Oxygen restriction increases the infective potential of Listeria monocytogenes in vitro in Caco-2 cells and in vivo in guinea pigs

<p>Abstract</p> <p>Background</p> <p>Listeria monocytogenes has been implicated in several food borne outbreaks as well as sporadic cases of disease. Increased understanding of the biology of this organism is important in the prevention of food borne listeriosis.</p> <p>The infectivity of <it>Listeria monocytogenes </it>ScottA, cultivated with and without oxygen restriction, was compared in <it>vitro </it>and <it>in vivo</it>. Fluorescent protein labels were applied to allow certain identification of <it>Listeria </it>cells from untagged bacteria in <it>in vivo </it>samples, and to distinguish between cells grown under different conditions in mixed infection experiments.</p> <p>Results</p> <p>Infection of Caco-2 cells revealed that <it>Listeria </it>cultivated under oxygen-restricted conditions were approximately 100 fold more invasive than similar cultures grown without oxygen restriction. This was observed for exponentially growing bacteria, as well as for stationary-phase cultures.</p> <p>Oral dosage of guinea pigs with <it>Listeria </it>resulted in a significantly higher prevalence (p < 0.05) of these bacteria in jejunum, liver and spleen four and seven days after challenge, when the bacterial cultures had been grown under oxygen-restricted conditions prior to dosage. Additionally, a 10–100 fold higher concentration of <it>Listeria </it>in fecal samples was observed after dosage with oxygen-restricted bacteria. These differences were seen after challenge with single <it>Listeria </it>cultures, as well as with a mixture of two cultures grown with and without oxygen restriction.</p> <p>Conclusion</p> <p>Our results show for the first time that the environmental conditions to which <it>L. monocytogenes </it>is exposed prior to ingestion are decisive for its <it>in vivo </it>infective potential in the gastrointestinal tract after passage of the gastric barrier. This is highly relevant for safety assessment of this organism in food.</p

Construction of a multiple fluorescence labelling system for use in co-invasion studies of Listeria monocytogenes

BACKGROUND: Existing virulence models are often difficult to apply for quantitative comparison of invasion potentials of Listeria monocytogenes. Well-to-well variation between cell-line based in vitro assays is practically unavoidable, and variation between individual animals is the cause of large deviations in the observed capacity for infection when animal models are used. One way to circumvent this problem is to carry out virulence studies as competition assays between 2 or more strains. This, however, requires invasion-neutral markers that enable easy discrimination between the different strains. RESULTS: A fluorescent marker system, allowing visualization and identification of single L. monocytogenes cells as well as colonies in a non-destructive manner, was developed. Five different fluorescent labels are available, and allowed simultaneous visual discrimination between three differently labelled strains at the single cell level by use of fluorescence microscopy. More than 90% of the L. monocytogenes host cells maintained the fluorescence tags for 40 generations. The fluorescence tags did not alter the invasive capacity of the L. monocytogenes cells in a traditional Caco-2 cell invasion assay, and visual discrimination between invaded bacteria carrying different fluorescent labels inside the cells was possible. CONCLUSION: The constructed fluorescent marker system is stable, easy to use, does not affect the virulence of L. monocytogenes in Caco-2 cell assays, and allows discrimination between differently labelled bacteria after internalization in these cells

EFSA Panel on Biological Hazards (BIOHAZ); Scientific Opinion on VTEC-seropathotype and scientific criteria regarding pathogenicity assessment

During 2007-2010, 13 545 confirmed human VTEC infections and 777 haemolytic uraemic syndrome (HUS) cases were reported in the EU; isolates from 85 % of cases were not fully serotyped and therefore could not be classified using the Karmali seropathotype concept. Seropathotype group D covered 5 % of isolates from fully serotyped cases; 14 cases (0.7 %) belonged to seropathotype group E, defined by Karmali et al. (2003) as non-human only. Isolates from around 27 % of cases could not be assigned. There were no HUS cases reported for the serotypes in groups D and E but 17 HUS cases could not be assigned. The health outcome was reported for only a fraction of confirmed cases. About 64 % of patients presented with only diarrhoea; VTEC infection resulted in HUS in around 10 % of cases. The new ISO/TS 13136:2012 standard improves the detection of VTEC in food. An alternative concept based on the detection of verocytotoxins alone or genes encoding such verocytotoxins does not provide a sound scientific basis on which to assess risk to the consumer because there is no single or combination of marker(s) that fully define a ‘pathogenic’ VTEC. Strains positive for verocytotoxin 2 gene(vtx2)- and eae (intimin production)- or [aaiC (secreted protein of EAEC) plus aggR (plasmid-encoded regulator)] genes are associated with higher risk of more severe illness than other virulence gene combinations. The 2011 O104:H4 outbreak demonstrated the difficulty of predicting the emergence of ‘new’ pathogenic VTEC types by screening only for the eae gene or by focusing on a restricted panel of serogroups. A molecular approach utilising genes encoding virulence characteristics additional to the presence of vtx genes has been proposed

