Development of a novel human intestinal model to elucidate the effect of anaerobic commensals on Escherichia coli infection.

The gut microbiota plays a crucial role in protecting against enteric infection. However, the underlying mechanisms are largely unknown owing to a lack of suitable experimental models. Although most gut commensals are anaerobic, intestinal epithelial cells require oxygen for survival. In addition, most intestinal cell lines do not produce mucus, which provides a habitat for the microbiota. Here, we have developed a microaerobic, mucus-producing vertical diffusion chamber (VDC) model and determined the influence of Limosilactobacillus reuteri and Ruminococcus gnavus on enteropathogenic Escherichia coli (EPEC) infection. Optimization of the culture medium enabled bacterial growth in the presence of mucus-producing T84/LS174T cells. Whereas L. reuteri diminished EPEC growth and adhesion to T84/LS174T and mucus-deficient T84 epithelia, R. gnavus only demonstrated a protective effect in the presence of LS174T cells. Reduced EPEC adherence was not associated with altered type III secretion pore formation. In addition, co-culture with L. reuteri and R. gnavus dampened EPEC-induced interleukin 8 secretion. The microaerobic mucin-producing VDC system will facilitate investigations into the mechanisms underpinning colonization resistance and aid the development of microbiota-based anti-infection strategies. This article has an associated First Person interview with the first author of the paper.

Determining Shiga Toxin-Producing Escherichia coli Interactions with Human Intestinal Epithelium in a Microaerobic Vertical Diffusion Chamber.

The environment in the human intestine is low in oxygen. This affects virulence gene expression of enteropathogens including Shiga toxin-producing E. coli (STEC) and enables mucosal colonization by oxygen-sensitive commensal bacteria. To simulate the oxygen-restricted milieu at the intestinal epithelium, we have developed a vertical diffusion chamber model (VDC) which allows infection of polarized human intestinal epithelia under microaerobic conditions. In this chapter, we present a detailed protocol for performing STEC infections in the VDC system and subsequent analysis of STEC pathogenesis.

Identification and characterisation of enteroaggregative Escherichia coli subtypes associated with human disease.

Enteroaggregative E. coli (EAEC) are a major cause of diarrhoea worldwide. Due to their heterogeneity and carriage in healthy individuals, identification of diagnostic virulence markers for pathogenic strains has been difficult. In this study, we have determined phenotypic and genotypic differences between EAEC strains of sequence types (STs) epidemiologically associated with asymptomatic carriage (ST31) and diarrhoeal disease (ST40). ST40 strains demonstrated significantly enhanced intestinal adherence, biofilm formation, and pro-inflammatory interleukin-8 secretion compared with ST31 isolates. This was independent of whether strains were derived from diarrhoea patients or healthy controls. Whole genome sequencing revealed differences in putative virulence genes encoding aggregative adherence fimbriae, E. coli common pilus, flagellin and EAEC heat-stable enterotoxin 1. Our results indicate that ST40 strains have a higher intrinsic potential of human pathogenesis due to a specific combination of virulence-related factors which promote host cell colonization and inflammation. These findings may contribute to the development of genotypic and/or phenotypic markers for EAEC strains of high virulence.

Advancing quality in sepsis management: a large-scale programme for improving sepsis recognition and management in the North West region of England.

OBJECTIVE: To evaluate the impact of a collaborative programme for the early recognition and management of patients admitted with sepsis in the northwest of England. SETTING: 14 hospitals in the northwest of England. INTERVENTION: A quality improvement programme (Advancing Quality (AQ) Sepsis) that promoted a sepsis care bundle including time-based recording of early warning scores, documenting systemic inflammatory response syndrome criteria and suspected source of infection, taking of blood cultures, measuring serum lactate levels, administration of intravenous antibiotics, administration of oxygen, fluid resuscitation, measurement of fluid balance and senior review. MAIN OUTCOME MEASURES: Inpatient mortality, 30-day readmission rates and duration of hospital ≥10 days. RESULTS: Data for 7776 patients were included in this study between 1 July 2014 and 29 December 2015. Participation in the AQ Sepsis programme was associated with a reduction in readmissions within 30 days (OR 0.81 (0.69-0.95)) and hospital stays over 10 days (OR 0.69 (0.60-0.78)). However, there was no reduction in mortality. Administration of a second litre of intravenous fluid within 2 hours, oxygen therapy and review by a senior clinician were associated with increased mortality. Starting a fluid balance chart within 4 hours was the only clinical process measure that did not affect mortality. Taking a blood culture sample, administering antibiotic therapy and measuring serum lactate within 3 hours of hospital arrival were all associated with reduced mortality (OR 0.69 (0.59-0.81), OR 0.77 (0.67-0.89) and OR 0.64 (0.54-0.77), respectively) and shorter hospitalisations (OR 0.58 (0.49-0.69), OR0.81 (0.70-0.94) and OR 0.54 (0.45-0.66), respectively). However, none of these measures had an impact on the risk of readmission to hospital within 30 days. CONCLUSIONS: The AQ Sepsis collaborative in northwest of England improved readmission and length of stay for patients admitted with sepsis but did not affect mortality. Further cost-effectiveness evaluation of the programme is needed.

