Disentangling the influence of reservoir abundance and pathogen shedding on zoonotic spillover of the Leptospira agent in urban informal settlements.

Rats are major reservoirs for pathogenic Leptospira, the bacteria causing leptospirosis, particularly in urban informal settlements. However, the impact of variation in rat abundance and pathogen shedding rates on spillover transmission to humans remains unclear. This study aimed to investigate how spatial variation in reservoir abundance and pathogen pressure affect Leptospira spillover transmission to humans in a Brazilian urban informal settlement. A longitudinal eco-epidemiological study was conducted from 2013 to 2014 to characterize the spatial distribution of rat abundance and Leptospira shedding rates in rats and determine the association with human infection risk in a cohort of 2,206 community residents. Tracking plates and live-trapping were used to measure rat abundance and quantify rat shedding status and load. In parallel, four sequential biannual serosurveys were used to identify human Leptospira infections. To evaluate the role of shedding on human risk, we built three statistical models for: (1) the relative abundance of rats, (2) the shedding rate by individual rats, and (3) human Leptospira infection, in which "total shedding", obtained by multiplying the predictions from those two models, was used as a risk factor. We found that Leptospira shedding was associated with older and sexually mature rats and varied spatially and temporally-higher at valley bottoms and with seasonal rainfall (December to March). The point estimate for "total shedding" by rat populations was positive, i.e., Leptospira infection risk increased with total shedding, but the association was not significant [odds ratio (OR) = 1.1; 95% confidence interval (CI): 0.9, 1.4]. This positive trend was mainly driven by rat abundance, rather than individual rat shedding (OR = 1.8; 95% CI: 0.6, 5.4 vs. OR = 1.0; 95% CI: 0.7, 1.4]. Infection risk was higher in areas with more vegetative land cover (OR = 2.4; 95% CI: 1.2, 4.8), and when floodwater entered the house (OR = 2.4; 95% CI: 1.6, 3.4). Our findings indicate that environmental and hydrological factors play a more significant role in Leptospira spillover than rat associated factors. Furthermore, we developed a novel approach combining several models to elucidate complex links between animal reservoir abundance, pathogen shedding and environmental factors on zoonotic spillover in humans that can be extended to other environmentally transmitted diseases.

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.

Volatile Fatty Acids Effective as Antibacterial Agents against Three Enteric Bacteria during Mesophilic Anaerobic Incubation.

The antibacterial effects of a selection of volatile fatty acids (acetic, propionic, butyric, valeric, and caproic acids) relevant to anaerobic digestion were investigated at 1, 2 and 4 g/L. The antibacterial effects were characterised by the dynamics of Enterococcus faecalis NCTC 00775, Escherichia coli JCM 1649 and Klebsiella pneumoniae A17. Mesophilic anaerobic incubation to determine the minimum bactericidal concentration (MBC) and median lethal concentration of the VFAs was carried out in Luria Bertani broth at 37 °C for 48 h. Samples collected at times 0, 3, 6, 24 and 48 h were used to monitor bacterial kinetics and pH. VFAs at 4 g/L demonstrated the highest bactericidal effect (p < 0.05), while 1 g/L supported bacterial growth. The VFA cocktail was the most effective, while propionic acid was the least effective. Enterococcus faecalis NCTC 00775 was the most resistant strain with the VFAs MBC of 4 g/L, while Klebsiella pneumoniae A17 was the least resistant with the VFAs MBC of 2 g/L. Allowing a 48 h incubation period led to more log decline in the bacterial numbers compared to earlier times. The VFA cocktail, valeric, and caproic acids at 4 g/L achieved elimination of the three bacteria strains, with over 7 log10 decrease within 48 h.

Effect of the Inoculum-to-Substrate Ratio on Putative Pathogens and Microbial Kinetics during the Batch Anaerobic Digestion of Simulated Food Waste.

The effects of the inoculum (anaerobic digestion effluent) to substrate (simulated food waste) ratio (ISR) 4.00 to 0.25 on putative pathogens and microbial kinetics during batch mesophilic anaerobic digestion were investigated. Red fluorescent protein labelled (RFPAKN132) Escherichia coli JM105 was introduced as a marker species, and together with the indigenous Clostridium sp., Enterococcus sp., Escherichia coli, and total coliforms were used to monitor pathogen death kinetics. Quantitative polymerase chain reaction was also used to estimate the bacterial, fungal, and methanogenic gene copies. All the ISRs eliminated E. coli and other coliforms (4 log10 CFU/mL), but ISR 0.25 achieved this within the shortest time (≤2 days), while ISR 1.00 initially supported pathogen proliferation. Up to 1.5 log10 CFU/mL of Clostridium was reduced by acidogenic conditions (ISR 0.25 and 0.50), while Enterococcus species were resistant to the digestion conditions. Fungal DNA was reduced (≥5 log10 copies/mL) and was undetectable in ISRs 4.00, 2.00, and 0.50 at the end of the incubation period. This study has demonstrated that ISR influenced the pH of the digesters during batch mesophilic anaerobic digestion, and that acidic and alkaline conditions achieved by the lower (0.50 and 0.25) and higher (4.00 and 2.00) ISRs, respectively, were critical to the sanitisation of waste.

Developing home cleaning intervention through community engagement to reduce infections and antimicrobial resistance in Ghanaian homes.

