encoded functions typical of bacteria associated with plants, such as activities of plant cell wall modification (
orf11
and
orf25
). In addition, a predicted high-affinity regulated system for the uptake of cobalt was identified (
orf19
to
orf21
[
orf19
-
21]
), which has a single database homolog on a plant-derived
Leuconostoc
plasmid and whose functionality was demonstrated following curing of pVF18. pVF21 and pVF22 encode additional metal transporters, which, along with
orf19
-
21
of pVF18, could enhance host ability to uptake growth-limiting amounts of biologically essential ions within the soil. In addition, vast regions from pVF50 and pVF21 share significant homology with the plant-derived lactococcal plasmid pGdh442, which is indicative of extensive horizontal gene transfer and recombination between these plasmids and suggests a common plant niche for their hosts. Phenotypes associated with these regions include glutamate dehydrogenase activity and Na
+
and K
+
transport. The presence of numerous plant-associated markers in
L. lactis
DPC3901 suggests a plant origin for the raw milk cheese isolate and provides for the first time the genetic basis to support the concept of the plant-milk transition for
Lactococcus
strains.
Detection of Viable Cryptosporidium parvum in Soil by Reverse Transcription–Real-Time PCR Targeting
hsp70
mRNA
Authors: Liang Zhanbei , Keeley Ann
Published: July 2011

Extraction of high-quality mRNA from
Cryptosporidium parvum
is a key step in PCR detection of viable oocysts in environmental samples. Current methods for monitoring oocysts are limited to water samples; therefore, the goal of this study was to develop a rapid and sensitive procedure for
Cryptosporidium
detection in soil samples. The efficiencies of five RNA extraction methods were compared (mRNA extraction with the Dynabeads mRNA Direct kit after chemical and physical sample treatments, and total RNA extraction methods using the FastRNA Pro Soil-Direct, PowerSoil Total RNA, E.Z.N.A. soil RNA, and Norgen soil RNA purification kits) for the direct detection of
Cryptosporidium
with oocyst-spiked sandy, loamy, and clay soils by using TaqMan reverse transcription-PCR. The study also evaluated the presence of inhibitors by synthesis and incorporation of an internal positive control (IPC) RNA into reverse transcription amplifications, used different facilitators (bovine serum albumin, yeast RNA, salmon DNA, skim milk powder, casein, polyvinylpyrrolidone, sodium hexametaphosphate, and
Salmonella enterica
serovar Typhi) to mitigate RNA binding on soil components, and applied various treatments (β-mercaptoethanol and bead beating) to inactivate RNase and ensure the complete lysis of oocysts. The results of spiking studies showed that
Salmonella
cells most efficiently relieved binding of RNA. With the inclusion of
Salmonella
during extraction, the most efficient mRNA method was Dynabeads, with a detection limit of 6 × 10
2
oocysts g
−1
of sandy soil. The most efficient total RNA method was PowerSoil, with detection limits of 1.5 × 10
2
, 1.5 × 10
3
, and 1.5 × 10
4
C. parvum
oocysts g
−1
soil for sandy, loamy, and clay samples, respectively.
Novel Pathway for the Degradation of 2-Chloro-4-Nitrobenzoic Acid by Acinetobacter sp. Strain RKJ12
Authors: Prakash Dhan , Kumar Ravi , Jain R. K. , Tiwary B. N.
Published: July 2011

