Reduced Bacterial Colony Count of Anaerobic Bacteria Is Associated with a Worsening in Lung Clearance Index and Inflammation in Cystic Fibrosis

Anaerobic bacteria have been identified in abundance in the airways of cystic fibrosis (CF) subjects. The impact their presence and abundance has on lung function and inflammation is unclear. The aim of this study was to investigate the relationship between the colony count of aerobic and anaerobic bacteria, lung clearance index (LCI), spirometry and C-Reactive Protein (CRP) in patients with CF. Sputum and blood were collected from CF patients at a single cross-sectional visit when clinically stable. Community composition and bacterial colony counts were analysed using extended aerobic and anaerobic culture. Patients completed spirometry and a multiple breath washout (MBW) test to obtain LCI. An inverse correlation between colony count of aerobic bacteria (n = 41, r = -0.35; p = 0.02), anaerobic bacteria (n = 41, r = -0.44, p = 0.004) and LCI was observed. There was an inverse correlation between colony count of anaerobic bacteria and CRP (n = 25, r = -0.44, p = 0.03) only. The results of this study demonstrate that a lower colony count of aerobic and anaerobic bacteria correlated with a worse LCI. A lower colony count of anaerobic bacteria also correlated with higher CRP levels. These results indicate that lower abundance of aerobic and anaerobic bacteria may reflect microbiota disruption and disease progression in the CF lung.

Relative Contribution of P5 and Hap Surface Proteins to Nontypable Haemophilus influenzae Interplay with the Host Upper and Lower Airways

Nontypable Haemophilus influenzae (NTHi) is a major cause of opportunistic respiratory tract disease, and initiates infection by colonizing the nasopharynx. Bacterial surface proteins play determining roles in the NTHi-airways interplay, but their specific and relative contribution to colonization and infection of the respiratory tract has not been addressed comprehensively. In this study, we focused on the ompP5 and hap genes, present in all H. influenzae genome sequenced isolates, and encoding the P5 and Hap surface proteins, respectively. We employed isogenic single and double mutants of the ompP5 and hap genes generated in the pathogenic strain NTHi375 to evaluate P5 and Hap contribution to biofilm growth under continuous flow, to NTHi adhesion, and invasion/phagocytosis on nasal, pharyngeal, bronchial, alveolar cultured epithelial cells and alveolar macrophages, and to NTHi murine pulmonary infection. We show that P5 is not required for bacterial biofilm growth, but it is involved in NTHi interplay with respiratory cells and in mouse lung infection. Mechanistically, P5NTHi375 is not a ligand for CEACAM1 or α5 integrin receptors. Hap involvement in NTHi375-host interaction was shown to be limited, despite promoting bacterial cell adhesion when expressed in H. influenzae RdKW20. We also show that Hap does not contribute to bacterial biofilm growth, and that its absence partially restores the deficiency in lung infection observed for the ΔompP5 mutant. Altogether, this work frames the relative importance of the P5 and Hap surface proteins in NTHi virulence.

HPV16 Down-Regulates the Insulin-Like Growth Factor Binding Protein 2 to Promote Epithelial Invasion in Organotypic Cultures

Cervical cancer is a multi-stage disease caused by human papillomaviruses (HPV) infection of cervical epithelial cells, but the mechanisms regulating disease progression are not clearly defined. Using 3-dimensional organotypic cultures, we demonstrate that HPV16 E6 and E7 proteins alter the secretome of primary human keratinocytes resulting in local epithelial invasion. Mechanistically, absence of the IGF-binding protein 2 (IGFBP2) caused increases in IGFI/II signalling and through crosstalk with KGF/FGFR2b/AKT, cell invasion. Repression of IGFBP2 is mediated by histone deacetylation at the IGFBP2 promoter and was reversed by treatment with histone deacetylase (HDAC) inhibitors. Our in vitro findings were confirmed in 50 invasive cancers and 79 cervical intra-epithelial neoplastic lesions caused by HPV16 infection, where IGFBP2 levels were reduced with increasing disease severity. In summary, the loss of IGFBP2 is associated with progression of premalignant disease, and sensitises cells to pro-invasive IGF signalling, and together with stromal derived factors promotes epithelial invasion.