Globally Antimicrobial Resistance (AMR) constitutes a health crisis, particularly in developing countries, where infectious disease are commonly fatal. There is clear evidence for microbial exposure and infection transmission within the home. Personal and environmental hygiene are the best ways of reducing household infections thus decreasing the need for antibiotics and consequently diminishing AMR. Despite this being an obvious step, research efforts to understand the home environment and its impact on AMR, cleaning and possible interventions on household cleaning are limited. We combined design and microbiology methods in an innovative mixed-method approach. A traditional survey design (n = 240), a design ethnography (n = 12), a co-design workshop and a pre-intervention microbiological dust sample analysis was undertaken to provide insights for codesign workshops in which new cleaning practices might be developed to minimise any AMR bacteria present in the household environments located in the Greater Accra Region of Ghana. Microbiological analysis of household dust showed that 36.6% of bacterial isolates detected were found to carry at least one resistance to the panel of antibiotics tested. Four scenarios were generated from an economic segmentation of the survey data. 50 ethnographic insights were 'presented' and descriptions of 12 bacteria species that showed resistance to one or more antibiotics (representing 176 bacterial isolates that showed resistance to one or more antibiotics found in the dust samples) were presented to the participants in a codesign workshop. An intervention, a new regime of cleaning practices agreed through the co-design workshop and practiced for thirty days, was made in (n = 7) households. The high prevalence of multidrug resistance observed in this study indicate the need for antibiotics surveillance program, not only in hospital settings but also in the household environment. There is, thus, an urgent need for targeting of interventions at the household level. Activating knowledge through community engagement in the research helps in increasing public perception and breaking down the scientist-public barrier.

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.

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.

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.

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.

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.

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.

Immobilization of Shewanella oneidensis MR-1 in diffusive gradients in thin films for determining metal bioavailability.

Assessing metal bioavailability in soil is important in modeling the effects of metal toxicity on the surrounding ecosystem. Current methods based on diffusive gradient thin films (DGTs) and Gel-Integrated Microelectrode are limited in their availability and sensitivity. To address this, Shewanella oneidensis, an anaerobic iron reducing bacterium, was incorporated into a thin layer of agarose to replace the polyacrylamide gel that is normally present in DGT to form biologically mobilizing DGT (BMDGT). Viability analysis revealed that 16-35% of the cells remained viable within the BMDGTs depending on the culturing conditions over a 20 h period with/without metals. Deployment of BMDGTs in standardized metal solutions showed significant differences to cell-free BMDGTs when cells grown in Luria Broth (LB) were incorporated into BMDGTs and deployed under anaerobic conditions. Deployment of these BMDGTs in hematite revealed no significant differences between BMDGTs and BMDGTs containing heat killed cells. Whether heat killed cells retain the ability to affect bioavailability is uncertain. This is the first study to investigate how a microorganism that was incorporated into a DGT device such as the metal reducing bacteria, S. oneidensis, may affect the mobility of metals.

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.

Mycobacterium avium Subspecies paratuberculosis: Human Exposure through Environmental and Domestic Aerosols.

Mycobacterium avium subspecies paratuberculosis (Map) causes Johne's disease in animals and is significantly associated with Crohn's disease (CD) in humans. Our previous studies have shown Map to be present in U.K. rivers due to land deposition from chronic livestock infection and runoff driven by rainfall. The epidemiology of CD in Cardiff showed a significant association with the River Taff, in which Map can be detected on a regular basis. We have previously hypothesized that aerosols from the river might influence the epidemiology of CD. In this preliminary study, we detected Map by quantitative PCR in one of five aerosol samples collected above the River Taff. In addition, we examined domestic showers from different regions in the U.K. and detected Map in three out of 30 independent samples. In detecting Map in river aerosols and those from domestic showers, this is the first study to provide evidence that aerosols are an exposure route for Map to humans and may play a role in the epidemiology of CD.

Honeybee nutrition is linked to landscape composition.

Declines in insect pollinators in Europe have been linked to changes in land use. Pollinator nutrition is dependent on floral resources (i.e., nectar and pollen), which are linked to landscape composition. Here, we present a stratified analysis of the nutritional composition of beebread in managed honeybee hives with a view to examining potential sources of variation in its nutritional composition. Specifically, we tested the hypothesis that beebread composition correlates with local land use and therefore available floral resources. The results demonstrated that the starch, lipid, and moisture contents of beebread are all highly conserved across hives, whereas levels of protein and nonreducing sugar increased as the year progressed, reducing sugars, however, decreased during the first half of the year and then increased toward the end. Local land use around hives was quantified using data from the Countryside Survey 2007 Land Cover Map. Bee-bread protein content was negatively correlated with increasing levels of arable and horticultural farmland surrounding hives and positively correlated with the cover of natural grasslands and broadleaf woodlands. Reducing sugar content was also positively correlated with the amount of broad-leaved woodland in a 3 Km² radius from the hives. Previous studies on a range of invertebrates, including honeybees, indicate that dietary protein intake may have a major impact on correlates of fitness, including longevity and immune function. The finding that beebread protein content correlates with land use suggests that landscape composition may impact on insect pollinator well-being and provides a link between landscape and the nutritional ecology of socially foraging insects in a way not previously considered.