The organismAcinetobactersp. RKJ12 is capable of utilizing 2-chloro-4-nitrobenzoic acid (2C4NBA) as a sole source of carbon, nitrogen, and energy. In the degradation of 2C4NBA by strain RKJ12, various metabolites were isolated and identified by a combination of chromatographic, spectroscopic, and enzymatic activities, revealing a novel assimilation pathway involving both oxidative and reductive catabolic mechanisms. The metabolism of 2C4NBA was initiated by oxidativeorthodehalogenation, leading to the formation of 2-hydroxy-4-nitrobenzoic acid (2H4NBA), which subsequently was metabolized into 2,4-dihydroxybenzoic acid (2,4-DHBA) by a mono-oxygenase with the concomitant release of chloride and nitrite ions. Stoichiometric analysis indicated the consumption of 1 mol O2per conversion of 2C4NBA to 2,4-DHBA, ruling out the possibility of two oxidative reactions. Experiments with labeled H218O and18O2indicated the involvement of mono-oxygenase-catalyzed initial hydrolytic dechlorination and oxidative denitration mechanisms. The further degradation of 2,4-DHBA then proceeds via reductive dehydroxylation involving the formation of salicylic acid. In the lower pathway, the organism transformed salicylic acid into catechol, which was mineralized by theorthoring cleavage catechol-1,2-dioxygenase tocis, cis-muconic acid, ultimately forming tricarboxylic acid cycle intermediates. Furthermore, the studies carried out on a 2C4NBA−derivative and a 2C4NBA+transconjugant demonstrated that the catabolic genes for the 2C4NBA degradation pathway possibly reside on the ∼55-kb transmissible plasmid present in RKJ12.
Time Course of Gene Expression during Porphyromonas gingivalis Strain ATCC 33277 Biofilm Formation
Authors: Yamamoto Reiko , Noiri Yuichiro , Yamaguchi Mikiyo , Asahi Yoko , Maezono Hazuki , Ebisu Shigeyuki
Published: July 2011

Chronological gene expression patterns of biofilm-forming cells are important to understand bioactivity and pathogenicity of biofilms. For
Porphyromonas gingivalis
ATCC 33277 biofilm formation, the number of genes differentially regulated by more than 1.5-fold was highest during the growth stage (312/2,090 genes), and some pathogen-associated genes were time-dependently controlled.
Nucleotide Polymorphism-Based Single-Tube Test for Robust Molecular Identification of All Currently Described Brucella Species
Authors: Wattiau Pierre , Whatmore Adrian M. , Van Hessche Mieke , Godfroid Jacques , Fretin David
Published: July 2011

Among the numerous molecular methods described during the last 20 years to identify
Brucella
, multiplexed amplification methods offer the cheapest and simplest technical solution for molecular identification. However, one disadvantage of such methods is their need to undergo technical revalidation each time a new marker is added to the system. Moreover, polymorphic markers cannot be assessed at the single-nucleotide level in these assays. Since new
Brucella
species are continuously being described, open methodologies able to accommodate new markers while preserving all other system parameters have an obvious advantage. We present a ligase chain reaction (LCR)-based method that simultaneously assesses multiple genetic markers at the single-nucleotide level. Most of the selected markers originate from a multilocus sequence typing (MLST) database that has been extensively validated on hundreds of different
Brucella
strains. When assayed on both reference and field strains, the method yields characteristic capillary electrophoresis profiles for each of the 10
Brucella
species described to date and displays discriminatory potential below the species level for some. Since the LCR methodology is insensitive to interference resulting from the use of multiple oligonucleotides in a single mixture, the way is open for smooth future updates of the proposed system. Such updates are inevitable, given the pending description of new
Brucella
species.
Distinct Responses in Ammonia-Oxidizing Archaea and Bacteria after Addition of Biosolids to an Agricultural Soil
Authors: Kelly John J. , Policht Katherine , Grancharova Tanya , Hundal Lakhwinder S.
Published: July 2011

The recently discovered ammonia-oxidizing archaea (AOA) have been suggested as contributors to the first step of nitrification in terrestrial ecosystems, a role that was previously assigned exclusively to ammonia-oxidizing bacteria (AOB). The current study assessed the effects of agricultural management, specifically amendment of soil with biosolids or synthetic fertilizer, on nitrification rates and copy numbers of archaeal and bacterial ammonia monooxygenase (
amoA
) genes. Anaerobically digested biosolids or synthetic fertilizer was applied annually for three consecutive years to field plots used for corn production. Biosolids were applied at two loading rates, a typical agronomic rate (27 Mg hectare
−1
year
−1
) and double the agronomic rate (54 Mg hectare
−1
year
−1
), while synthetic fertilizer was applied at an agronomic rate typical for the region (291 kg N hectare
−1
year
−1
). Both biosolids amendments and synthetic fertilizer increased soil N and corn yield, but only the biosolids amendments resulted in significant increases in nitrification rates and increases in the copy numbers of archaeal and bacterial
amoA
genes. In addition, only archaeal
amoA
gene copy numbers increased in response to biosolids applied at the typical agronomic rate and showed a significant correlation with nitrification rates. Finally, copy numbers of archaeal
amoA
genes were significantly higher than copy numbers of bacterial
amoA
genes for all treatments. These results implicate AOA as being primarily responsible for the increased nitrification observed in an agricultural soil amended with biosolids. These results also support the hypothesis that physiological differences between AOA and AOB may enable them to occupy distinct ecological niches.
Effects of Animal Diet, Manure Application Rate, and Tillage on Transport of Microorganisms from Manure-Amended Fields
Authors: Durso Lisa M. , Gilley John E. , Marx David B. , Woodbury Bryan L.
Published: July 2011

