Salad leaf juices enhance Salmonella growth, fresh produce colonisation and virulence

Abstract: We show in this report that traces of juices released from salad leaves as they became damaged can significantly enhance Salmonella enterica salad leaf colonisation. Salad juices in water increased Salmonella growth by 110% over the un-supplemented control, and in host-like serum based media by more than 2400-fold over controls. In serum based media salad juices induced growth of Salmonella via provision of Fe from transferrin, and siderophore production was found to be integral to the growth induction process. Other aspects relevant to salad leaf colonisation and retention were enhanced, such as motility and biofilm formation, which increased over controls by >220% and 250% respectively; direct attachment to salad leaves increased by >350% when a salad leaf juice was present. In terms of growth and biofilm formation the endogenous salad leaf microbiota was largely unresponsive to leaf juice, suggesting that Salmonella gains a marked advantage from fluids released from salad leaf damage. Salad leaf juices also enhanced pathogen attachment to the salad bag plastic. Over 5 days refrigeration (a typical storage time for bagged salad leaves) even traces of juice within the salad bag fluids increased Salmonella growth in water by up to 280-fold over control cultures, as well as enhancing salad bag colonisation, which could be an unappreciated factor in pathogen fresh produce retention. Collectively, this study shows that exposure to salad leaf juice may contribute to the persistence of Salmonella on salad leaves, and strongly emphasizes the importance of ensuring the microbiological safety of fresh produce. Importance: Salad leaves are an important part of a healthy diet, but in recent years have been associated with a growing risk of food poisoning from bacterial pathogens such as Salmonella enterica. Although this is considered a significant public health problem, very little is known about what happens to the behaviour of the Salmonella when in the actual salad bag. We show that juices released from the cut-ends of the salad leaves enabled the Salmonella to grow in water, even when it was refrigerated. Salad juice exposure also helped the Salmonella cells to attach to the salad leaves so strongly that washing could not remove them. Collectively, this study shows that exposure to even traces of salad leaf juice may contribute to the persistence of Salmonella on salad leaves as well as priming it for establishing an infection in the consumer

Ex vivo perfusion of isolated human liver segments: the development of a novel model for ethical, translational research

Background Ex vivo perfusion techniques for human organs, in particular for the human liver, have been extensively studied for decades. Although ex vivo perfusion of human organs has been widely studied in the context of organ preservation and transplantation, it has also proven to be an invaluable tool in the development of novel models for translational pre-clinical research. Not only do these models allow more accurate study of human organ response to noxious external stimuli, but they also represent a far more ethical alternative to live animal experimentation.Although split-liver perfusion of the left or right hepatic lobe is well described in literature, ex vivo perfusion of isolated liver segments has not been previously attempted. A successful hepatic segmental perfusion model would provide a unique opportunity to study inflammation, response to infection and novel therapeutic approaches.The overall aim of this study was to establish an experimental ex vivo hepatic perfusion model on surgically resected human liver segments as a platform to evaluate and study organ preservation and function. The development of an ex vivo perfusion model of human liver segments would produce the ideal platform to study treatment effects without needing to sacrifice animals. Methods Patients were recruited as part of the TIMOLD (Tissue Models for Liver Disease) clinical trial (ClinicalTrials.gov Identifier: NCT05255042; 8/9/2021). Patients recruited were those aged eighteen or over undergoing elective liver resections at a single hepatobiliary unit. Nine human liver segments were retrieved following hemi-hepatectomy for colorectal liver metastases or hepatocellular carcinoma. A healthy segment was resected from the diseased specimen in theatre and a segmental hepatic artery and portal vein was cannulated immediately and flushed with ice cold heparinised preservation solution. Four segments were perfused with 600ml O negative expired red blood cells (HL-RBC) and five segments were perfused with 250ml Oxyglobin diluted with 250ml Volplex (HL-OXY). All segments were administered several drugs to provide metabolic support and optimise perfusion owing to their anticoagulant, antioxidant, and anti-inflammatory properties. Segments were perfused on a bespoke normothermic machine perfusion circuit for 4–6 hours. Perfusion parameters were monitored throughout perfusion and hourly hepatic venous blood gases were taken to monitor glucose and lactate levels. Hourly core biopsies were also taken for H&E staining and interpreted by a consultant histopathologist. Data is reported as the median (range) and statistical analysis has been performed using the Mann-Whitney U test on Graphpad Prism. Results HL-RBC (n=4) had a median pre-perfusion weight of 216 (146–1330) grams whilst HL-OXY (n=5) had a median weight of 269(128–367) grams (p>0.05). HL-RBC segments had a median warm ischaemic (WI) time of 76.5(68–81) minutes and a median cold ischaemic (CI) time of 97 (35–119) minutes. HL-OXY segments had a median WI time of 5 (3–80) minutes and a median CI time of 38 (28–63) minutes (p>0.05). Median portal venous resistance was lower for the HL-OXY group (0.076mmHg/ml/min) in comparison to the HL-RBC group (0.62mmHg/ml/min); p0.05). HL-OXY had a lower median lactate of 4.54mmol/L in comparison to HL-RBC (14.7mmol/L) throughout perfusion (p Conclusions We present here, for the first time, the development of a successful ex vivo isolated hepatic segmental perfusion model. We demonstrate, in the development of this model, that bovine haemoglobin glutamer-200 (Oxyglobin) is a superior oxygen carrier to expired O negative red blood cells in the ex vivo perfusion of isolated human hepatic segments. The HL-OXY group demonstrated a significantly lower portal venous resistance in comparison to HL-RBC, although there was no difference in arterial resistance. Furthermore, HL-OXY segments had lower median glucose and lactate levels during perfusion compared to HL-RBC segments. 4 of 5 segments perfused with Oxyglobin demonstrated viable hepatocytes on histopathological analysis after 6 hours of perfusion. The segment demonstrating early necrosis was one with established cirrhosis on pre-perfusion histology. We describe a protocol for the successful ex vivo perfusion of non-cirrhotic human hepatic segments. Human liver is routinely resected in tertiary hepatobiliary units and much of the specimen discarded. The paradigm presented here identifies a readily available ethical source of human liver with minimal warm ischaemia. This facilitates optimal conditions for translational research, is cost-effective and avoids animal experimentation.</p

