Nutritional composition of honey bee food stores vary with floral composition.

Sufficiently diverse and abundant resources are essential for generalist consumers, and form an important part of a suite of conservation strategies for pollinators. Honey bees are generalist foragers and are dependent on diverse forage to adequately meet their nutritional needs. Through analysis of stored pollen (bee bread) samples obtained from 26 honey bee (Apis mellifera L.) hives across NW-England, we quantified bee bread nutritional content and the plant species that produced these stores from pollen. Protein was the most abundant nutrient by mass (63%), followed by carbohydrates (26%). Protein and lipid content (but not carbohydrate) contributed significantly to ordinations of floral diversity, linking dietary quality with forage composition. DNA sequencing of the ITS2 region of the nuclear ribosomal DNA gene identified pollen from 89 distinct plant genera, with each bee bread sample containing between 6 and 35 pollen types. Dominant genera included dandelion (Taraxacum), which was positively correlated with bee bread protein content, and cherry (Prunus), which was negatively correlated with the amount of protein. In addition, proportions of amino acids (e.g. histidine and valine) varied as a function of floral species composition. These results also quantify the effects of individual plant genera on the nutrition of honey bees. We conclude that pollens of different plants act synergistically to influence host nutrition; the pollen diversity of bee bread is linked to its nutrient content. Diverse environments compensate for the loss of individual forage plants, and diversity loss may, therefore, destabilize consumer communities due to restricted access to alternative resources.

The rulB gene of plasmid pWW0 is a hotspot for the site-specific insertion of integron-like elements found in the chromosomes of environmental Pseudomonas fluorescens group bacteria.

The rulAB operon of Pseudomonas spp. confers fitness traits on the host and has been suggested to be a hotspot for insertion of mobile elements that carry avirulence genes. Here, for the first time, we show that rulB on plasmid pWW0 is a hotspot for the active site-specific integration of related integron-like elements (ILEs) found in six environmental pseudomonads (strains FH1-FH6). Integration into rulB on pWW0 occurred at position 6488 generating a 3 bp direct repeat. ILEs from FH1 and FH5 were 9403 bp in length and contained eight open reading frames (ORFs), while the ILE from FH4 was 16 233 bp in length and contained 16 ORFs. In all three ILEs, the first 5.1 kb (containing ORFs 1-4) were structurally conserved and contained three predicted site-specific recombinases/integrases and a tetR homologue. Downstream of these resided ORFs of the 'variable side' with structural and sequence similarity to those encoding survival traits on the fitness enhancing plasmid pGRT1 (ILE(FH1) and ILE(FH5)) and the NR-II virulence region of genomic island PAGI-5 (ILE(FH4)). Collectively, these ILEs share features with the previously described type III protein secretion system effector ILEs and are considered important to host survival and transfer of fitness enhancing and (a)virulence genes between bacteria.

Polycyclic aromatic hydrocarbon degradation of phytoplankton-associated Arenibacter spp. and description of Arenibacter algicola sp. nov., an aromatic hydrocarbon-degrading bacterium.

Pyrosequencing of the bacterial community associated with a cosmopolitan marine diatom during enrichment with crude oil revealed several Arenibacter phylotypes, of which one (OTU-202) had become significantly enriched by the oil. Since members of the genus Arenibacter have not been previously shown to degrade hydrocarbons, we attempted to isolate a representative strain of this genus in order to directly investigate its hydrocarbon-degrading potential. Based on 16S rRNA sequencing, one isolate (designated strain TG409(T)) exhibited >99% sequence identity to three type strains of this genus. On the basis of phenotypic and genotypic characteristics, strain TG409(T) represents a novel species in the genus Arenibacter, for which the name Arenibacter algicola sp. nov. is proposed. We reveal for the first time that polycyclic aromatic hydrocarbon (PAH) degradation is a shared phenotype among members of this genus, indicating that it could be used as a taxonomic marker for this genus. Kinetic data for PAH mineralization rates showed that naphthalene was preferred to phenanthrene, and its mineralization was significantly enhanced in the presence of glass wool (a surrogate for diatom cell surfaces). During enrichment on hydrocarbons, strain TG409(T) emulsified n-tetradecane and crude oil, and cells were found to be preferentially attached to oil droplets, indicating an ability by the strain to express cell surface amphiphilic substances (biosurfactants or bioemulsifiers) as a possible strategy to increase the bioavailability of hydrocarbons. This work adds to our growing knowledge on the diversity of bacterial genera in the ocean contributing to the degradation of oil contaminants and of hydrocarbon-degrading bacteria found living in association with marine eukaryotic phytoplankton.