Manure from cattle fed distillers' grain or corn diets was applied to fields, and the fields were subjected to rainfall simulation tests. Manure was added at three rates on till and no-till plots. Correlations between microbial transport and runoff characteristics were identified. Results indicate that diet affects phage but not bacterial transport from manure-amended fields.
Effect of Dietary Supplementation with a Saccharomyces cerevisiae Mannan Oligosaccharide on the Bacterial Community Structure of Broiler Cecal Contents
Authors: Corrigan A. , Horgan K. , Clipson N. , Murphy R. A.
Published: July 2011

This study investigated the effects of dietary supplementation with a prebiotic mannan oligosaccharide (MOS) on broiler performance, bacterial community structure, and phylogenetic populations of cecal contents. Bird performance data were collected, and cecal samples were extracted from randomly caught poults from each treatment group every 7 days from hatching to the age of 42 days. Weight gain, feed consumption, and feed efficiency ratios did not differ significantly between groups. Automated ribosomal intergenic spacer analysis (ARISA) of the bacterial communities in birds receiving MOS-supplemented diets indicated that dietary supplementation with MOS at either of 2 levels significantly altered the bacterial community structure from that of the control group on all sample days. The phylogenetic identities of bacteria contained within the cecum were determined by constructing a 16S rRNA gene clone library. A total of 594 partial 16S rRNA gene sequences from the cecal contents were analyzed and compared for the three dietary treatments. The dominant bacteria of the cecum belonged to three phyla,
Firmicutes
,
Bacteroidetes
, and
Proteobacteria
; of these,
Firmicutes
were the most dominant in all treatment groups. Statistical analysis of the bacterial 16S rRNA gene clone libraries showed that the compositions of the clone libraries from broilers receiving MOS-supplemented diets were, in most cases, significantly different from that of the control group. It can be concluded that in this trial MOS supplementation significantly altered the cecal bacterial community structure.
Robust Linuron Degradation in On-Farm Biopurification Systems Exposed to Sequential Environmental Changes
Authors: Sniegowski Kristel , Bers Karolien , Ryckeboer Jaak , Jaeken Peter , Spanoghe Pieter , Springael Dirk
Published: July 2011

On-farm biopurification systems (BPS) treat pesticide-contaminated wastewater of farms through biodegradation. Adding pesticide-primed soil has been shown to be beneficial for the establishment of pesticide-degrading populations in BPS. However, no data exist on the response of pesticide-degrading microbiota, either endogenous or introduced with pesticide-primed soil, when BPS are exposed to expected less favorable environmental conditions like cold periods, drought periods, and periods without a pesticide supply. Therefore, the response of microbiota mineralizing the herbicide linuron in BPS microcosm setups inoculated either with a linuron-primed soil or a nonprimed soil to a sequence of such less favorable conditions was examined. A period without linuron supply or a drought period reduced the size of the linuron-mineralizing community in both setups. The most severe effect was recorded for the setup containing nonprimed soil, in which stopping the linuron supply decreased the linuron degradation capacity to nondetectable levels. In both systems, linuron mineralization rapidly reestablished after conventional operation conditions were restored. A cold period and feeding with a pesticide mixture did not affect linuron mineralization. The changes in the linuron-mineralizing capacity in microcosms containing primed soil were associated with the dynamics of a particular
Variovorax
phylotype that previously had been associated with linuron mineralization. This study suggests that the pesticide-mineralizing community in BPS is robust in stress situations imposed by changes in environmental conditions expected to occur on farms. Moreover, it suggests that, in cases where effects do occur, recovery is rapid after restoring conventional operation conditions.
Identification of New Single Nucleotide Polymorphism-Based Markers for Inter- and Intraspecies Discrimination of Obligate Bacterial Parasites (Pasteuria spp.) of Invertebrates
Authors: Mauchline Tim H. , Knox Rachel , Mohan Sharad , Powers Stephen J. , Kerry Brian R. , Davies Keith G. , Hirsch Penny R.
Published: July 2011