Phase-variable methylation and epigenetic regulation by type I restriction-modification systems.

Epigenetic modifications in bacteria, such as DNA methylation, have been shown to affect gene regulation, thereby generating cells that are isogenic but with distinctly different phenotypes. Restriction-modification (RM) systems contain prototypic methylases that are responsible for much of bacterial DNA methylation. This review focuses on a distinctive group of type I RM loci that , through phase variation, can modify their methylation target specificity and can thereby switch bacteria between alternative patterns of DNA methylation. Phase variation occurs at the level of the target recognition domains of the hsdS (specificity) gene via reversible recombination processes acting upon multiple hsdS alleles. We describe the global distribution of such loci throughout the prokaryotic kingdom and highlight the differences in loci structure across the various bacterial species. Although RM systems are often considered simply as an evolutionary response to bacteriophages, these multi-hsdS type I systems have also shown the capacity to change bacterial phenotypes. The ability of these RM systems to allow bacteria to reversibly switch between different physiological states, combined with the existence of such loci across many species of medical and industrial importance, highlights the potential of phase-variable DNA methylation to act as a global regulatory mechanism in bacteria

Dissemination of Novel Antimicrobial Resistance Mechanisms through the Insertion Sequence Mediated Spread of Metabolic Genes.

The widely used biocide triclosan selectively targets FabI, the NADH-dependent trans-2-enoyl-acyl carrier protein (ACP) reductase, which is also an important target for the development of narrow spectrum antibiotics. The analysis of triclosan resistant Staphylococcus aureus isolates had previously shown that in about half of the strains, the mechanism of triclosan resistance consists on the heterologous duplication of the triclosan target gene due to the acquisition of an additional fabI allele derived from Staphylococcus haemolyticus (sh-fabI). In the current work, the genomic sequencing of 10 of these strains allowed the characterization of two novel composite transposons TnSha1 and TnSha2 involved in the spread of sh-fabI. TnSha1 harbors one copy of IS1272, whereas TnSha2 is a 11.7 kb plasmid carrying TnSha1 present either as plasmid or in an integrated form generally flanked by two IS1272 elements. The target and mechanism of integration for IS1272 and TnSha1 are novel and include targeting of DNA secondary structures, generation of blunt-end deletions of the stem-loop and absence of target duplication. Database analyses showed widespread occurrence of these two elements in chromosomes and plasmids, with TnSha1 mainly in S. aureus and with TnSha2 mainly in S. haemolyticus and S. epidermidis. The acquisition of resistance by means of an insertion sequence-based mobilization and consequent duplication of drug-target metabolic genes, as observed here for sh-fabI, is highly reminiscent of the situation with the ileS2 gene conferring mupirocin resistance, and the dfrA and dfrG genes conferring trimethoprim resistance both of which are mobilized by IS257. These three examples, which show similar mechanisms and levels of spread of metabolic genes linked to IS elements, highlight the importance of this genetic strategy for recruitment and rapid distribution of novel resistance mechanisms in staphylococci