Visualisation of Mycobacterium avium subsp. paratuberculosis in cultured cells, infected sheep and human tissue sections using fluorescent in situ hybridization (FISH).

We describe the development, testing and specificity of a modified oligonucleotide probe for the specific detection of Mycobacterium avium subsp. paratuberculosis (MAP) in culture and in infected tissue using fluorescent in situ hybridisation and confocal microscopy. The detection of MAP in both animal and human tissue using our modified probe allows for a more rapid diagnosis of MAP infection compared to the more often applied detection methods of culture and PCR and has the potential for quantification of cellular abundance. This approach would enable earlier treatment intervention and therefore the potential for reduced morbidity.

Mycobacterium avium subspecies paratuberculosis is widely distributed in British soils and waters: implications for animal and human health.

In the first comprehensive geographical survey of distribution in Great Britain, Mycobacterium avium ssp. paratuberculosis (MAP) was detected in 115 of 1092 (10.5%) soil cores, in the range of 5 × 10(2) to 3 × 10(6) MAP cell equivalents (CE) g(-1) wet weight soil with the majority of the positive PCR reactions (n = 75; 65%) occurring around the limit of detection (500-5000 CE g(-1) wet weight soil). The distribution of MAP significantly increased from North to South and was significantly correlated with increasing cattle numbers over the same longitudinal axis. Similarly MAP occurrence significantly increased towards easterly latitudes although none of the parameters measured were associated. Comparisons of land use indicated that MAP was widely distributed in both farming and non-farming areas. Soil core samples taken from the rivers Wyre and Douglas catchments (Lancashire, UK) and river Tywi (South Wales) were negative for MAP. However, river monitoring showed a consistent presence of MAPs throughout those catchments over a 6-month period. We concluded that MAP is widely distributed within and outside the confines of the farming environment; its geographical distribution is wider than originally anticipated and; monitoring rivers describes the MAP status of catchment better than individual soil samples.

Implementation of a quantitative real-time PCR assay for the detection of Mycobacterium immunogenum in metalworking fluids.

The bacterium Mycobacterium immunogenum has been implicated in causing the lung condition hypersensitivity pneumonitis (HP) in factory workers exposed to colonized metalworking fluids (MWFs). M. immunogenum-specific, real-time quantitative PCR detection technique (MiRT-qPCR) was implemented on a large scale to 363 MWFs of varying types, originating from the United States and Europe, that had been in use for between 30 days and 1 year. In MWFs that contained between 10(3) and 10(9) culturable general heterotrophs mL(-1) the technique detected between 5 and 2 × 10(6) mL(-1) M. immunogenum cell equivalents (CE) in 12.2% (23 of 189) of U.S. samples and between 8 and 6 × 10(5) mL(-1) CE in 39.1% (68 of 174) of samples from Europe. In contrast, only three cultured presumptive mycobacterial isolates across all samples were confirmed as M. immunogenum. Implementation of the assay demonstrated its practicality and further emphasized the limitations of using cultivation alone. Interestingly, no M. immunogenum were detected in mineral oil-based Bio-Concept MWFs from the United States, while it was more commonly detected in used MWFs based on formaldehyde-releasing biocides than in MWFs free of formaldehyde-depot biocides.

Diversity and temporal stability of bacterial communities in a model passerine bird, the zebra finch.