Protein-encoding and 16S rRNA genes ofPasteuria penetranspopulations from a wide range of geographic locations were examined. Most interpopulation single nucleotide polymorphisms (SNPs) were detected in the 16S rRNA gene. However, in order to fully resolve all populations, these were supplemented with SNPs from protein-encoding genes in a multilocus SNP typing approach. Examination of individual 16S rRNA gene sequences revealed the occurrence of “cryptic” SNPs which were not present in the consensus sequences of anyP. penetranspopulation. Additionally, hierarchical cluster analysis separatedP. penetrans16S rRNA gene clones into four groups, and one of which contained sequences from the most highly passaged population, demonstrating that it is possible to manipulate the population structure of this fastidious bacterium. The other groups were made from representatives of the other populations in various proportions. Comparison of sequences among threePasteuriaspecies, namely,P. penetrans,P. hartismeri, andP. ramosa, showed that the protein-encoding genes provided greater discrimination than the 16S rRNA gene. From these findings, we have developed a toolbox for the discrimination ofPasteuriaat both the inter- and intraspecies levels. We also provide a model to monitor genetic variation in other obligate hyperparasites and difficult-to-culture microorganisms.
Enzymatic Properties and Substrate Specificity of the Trehalose Phosphorylase from Caldanaerobacter subterraneus
Authors: Van der Borght Jef , Chen Chao , Hoflack Lieve , Van Renterghem Lucas , Desmet Tom , Soetaert Wim
Published: July 2011

A putative glycoside phosphorylase fromCaldanaerobacter subterraneussubsp.pacificuswas recombinantly expressed inEscherichia coli, after codon optimization and chemical synthesis of the encoding gene. The enzyme was purified by His tag chromatography and was found to be specifically active toward trehalose, with an optimal temperature of 80°C. In addition, no loss of activity could be detected after 1 h of incubation at 65°C, which means that it is the most stable trehalose phosphorylase reported so far. The substrate specificity was investigated in detail by measuring the relative activity on a range of alternative acceptors, applied in the reverse synthetic reaction, and determining the kinetic parameters for the best acceptors. These results were rationalized based on the enzyme-substrate interactions observed in a homology model with a docked ligand. The specificity for the orientation of the acceptor's hydroxyl groups was found to decrease in the following order: C-3 > C-2 > C-4. This results in a particularly high activity on the monosaccharidesd-fucose,d-xylose,l-arabinose, andd-galactose, as well as onl-fucose. However, determination of the kinetic parameters revealed that these acceptors bind less tightly in the active site than the natural acceptord-glucose, resulting in drastically increasedKmvalues. Nevertheless, the enzyme's high thermostability and broad acceptor specificity make it a valuable candidate for industrial disaccharide synthesis.
Role of Glucosyltransferase B in Interactions of Candida albicans with Streptococcus mutans and with an Experimental Pellicle on Hydroxyapatite Surfaces
Authors: Gregoire S. , Xiao J. , Silva B. B. , Gonzalez I. , Agidi P. S. , Klein M. I. , Ambatipudi K. S. , Rosalen P. L. , Bauserman R. , Waugh R. E. , Koo H.
Published: July 2011