Exhaled SARS-CoV-2 quantified by face-mask sampling in hospitalised patients with covid-19

BackgroundHuman to human transmission of SARS-CoV-2 is driven by the respiratory route but little is known about the pattern and quantity of virus output from exhaled breath. We have previously shown that face-mask sampling (FMS) can detect exhaled tubercle bacilli and have adapted its use to quantify exhaled SARS-CoV-2 RNA in patients admitted to hospital with Coronavirus Disease-2019 (COVID-19).MethodsBetween May and December 2020, we took two concomitant FMS and nasopharyngeal samples (NPS) over two days, starting within 24 hours of a routine virus positive NPS in patients hospitalised with COVID-19, at University Hospitals of Leicester NHS Trust, UK. Participants were asked to wear a modified duckbilled facemask for 30 minutes, followed by a nasopharyngeal swab. Demographic, clinical, and radiological data, as well as International Severe Acute Respiratory and emerging Infections Consortium (ISARIC) mortality and deterioration scores were obtained. Exposed masks were processed by removal, dissolution and analysis of sampling matrix strips fixed within the mask by RT-qPCR. Viral genome copy numbers were determined and results classified as Negative; Low: ≤999 copies; Medium: 1,000-99,999 copies and High ≥ 100,000 copies per strip for FMS or per 100µl for NPS.Results102 FMS and NPS were collected from 66 routinely positive patients; median age: 61 (IQR 49 - 77), of which FMS was positive in 38% of individuals and concomitant NPS was positive in 50%. Positive FMS viral loads varied over five orders of magnitude (6 genome copies/strip); 21 (32%) patients were asymptomatic at the time of sampling. High FMS viral load was associated with respiratory symptoms at time of sampling and shorter interval between sampling and symptom onset (FMS High: median (IQR) 2 days (2-3) vs FMS Negative: 7 days (7-10), p=0.002). On multivariable linear regression analysis, higher FMS viral loads were associated with higher ISARIC mortality (Medium FMS vs Negative FMS gave an adjusted coefficient of 15.7, 95% CI 3.7-27.7, p=0.01) and deterioration scores (High FMS vs Negative FMS gave an adjusted coefficient of 37.6, 95% CI 14.0 to 61.3, p=0.002), while NPS viral loads showed no significant association.ConclusionWe demonstrate a simple and effective method for detecting and quantifying exhaled SARS-CoV-2 in hospitalised patients with COVID-19. Higher FMS viral loads were more likely to be associated with developing severe disease compared to NPS viral loads. Similar to NPS, FMS viral load was highest in early disease and in those with active respiratory symptoms, highlighting the potential role of FMS in understanding infectivity

A random six-phase switch regulates pneumococcal virulence via global epigenetic changes.

Streptococcus pneumoniae (the pneumococcus) is the world's foremost bacterial pathogen in both morbidity and mortality. Switching between phenotypic forms (or 'phases') that favour asymptomatic carriage or invasive disease was first reported in 1933. Here, we show that the underlying mechanism for such phase variation consists of genetic rearrangements in a Type I restriction-modification system (SpnD39III). The rearrangements generate six alternative specificities with distinct methylation patterns, as defined by single-molecule, real-time (SMRT) methylomics. The SpnD39III variants have distinct gene expression profiles. We demonstrate distinct virulence in experimental infection and in vivo selection for switching between SpnD39III variants. SpnD39III is ubiquitous in pneumococci, indicating an essential role in its biology. Future studies must recognize the potential for switching between these heretofore undetectable, differentiated pneumococcal subpopulations in vitro and in vivo. Similar systems exist in other bacterial genera, indicating the potential for broad exploitation of epigenetic gene regulation