Influence of filter ventilation on the chemical composition of cigarette mainstream smoke.

Total yields of cigarette smoke constituents are greatly influenced by smoking behaviour, the tobacco blend as well as a variety of cigarette design parameters. Thereby, filter ventilation, i.e. diluting the smoke by providing a zone of microscopic holes around the circumference of the filter is one method to reduce the yield of 'tar' and other smoke compounds. However, little is known how these design variations influence the combustion conditions, and therefore, the overall chemical pattern of the smoke. In this paper single photon ionization-time-of-flight mass spectrometry (SPI-TOFMS) is used to characterize and compare cigarettes on a puff-by-puff basis, which differ only in filter ventilation magnitude. The research cigarettes investigated were made from Virginia tobacco and featured filter ventilations of 0% (no ventilation), 35%, and 70%. The cigarettes were smoked under two different puffing regimes, one using the puffing parameters of the conventional International Organization for Standardization (ISO) smoking regime and a more intense smoking condition. Results show that every variation entails a change of the chemical pattern, whereby, in general, cigarettes with 0% filter ventilation as well as the intense smoking regime lead to a more complete combustion compared to the ISO smoking conditions and the high ventilated cigarettes. Changes in the overall patterns can also be observed during the smoking for individual puffs. Some substances dominate the first puff, some species are more pronounced in the middle puffs, whereas others are preferably formed in the last puffs. This demonstrates the high complexity of the occurring processes. Results might help to understand the formation and decomposition reactions taking place when a cigarette is smoked and offer scope for targeted reduction strategies for specific toxicants or groups of toxicants in the smoke.

Simultaneous on-line size and chemical analysis of gas phase and particulate phase of cigarette mainstream smoke.

This paper describes the combined set-up of on-line chemical analysis of gas phase by single-photon ionisation/resonance enhanced multiphoton ionisation-time-of-flight mass spectrometry (SPI/REMPI-TOFMS) and on-line particle size analysis by differential electrical mobility particle spectrometry (DMS 500) for the investigation of fresh cigarette mainstream smoke. SPI is well suited for the investigation of a great variety of organic species, whereas REMPI is highly sensitive for aromatic compounds. Gas phase measurements of filtered and unfiltered smoke are possible with the SPI/REMPI-TOFMS in order to determine the influence of the presence of particles on the chemical composition of the gas phase. Initial results are shown for the characterisation and comparison of three pure Virginia tobacco research cigarettes having filter ventilations of 0%, i.e. no filter ventilation, 35% and 70% ventilation. The three cigarette types are smoked under two different smoking regimes, a standard regime using puff parameters equivalent to the conventional International Standard Organisation regime and a more intense smoking regime. For the gas phase, qualitative puff-by-puff resolved yields of three selected compounds (acetaldehyde, phenol and styrene) are shown and compared. For particulate matter, particle number, count median diameter and total surface area are illustrated on a puff-by-puff basis. Yields of the chemicals analysed, puff number and surface area are in good agreement with the intensity of the smoking regime and the dilution of smoke by filter ventilation. However, gaseous compounds are influenced differently, depending whether an absolute particle filter is present or not, i.e. they can be totally removed (phenol), partially removed (styrene) or not affected (acetaldehyde). For particle analysis, the count median diameter decreases from puff to puff and is strongly dependent on the smoking regime and ventilation rate. Thereby, 0% ventilated cigarettes smoked under the intense regime result in the smallest count median diameters of ca. 180 nm, whereas 70% ventilated cigarettes smoked with a standard regime lead to the largest values of up to 280 nm. As particle diameter increases, particle number decreases as a consequence of increasing time for particle coagulation.