The composition and dynamics of the gastrointestinal bacterial communities in birds is determined by both host-specific and environmental exposure factors yet these are poorly understood. We selected the zebra finch, Taeniopygia guttata, as the host species to examine the diversity and temporal stability of the faecal microflora in a bird, owing to its importance as a model organism in avian ecology, neuroscience and evolution studies. The stability of the gut bacterial community of individual male and female zebra finches was assessed through repeat faecal sampling via culture and temperature gradient gel electrophoresis and partial sequencing of PCR-amplified eubacterial 16S rRNA gene products. Nineteen bacterial genera were detected across all samples (n = 99), with each bird carrying on average six operational taxonomic units. Using a novel statistical approach, we showed that bacterial assemblages and community richness varied between individual birds but remained stable over time within individuals. Neither the composition nor richness of bacterial communities differed significantly between the sexes. Our results show that zebra finches housed together under controlled conditions show consistent variation between individuals in their gut microflora that is not attributable to differences in host exposure to environmental microbial sources. Future studies could usefully explore the origin of this individual-specific variation and its consequences for host fitness and sexual selection.

Presence of Mycobacterium avium Subspecies paratuberculosis Monitored Over Varying Temporal and Spatial Scales in River Catchments: Persistent Routes for Human Exposure.

Mycobacterium avium subspecies paratuberculosis (Map) was monitored by quantitative PCR over a range of temporal and spatial scales in the River Tywi catchment. This study shows the persistence of Map over a 10-year period with little change, which correlates with the recognised levels of Johne's disease in British herds over that period (aim 1). Map was quantified within the river at up to 108 cell equivalents L-1 and was shown to be consistently present when monitored over finer timescales (aim 4). Small wastewater treatment plants where the ingress of human-associated Map might be expected had no significant effect (aim 2). Map was found for the first time to be located in natural river foams providing another route for spread via aerosols (aim 5). This study provides evidence for the environmental continuum of Map from the grazing infected animal via rain driven runoff through field drains and streams into main rivers; with detection at a high frequency throughout the year. Should Map need to be monitored in the future, we recommend that weekly or monthly sampling from a fixed location on a river will capture an adequate representation of the flow dynamics of Map in a catchment (aim 3). The human exposure to Map during this process and its impact on human health remains unquantified.

Detection of Mycobacterium immunogenum by real-time quantitative Taqman PCR.

A quantitative real-time 5'-nuclease (Taqman) PCR technique was developed to specifically detect Mycobacterium immunogenum. rpoB-specific primers and Taqman probe were evaluated for detection of M. immunogenum DNA extracted from pure cultures and from industrial metal working fluids (MWFs). Specificity was confirmed and the sensitivity of detection of M. immunogenum genomic DNA was shown to be approximately 9 fg (2 cell equivalents). When tested on industrial metal working fluids from the UK and USA from which no M. immunogenum CFU were recovered, the assay detected between 3.4x10(1) and 1.9x10(4) cell equivalents (CE) per ml, and increased the detection rate over culture to 37.5% (12 of 32 samples). This assay provides a specific, sensitive and rapid method for the detection of M. immunogenum and is applicable within industry for the early detection of this human pathogen and to the possible prevention of hypersensitivity pneumonitis (HP) in workers.

Bacterial communities associated with honeybee food stores are correlated with land use.