The paradox of invasion: Reeves’ muntjac deer invade the British Isles from a limited number of founding females

High levels of genetic diversity and high propagule pressure are favoured by conservation biologists as the basis for successful reintroductions and ensuring the persistence of populations. However, invasion ecologists recognize the ‘paradox of invasion’, as successful species introductions may often be characterized by limited numbers of individuals and associated genetic bottlenecks. In the present study, we used a combination of high-resolution nuclear and mitochondrial genetic markers to investigate the invasion history of Reeves' muntjac deer in the British Isles. This invasion has caused severe economic and ecological damage, with secondary spread currently a concern throughout Europe and potentially globally. Microsatellite analysis based on eight loci grouped all 176 introduced individuals studied from across the species' range in the UK into one genetic cluster, and seven mitochondrial D-loop haplotypes were recovered, two of which were present at very low frequency and were related to more common haplotypes. Our results indicate that the entire invasion can be traced to a single founding event involving a low number of females. These findings highlight the fact that even small releases of species may, if ignored, result in irreversible and costly invasion, regardless of initial genetic diversity or continual genetic influx.

A small supramolecular system which emulates the unidirectional, path-selective photoinduced electron transfer (PET) of the bacterial photosynthetic reaction centre (PRC)

The singlet excited state of the 4-aminonaphthalimide fluorophore in 1a and 1b directs electron transfer from intramolecular but external amine groups along only one of two available paths.

Mineralisation of 2,4-dichlorophenol and glucose placed into the same or different hydrological domains as a bacterial inoculant

The spatial location of microorganisms in the soil three-dimensional structure with respect to their substrates plays an important role in the persistence and turnover of natural and xenobiotic organic compounds. To study the effect of spatial location on the mineralisation of ¹⁴C-2,4-dichlorophenol (2,4-DCP, 0.15 or 0.31 μmol g^-1) and ¹⁴C-glucose (2.77 μmol g^-1), columns packed with autoclaved soil aggregates (2-5 mm) were used. Using a chloride tracer of water movement, the existence of 'immobile' water, which was by-passed by preferentially flowing 'mobile' water, was demonstrated. By manipulation of the soil moisture content, the substrates were putatively placed to these conceptual hydrological domains (immobile and mobile water). Leaching studies revealed that approximately 1.7 (glucose) and 3.4 (2.4-DCP) times the amount of substrate placed in mobile water was recovered in the first 4 fractions of leachate when compared to substrate placed in immobile water. The marked difference in the breakthrough curves was taken as evidence of successful substrate placement. The 2,4-DCP degrading bacterium, Burkholderia sp. RASCc2, was inoculated in mobile water (1.8-5.2 × 10⁷ cells g^-1 soil) and parameters (asymptote, time at maximum rate, calculated maximum rate) describing the mineralisation kinetics of 2,4-DCP and glucose previously added to immobile or mobile water domains were compared, For glucose, there was no significant effect (P > 0.1) of substrate placement on any of the mineralisation parameters. However, substrate placement had a significant effect (P < 0.05) on parameters describing 2,4-DCP mineralisation. In particular, 2,4-DCP added in mobile water was mineralised with a greater maximum rate and with a reduced time at maximum rate when compared to 2,4-DCP added to immobile water. The difference in response between the two test substrates may reflect the importance of sorption in controlling the spatial bioavailability of compounds in soil. © 2002 Elsevier Science Ltd. All rights reserved.

Assessment of toxicological interactions of benzene and its primary degradation products (catechol and phenol) using a lux-modified bacterial bioassay

A bacterial bioassay has been developed to assess the relative toxicities of xenobiotics commonly found in contaminated soils, rivers, waters, and ground waters. The assay utilized decline in luminescence of lux- marked Pseudomonas fluorescens on exposure to xenobiotics. Pseudomonas fluorescens is a common bacterium in the terrestrial environment, providing environmental relevance to soil, river, and ground water systems. Three principal environmental contaminants associated with benzene degradation were exposed to the luminescence-marked bacterial biosensor to assess their toxicity individually and in combination. Median effective concentration (EC50) values for decline in luminescence were determined for benzene, catechol, and phenol and were found to be 39.9, 0.77, and 458.6 mg/L, respectively. Catechol, a fungal and bacterial metabolite of benzene, was found to be significantly more toxic to the biosensor than was the parent compound benzene, showing that products of xenobiotic biodegradation may be more toxic than the parent compounds. Combinations of parent compounds and metabolites were found to be significantly more toxic to the bioassay than were the individual compounds themselves. Development of this bioassay has provided a rapid screening system suitable for assessing the toxicity of xenobiotics commonly found in contaminated soil, river, and ground-water environments. The assay can be utilized over a wide pH range and is therefore more applicable to such environmental systems than bioluminescence-based bioassays that utilize marine organisms and can only be applied over a limited pH and salinity range.