Blood neutrophil to lymphocyte ratio is associated with 90-day mortality and 60-day readmission in Gram negative bacteremia: a multi-center cohort study

Abstract Introduction The Neutrophil-Lymphocyte Ratio (NLR) in blood has demonstrated its capability to predict bacteremia in emergency departments, and its association with mortality has been established in patients with sepsis in intensive care units. However, its potential concerning mortality and readmission in patients with Gram-negative bacteremia (GNB) is unexplored. Methods This retrospective cohort study included patients with GNB between 2018 and 2022 from six hospitals in the Capital Region of Denmark. Patients who were immunosuppressed or had missing NLR values on the day of blood culture were excluded. Logistic regression models were used to analyze the association between NLR levels and 90-day all-cause mortality, while the logit link interpretation of the cumulative incidence function was used to assess the association between NLR levels and 60-day readmission. Associations were quantified as odds ratios (OR) with corresponding 95% confidence intervals (CI). Results The study included 1763 patients with a median age was 76.8 years and 51.3% were female. The median NLR was 17.3 and 15.8% of patients had a quick sequential organ failure assessment score of two or three. Urinary tract infection (UTI) was the most frequent focus and Escherichia coli the most frequent pathogen. Statistically significant differences in median NLR were found by age group and pathogen, and for patients with or without hypertension, liver disease, chronic obstructive pulmonary disease, dementia, and alcohol abuse. 378 patients (21.4%) died before 90 days. 526 (29.8%) patients were readmitted to the hospital within 60 days. For each doubling of the NLR, the OR for all-cause 90-day mortality was 1.15 (95% CI, 1.04–1.27) and 1.12 (95% CI, 1.02–1.24) for 60-day readmission. Analysis of subgroups did not show statistically significant differences between groups in relation to the association between NLR and mortality. The discriminatory ability of NLR for mortality was limited and comparable to blood neutrophil or lymphocyte count, producing receiver operating characteristic curves with an area under the curve of 0.59 (95% CI, 0.56–0.63), 0.60 (95% CI, 0.56–0.65) and 0.53 (95% CI, 0.49–0.56), respectively. Conclusion Blood neutrophil-lymphocyte ratio was associated with 90-day all-cause mortality and 60-day readmission in patients with GNB. However, the ratio has limited ability in predicting mortality or readmission

Temporal trends in 90-day survival of hospitalised individuals during two years of the COVID-19 pandemic in Denmark

Mortality rates peaked early in the COVID-19 pandemic and then declined. Possible explanations are pharmacological and non-pharmacological treatments, vaccines and changing demographics. We sought to evaluate temporal trends in clinical characteristics and survival of patients hospitalised with COVID-19 during the first two years of the pandemic in Denmark. In this observational study, we included all adults with COVID-19 consecutively admitted to three hospitals in Copenhagen, Denmark, from March 2020 through March 2022. The primary outcome was overall survival up to day 90 from admission. We used multivariable Cox proportional hazards models to estimate the association of survival within five consecutive time-periods, based on admission date, adjusted for baseline characteristics, vaccination status, remdesivir and dexamethasone treatment. In 1630 included patients, the median age [IQR] was 68 [52, 79] years, 56.6% were men and 86.2% had comorbidity. Clinical characteristics changed over time. The crude 90-day mortality rate peaked in March–June 2020 with 28.9%, decreased from July 2020 to 17.5%, and increased again in January-March 2022 to 28.6%. Lower hazard ratios for death were observed in individuals admitted from July 2020 and persisted after adjusting for baseline characteristics. Adjusting for vaccination, remdesivir treatment and dexamethasone treatment attenuated the association in patients requiring low-flow oxygen. Our study suggests lower hazard rates for mortality in patients hospitalised with COVID-19 from July 2020 compared to March-June 2020, mainly driven by lower mortality in patients with a need of oxygen at baseline.</p