Candida albicans
and mutans streptococci are frequently detected in dental plaque biofilms from toddlers afflicted with early childhood caries. Glucosyltransferases (Gtfs) secreted by
Streptococcus mutans
bind to saliva-coated apatite (sHA) and to bacterial surfaces, synthesizing exopolymers
in situ
, which promote cell clustering and adherence to tooth enamel. We investigated the potential role Gtfs may play in mediating the interactions between
C. albicans
SC5314 and
S. mutans
UA159, both with each other and with the sHA surface. GtfB adhered effectively to the
C. albicans
yeast cell surface in an enzymatically active form, as determined by scintillation spectroscopy and fluorescence imaging. The glucans formed on the yeast cell surface were more susceptible to dextranase than those synthesized in solution or on sHA and bacterial cell surfaces (
P
< 0.05), indicating an elevated α-1,6-linked glucose content. Fluorescence imaging revealed that larger numbers of
S. mutans
cells bound to
C. albicans
cells with glucans present on their surface than to yeast cells without surface glucans (uncoated). The glucans formed
in situ
also enhanced
C. albicans
interactions with sHA, as determined by a novel single-cell micromechanical method. Furthermore, the presence of glucan-coated yeast cells significantly increased the accumulation of
S. mutans
on the sHA surface (versus
S. mutans
incubated alone or mixed with uncoated
C. albicans
;
P
< 0.05). These data reveal a novel cross-kingdom interaction that is mediated by bacterial GtfB, which readily attaches to the yeast cell surface. Surface-bound GtfB promotes the formation of a glucan-rich matrix
in situ
and may enhance the accumulation of
S. mutans
on the tooth enamel surface, thereby modulating the development of virulent biofilms.
Self-Inducible Bacillus subtilis Expression System for Reliable and Inexpensive Protein Production by High-Cell-Density Fermentation
Authors: Wenzel Marian , Müller Alexander , Siemann-Herzberg Martin , Altenbuchner Josef
Published: July 2011

A novel technically compliant expression system was developed for heterologous protein production in
Bacillus subtilis
with the aim of increasing product yields at the same time as decreasing production costs. Standard systems involve the positively regulated
manP
promoter of the mannose operon, which led to relatively high product yields of 5.3% (5.3 g enhanced green fluorescent protein [eGFP] per 100 g cell dry weight [CDW]) but required large quantities of mannose to induce the reactions, thus rendering the system's technical application rather expensive. To improve this situation, mutant
B. subtilis
strains were used: the Δ
manA
(mannose metabolism) strain TQ281 and the Δ
manP
(mannose uptake) strain TQ356. The total amount of inducer could be reduced with TQ281, which, however, displayed sensitivity to mannose. An inducer-independent self-induction system was developed with TQ356 to further improve the cost efficiency and product yield of the system, in which glucose prevents induction by carbon catabolite repression. To create optimal self-induction conditions, a glucose-limited process strategy, namely, a fed-batch process, was utilized as follows. The initiation of self-induction at the beginning of the glucose-restricted transition phase between the batch and fed-batch phase of fermentation and its maintenance throughout the glucose-limiting fed-batch phase led to a nearly 3-fold increase of product yield, to 14.6%. The novel
B. subtilis
self-induction system thus makes a considerable contribution to improving product yield and reducing the costs associated with its technical application.
Use of Acid Treatment and a Selective Medium To Enhance the Recovery of Francisella tularensis from Water
Authors: Humrighouse B. W. , Adcock N. J. , Rice E. W.
Published: July 2011

Francisella tularensis
has been associated with naturally occurring waterborne outbreaks and is also of interest as a potential biological weapon. Recovery of this pathogen from water using cultural methods is challenging due to the organism's fastidious growth requirements and interference by indigenous bacteria. A 15-min acid treatment procedure prior to culture on a selective agar was evaluated for recovery of
F. tularensis
from seeded water samples. Mean levels of reduction of virulent strains of
F. tularensis
subsp
. holarctica
and subsp.
tularensis
were less than 20% following acid treatment. The attenuated live vaccine strain (LVS) was less resistant to acid exposure. The acid treatment procedure coupled with plating on cystine heart agar with rabbit blood and antibiotics (CHARBab) allowed the isolation of
F. tularensis
seeded into five natural water samples.
Coproduction of Acetaldehyde and Hydrogen during Glucose Fermentation by Escherichia coli
Authors: Zhu Huilin , Gonzalez Ramon , Bobik Thomas A.
Published: July 2011