72 Potential for airborne bacterial contamination in an outpatient respiratory clinic

Slug-dependent upregulation of L1CAM is responsible for the increased invasion potential of pancreatic cancer cells following long-term 5-FU treatment

BACKGROUND: Pancreatic adenocarcinoma is a lethal disease with 5-year survival of less than 5%. 5-fluorouracil (5-FU) is a principal first-line therapy, but treatment only extends survival modestly and is seldom curative. Drug resistance and disease recurrence is typical and there is a pressing need to overcome this. To investigate acquired 5-FU resistance in pancreatic adenocarcinoma, we established chemoresistant monoclonal cell lines from the Panc 03.27 cell line by long-term exposure to increasing doses of 5-FU.

RESULTS: 5-FU-resistant cell lines exhibited increased expression of markers associated with multidrug resistance explaining their reduced sensitivity to 5-FU. In addition, 5-FU-resistant cell lines showed alterations typical for an epithelial-to-mesenchymal transition (EMT), including upregulation of mesenchymal markers and increased invasiveness. Microarray analysis revealed the L1CAM pathway as one of the most upregulated pathways in the chemoresistant clones, and a significant upregulation of L1CAM was seen on the RNA and protein level. In pancreatic cancer, expression of L1CAM is associated with a chemoresistant and migratory phenotype. Using esiRNA targeting L1CAM, or by blocking the extracellular part of L1CAM with antibodies, we show that the increased invasiveness observed in the chemoresistant cells functionally depends on L1CAM. Using esiRNA targeting β-catenin and/or Slug, we demonstrate that in the chemoresistant cell lines, L1CAM expression depends on Slug rather than β-catenin.

CONCLUSION: Our findings establish Slug-induced L1CAM expression as a mediator of a chemoresistant and migratory phenotype in pancreatic adenocarcinoma cells.

MT1-MMP regulates urothelial cell invasion via transcriptional regulation of Dickkopf-3

Membrane type-1 matrix metalloproteinase (MT1-MMP) is a zinc-binding endopeptidase, which plays a crucial role in tumour growth, invasion and metastasis. We have shown previously that MT1-MMP has higher expression levels in the human urothelial cell carcinoma (UCC) tissue. We show here that siRNA against MT1-MMP blocks invasion in UCC cell lines. Invasion is also blocked by broad-spectrum protease and MMP inhibitors including tissue inhibitor of metalloproteinase-1 and -2. Membrane type-1-MMP can also regulate transcription. We have used expression arrays to identify genes that are differentially transcribed when siRNA is used to suppress MT1-MMP expression. Upon MT1-MMP knockdown, Dickkopf-3 (DKK3) expression was highly upregulated. The stability of DKK3 mRNA was unaffected under these conditions, suggesting transcriptional regulation of DKK3 by MT1-MMP. Dickkopf-3 has been previously shown to inhibit invasion. We confirm that the overexpression of DKK3 leads to decreased invasive potential as well as delayed wound healing. We show for the first time that the effects of MT1-MMP on cell invasion are mediated in part through changes in DKK3 gene transcription.

Characterizing Aquifer Heterogeneity Using Bacterial and Bacteriophage Tracers

Gravel aquifers act as important potable water sources in central western Europe yet they are subject to numerous contamination pressures. Compositional and textural heterogeneity makes protection zone delineation around groundwater supplies in these units challenging; artificial tracer testing aids characterization. This paper reappraises previous tracer test results in light of new geological and microbiological data. Comparative passive gradient testing, using a fluorescent solute (Uranine), virus (H40/1 bacteriophage), and comparably sized bacterial tracers Escherichia coli and Pseudomonas putida, was used to investigate a calcareous gravel aquifer’s ability to remove microbiological contaminants at a test site near Munich, Germany. Test results revealed E. coli relative recoveries could exceed those of H40/1 at monitoring wells 10 m and 20 m from an injection well by almost four times; P. putida recoveries varied by a factor of up to three between wells. Application of filtration theory suggested greater attenuation of H40/1 relative to similarly charged E. coli occurred due to differences in microorganism size, while estimated collision efficiencies appeared comparable. By contrast, more positively charged P. putida experienced greater attenuation at one monitoring point, while lower attenuation rates at the second location indicated the influence of geochemical heterogeneity. Test findings proved consistent with observations from nearby fresh outcrops that suggested thin open framework gravel beds dominated mass transport in the aquifer, while discrete intervals containing stained clasts reflect localized geochemical heterogeneity. Study results highlight the utility of reconciling outcrop observations with artificial tracer test responses, using microbiological tracers with well-defined properties, to characterize aquifer heterogeneity.