Microbial communities, associated with almost all metazoans, can be inherited from the environment. Although the honeybee (Apis mellifera L.) gut microbiome is well documented, studies of the gut focus on just a small component of the bee microbiome. Other key areas such as the comb, propolis, honey, and stored pollen (bee bread) are poorly understood. Furthermore, little is known about the relationship between the pollinator microbiome and its environment. Here we present a study of the bee bread microbiome and its relationship with land use. We estimated bacterial community composition using both Illumina MiSeq DNA sequencing and denaturing gradient gel electrophoresis (DGGE). Illumina was used to gain a deeper understanding of precise species diversity across samples. DGGE was used on a larger number of samples where the costs of MiSeq had become prohibitive and therefore allowed us to study a greater number of bee breads across broader geographical axes. The former demonstrates bee bread comprises, on average, 13 distinct bacterial phyla; Bacteroidetes, Firmicutes, Alpha-proteobacteria, Beta-proteobacteria, and Gamma-proteobacteria were the five most abundant. The most common genera were Pseudomonas, Arsenophonus, Lactobacillus, Erwinia, and Acinetobacter. DGGE data show bacterial community composition and diversity varied spatially and temporally both within and between hives. Land use data were obtained from the 2007 Countryside Survey. Certain habitats, such as improved grasslands, are associated with low diversity bee breads, meaning that these environments may be poor sources of bee-associated bacteria. Decreased bee bread bacterial diversity may result in reduced function within hives. Although the dispersal of microbes is ubiquitous, this study has demonstrated landscape-level effects on microbial community composition.

Sequence Variation in Multidrug-Resistant Plasmid pLUH01, Isolated from Human Nasopharyngeal Swabs.

Three variants of the multidrug-resistant plasmid pLUH01 were assembled by deep sequencing from nasopharyngeal swabs. All have a 21-bp deletion in the RS14515 hypothetical gene. Variants 1 through 3 have 2, 6, and 3 nucleotide substitutions, respectively, compared to the pLUH01 reference genome. We named the new plasmid variants pLUH01/Lancaster/2015/1 to pLUH01/Lancaster/2015/3.

Genome Sequence of Human Papillomavirus 23 Strain HPV-23/Lancaster/2015.

The genome of human papillomavirus type 23 (HPV-23; family Papillomaviridae, genus Betapapillomavirus, species Betapapillomavirus 2, type 23) was assembled by deep sequencing from nasopharyngeal swabs. The assembled genome is 2.7% divergent over its full length from the single complete genome of HPV-23 in GenBank (accession no. U31781). We named the strain HPV-23/Lancaster/2015.

Influenza C in Lancaster, UK, in the winter of 2014-2015.

Influenza C is not included in the annual seasonal influenza vaccine, and has historically been regarded as a minor respiratory pathogen. However, recent work has highlighted its potential role as a cause of pneumonia in infants. We performed nasopharyngeal or nasal swabbing and/or serum sampling (n = 148) in Lancaster, UK, over the winter of 2014-2015. Using enzyme-linked immunosorbent assay (ELISA), we obtain seropositivity of 77%. By contrast, only 2 individuals, both asymptomatic adults, were influenza C-positive by polymerase chain reaction (PCR). Deep sequencing of nasopharyngeal samples produced partial sequences for 4 genome segments in one of these patients. Bayesian phylogenetic analysis demonstrated that the influenza C genome from this individual is evolutionarily distant to those sampled in recent years and represents a novel genome constellation, indicating that it may be a product of a decades-old reassortment event. Although we find no evidence that influenza C was a significant respiratory pathogen during the winter of 2014-2015 in Lancaster, we confirm previous observations of seropositivity in the majority of the population. (170 words).

Genome Sequence of Human Rhinovirus A22, Strain Lancaster/2015.

The genome of human rhinovirus A22 (HRV-A22) was assembled by deep sequencing RNA samples from nasopharyngeal swabs. The assembled genome is 8.7% divergent from the HRV-A22 reference strain over its full length, and it is only the second full-length genome sequence for HRV-A22. The new strain is designated strain HRV-A22/Lancaster/2015.

Nasopharyngeal metagenomic deep sequencing data, Lancaster, UK, 2014-2015.