Escherichia coli
K-12 strain MG1655 was engineered to coproduce acetaldehyde and hydrogen during glucose fermentation by the use of exogenous acetyl-coenzyme A (acetyl-CoA) reductase (for the conversion of acetyl-CoA to acetaldehyde) and the native formate hydrogen lyase. A putative acetaldehyde dehydrogenase/acetyl-CoA reductase from
Salmonella enterica
(SeEutE) was cloned, produced at high levels, and purified by nickel affinity chromatography.
In vitro
assays showed that this enzyme had both acetaldehyde dehydrogenase activity (68.07 ± 1.63 μmol min
−1
mg
−1
) and the desired acetyl-CoA reductase activity (49.23 ± 2.88 μmol min
−1
mg
−1
). The
eutE
gene was engineered into an
E. coli
mutant lacking native glucose fermentation pathways (Δ
adhE
, Δ
ackA
-
pta
, Δ
ldhA
, and Δ
frdC
). The engineered strain (ZH88) produced 4.91 ± 0.29 mM acetaldehyde while consuming 11.05 mM glucose but also produced 6.44 ± 0.26 mM ethanol. Studies showed that ethanol was produced by an unknown alcohol dehydrogenase(s) that converted the acetaldehyde produced by SeEutE to ethanol. Allyl alcohol was used to select for mutants with reduced alcohol dehydrogenase activity. Three allyl alcohol-resistant mutants were isolated; all produced more acetaldehyde and less ethanol than ZH88. It was also found that modifying the growth medium by adding 1 g of yeast extract/liter and lowering the pH to 6.0 further increased the coproduction of acetaldehyde and hydrogen. Under optimal conditions, strain ZH136 converted glucose to acetaldehyde and hydrogen in a 1:1 ratio with a specific acetaldehyde production rate of 0.68 ± 0.20 g h
−1
g
−1
dry cell weight and at 86% of the maximum theoretical yield. This specific production rate is the highest reported thus far and is promising for industrial application. The possibility of a more efficient “no-distill” ethanol fermentation procedure based on the coproduction of acetaldehyde and hydrogen is discussed.
Lachnospiraceae and Bacteroidales Alternative Fecal Indicators Reveal Chronic Human Sewage Contamination in an Urban Harbor
Authors: Newton Ryan J. , VandeWalle Jessica L. , Borchardt Mark A. , Gorelick Marc H. , McLellan Sandra L.
Published: July 2011

The complexity of fecal microbial communities and overlap among human and other animal sources have made it difficult to identify source-specific fecal indicator bacteria. However, the advent of next-generation sequencing technologies now provides increased sequencing power to resolve microbial community composition within and among environments. These data can be mined for information on source-specific phylotypes and/or assemblages of phylotypes (i.e., microbial signatures). We report the development of a new genetic marker for human fecal contamination identified through microbial pyrotag sequence analysis of the V6 region of the 16S rRNA gene. Sequence analysis of 37 sewage samples and comparison with database sequences revealed a human-associated phylotype within the
Lachnospiraceae
family, which was closely related to the genus
Blautia
. This phylotype, termed Lachno2, was on average the second most abundant fecal bacterial phylotype in sewage influent samples from Milwaukee, WI. We developed a quantitative PCR (qPCR) assay for Lachno2 and used it along with the qPCR-based assays for human
Bacteroidales
(based on the HF183 genetic marker), total
Bacteroidales
spp., and enterococci and the conventional
Escherichia coli
and enterococci plate count assays to examine the prevalence of fecal and human fecal pollution in Milwaukee's harbor. Both the conventional fecal indicators and the human-associated indicators revealed chronic fecal pollution in the harbor, with significant increases following heavy rain events and combined sewer overflows. The two human-associated genetic marker abundances were tightly correlated in the harbor, a strong indication they target the same source (i.e., human sewage). Human adenoviruses were routinely detected under all conditions in the harbor, and the probability of their occurrence increased by 154% for every 10-fold increase in the human indicator concentration. Both Lachno2 and human
Bacteroidales
increased specificity to detect sewage compared to general indicators, and the relationship to a human pathogen group suggests that the use of these alternative indicators will improve assessments for human health risks in urban waters.
Biocontrol of Listeria monocytogenes and Escherichia coli O157:H7 in Meat by Using Phages Immobilized on Modified Cellulose Membranes
Authors: Anany H. , Chen W. , Pelton R. , Griffiths M. W.
Published: July 2011

The ability of phages to specifically interact with and lyse their host bacteria makes them ideal antibacterial agents. The range of applications of bacteriophage can be extended by their immobilization on inert surfaces. A novel method for the oriented immobilization of bacteriophage has been developed. The method was based on charge differences between the bacteriophage head, which exhibits an overall net negative charge, and the tail fibers, which possess an overall net positive charge. Hence, the head would be more likely to attach to positively charged surfaces, leaving the tails free to capture and lyse bacteria. Cellulose membranes modified so that they had a positive surface charge were used as the support for phage immobilization. It was established that the number of infective phages immobilized on the positively charged cellulose membranes was significantly higher than that on unmodified membranes. Cocktails of phages active against
Listeria
or
Escherichia coli
immobilized on these membranes were shown to effectively control the growth of
L. monocytogenes
and
E. coli
O157:H7 in ready-to-eat and raw meat, respectively, under different storage temperatures and packaging conditions. The phage storage stability was investigated to further extend their industrial applications. It was shown that lyophilization can be used as a phage-drying method to maintain their infectivity on the newly developed bioactive materials. In conclusion, utilizing the charge difference between phage heads and tails provided a simple technique for oriented immobilization applicable to a wide range of phages and allowed the retention of infectivity.
Sources of Bacteria in Outdoor Air across Cities in the Midwestern United States
Authors: Bowers Robert M. , Sullivan Amy P. , Costello Elizabeth K. , Collett Jeff L. , Knight Rob , Fierer Noah
Published: July 2011

Bacteria are abundant in the atmosphere, where they often represent a major portion of the organic aerosols. Potential pathogens of plants and livestock are commonly dispersed through the atmosphere, and airborne bacteria can have important effects on human health as pathogens or triggers of allergic asthma and seasonal allergies. Despite their importance, the diversity and biogeography of airborne microorganisms remain poorly understood. We used high-throughput pyrosequencing to analyze bacterial communities present in the aerosol fraction containing fine particulate matter of ≤2.5 μm from 96 near-surface atmospheric samples collected from cities throughout the midwestern United States and found that the communities are surprisingly diverse and strongly affected by the season. We also directly compared the airborne communities to those found in hundreds of samples representing potential source environments. We show that, in addition to the more predictable sources (soils and leaf surfaces), fecal material, most likely dog feces, often represents an unexpected source of bacteria in the atmosphere at more urbanized locations during the winter. Airborne bacteria are clearly an important, but understudied, component of air quality that needs to be better integrated into efforts to measure and model pollutants in the atmosphere.
Sequencing and Transcriptional Analysis of the Biosynthesis Gene Cluster of Putrescine-Producing Lactococcus lactis
Authors: Ladero Victor , Rattray Fergal P. , Mayo Baltasar , Martín María Cruz , Fernández María , Alvarez Miguel A.
Published: July 2011

Lactococcus lactis
is a prokaryotic microorganism with great importance as a culture starter and has become the model species among the lactic acid bacteria. The long and safe history of use of
L. lactis
in dairy fermentations has resulted in the classification of this species as GRAS (General Regarded As Safe) or QPS (Qualified Presumption of Safety). However, our group has identified several strains of
L. lactis
subsp.
lactis
and
L. lactis
subsp.
cremoris
that are able to produce putrescine from agmatine via the agmatine deiminase (AGDI) pathway. Putrescine is a biogenic amine that confers undesirable flavor characteristics and may even have toxic effects. The AGDI cluster of
L. lactis
is composed of a putative regulatory gene,
aguR
, followed by the genes (
aguB
,
aguD
,
aguA
, and
aguC
) encoding the catabolic enzymes. These genes are transcribed as an operon that is induced in the presence of agmatine. In some strains, an insertion (IS) element interrupts the transcription of the cluster, which results in a non-putrescine-producing phenotype. Based on this knowledge, a PCR-based test was developed in order to differentiate nonproducing
L. lactis
strains from those with a functional AGDI cluster. The analysis of the AGDI cluster and their flanking regions revealed that the capacity to produce putrescine via the AGDI pathway could be a specific characteristic that was lost during the adaptation to the milk environment by a process of reductive genome evolution.