Nasopharyngeal swabs were taken from volunteers attending a general medical practice and a general hospital in Lancaster, UK, and at Lancaster University, in the winter of 2014-2015. 51 swabs were selected based on high RNA yield and allocated to deep sequencing pools as follows: patients with chronic obstructive pulmonary disease; asthmatics; adults with no respiratory symptoms; adults with feverish respiratory symptoms; adults with respiratory symptoms and presence of antibodies against influenza C; paediatric patients with respiratory symptoms (2 pools); adults with influenza C infection (2 pools), giving a total of 9 pools. Illumina sequencing was performed, with data yields per pool in the range of 345.6 megabases to 14 gigabases after removal of reads aligning to the human genome. The data were deposited in the Sequence Read Archive at NCBI, and constitute a resource for study of the viral, bacterial and fungal metagenome of the human nasopharynx in healthy and diseased states and comparison with other metagenomic studies on the human respiratory tract.

Genome Sequence of Human Papillomavirus Type 20, Strain HPV-20/Lancaster/2015.

The genome sequence of human papillomavirus type 20 (HPV-20; family Papillomaviridae, genus Betapapillomavirus, species Betapapillomavirus 1, type 20) was assembled by deep sequencing from nasopharyngeal swabs. The assembled genome is 0.37% divergent over its full length from the single complete genome of HPV-20 in GenBank (U31778). We named the strain HPV-20/Lancaster/2015.

Monitoring bacterial pathogens in the environment: advantages of a multilayered approach.

The application of advanced and highly sensitive molecular techniques to the detection of specific bacteria in the freshwater environment is limited, in the first instance, by sampling strategy and sample quality. Further combinations of molecular methods and techniques from apparently unrelated disciplines will ultimately shape the monitoring techniques of the future.

Development of a Real-Time qPCR Assay for Quantification of Covert Baculovirus Infections in a Major African Crop Pest.

Many pathogens and parasites are present in host individuals and populations without any obvious signs of disease. This is particularly true for baculoviruses infecting lepidopteran hosts, where studies have shown that covert persistent viral infections are almost ubiquitous in many species. To date, the infection intensity of covert viruses has rarely been quantified. In this study, we investigated the dynamics of a covert baculovirus infection within the lepidopteran crop pest Spodoptera exempta. A real-time quantitative polymerase chain reaction (qPCR) procedure using a 5' nuclease hydrolysis (TaqMan) probe was developed for specific detection and quantification of Spodoptera exempta nucleopolyhedrovirus (SpexNPV). The qPCR assay indicated that covert baculovirus dynamics varied considerably over the course of the host life-cycle, with infection load peaking in early larval instars and being lowest in adults and final-instar larvae. Adult dissections indicated that, contrary to expectation, viral load aggregation was highest in the head, wings and legs, and lowest in the thorax and abdomen. The data presented here have broad implications relating to our understanding of transmission patterns of baculoviruses and the role of covert infections in host-pathogen dynamics.

Life history correlates of fecal bacterial species richness in a wild population of the blue tit Cyanistes caeruleus.

Very little is known about the normal gastrointestinal flora of wild birds, or how it might affect or reflect the host's life-history traits. The aim of this study was to survey the species richness of bacteria in the feces of a wild population of blue tits Cyanistes caeruleus and to explore the relationships between bacterial species richness and various life-history traits, such as age, sex, and reproductive success. Using PCR-TGGE, 55 operational taxonomic units (OTUs) were identified in blue tit feces. DNA sequencing revealed that the 16S rRNA gene was amplified from a diverse range of bacteria, including those that shared closest homology with Bacillus licheniformis, Campylobacter lari, Pseudomonas spp., and Salmonella spp. For adults, there was a significant negative relationship between bacterial species richness and the likelihood of being detected alive the following breeding season; bacterial richness was consistent across years but declined through the breeding season; and breeding pairs had significantly more similar bacterial richness than expected by chance alone. Reduced adult survival was correlated with the presence of an OTU most closely resembling C. lari; enhanced adult survival was associated with an OTU most similar to Arthrobacter spp. For nestlings, there was no significant change in bacterial species richness between the first and second week after hatching, and nestlings sharing the same nest had significantly more similar bacterial richness. Collectively, these results provide compelling evidence that bacterial species richness was associated with several aspects of the life history of their hosts.