In vivo single-cell high-dimensional mass cytometry analysis to track the interactions between Klebsiella pneumoniae and myeloid cells.

In vivo single-cell approaches have transformed our understanding of the immune populations in tissues. Mass cytometry (CyTOF), that combines the resolution of mass spectrometry with the ability to conduct multiplexed measurements of cell molecules at the single cell resolution, has enabled to resolve the diversity of immune cell subsets, and their heterogeneous functionality. Here we assess the feasibility of taking CyTOF one step further to immuno profile cells while tracking their interactions with bacteria, a method we term Bac-CyTOF. We focus on the pathogen Klebsiella pneumoniae interrogating the pneumonia mouse model. Using Bac-CyTOF, we unveil the atlas of immune cells of mice infected with a K. pneumoniae hypervirulent strain. The atlas is characterized by a decrease in the populations of alveolar and monocyte-derived macrophages. Conversely, neutrophils, and inflammatory monocytes are characterized by an increase in the subpopulations expressing markers of less active cells such as the immune checkpoint PD-L1. These are the cells infected. We show that the type VI secretion system (T6SS) contributes to shape the lung immune landscape. The T6SS governs the interaction with monocytes/macrophages by shifting Klebsiella from alveolar macrophages to interstitial macrophages and limiting the infection of inflammatory monocytes. The lack of T6SS results in an increase of cells expressing markers of active cells, and a decrease in the subpopulations expressing PD-L1. By probing Klebsiella, and Acinetobacter baumannii strains with limited ability to survive in vivo, we uncover that a heightened recruitment of neutrophils, and relative high levels of alveolar macrophages and eosinophils and the recruitment of a characteristic subpopulation of neutrophils are features of mice clearing infections. We leverage Bac-CyTOF-generated knowledge platform to investigate the role of the DNA sensor STING in Klebsiella infections. sting-/- infected mice present features consistent with clearing the infection including the reduced levels of PD-L1. STING absence facilitates Klebsiella clearance.

The Role of Wildlife and Pests in the Transmission of Pathogenic Agents to Domestic Pigs: A Systematic Review.

Wild animals and pests are important reservoirs and vectors of pathogenic agents that can affect domestic pigs. Rapid globalization, anthropogenic factors, and increasing trends toward outdoor pig production facilitate the contact between domestic pigs and wildlife. However, knowledge on the transmission pathways between domestic pigs and the aforementioned target groups is limited. The present systematic review aims to collect and analyze information on the roles of different wild animal species and pests in the spread of pathogens to domesticated pigs. Overall, 1250 peer-reviewed manuscripts published in English between 2010 and 2022 were screened through the PRISMA framework using PubMed, Scopus, and Web of Science databases. A total of 84 studies reporting possible transmission routes of different pathogenic agents were included. A majority of the studies (80%) focused on the role of wild boars in the transmission of pathogenic agents to pig farms. Studies involving the role of rodents (7%), and deer (6%) were the next most frequent, whereas the role of insects (5%), wild carnivores (5%), wild birds (4%), cats (2%), and badgers (1%) were less available. Only 3.5% of studies presented evidence-based transmission routes from wildlife to domestic pigs. Approximately 65.5% of the included studies described possible risks/risk factors for pathogens' transmission based on quantitative data, whereas 31% of the articles only presented a hypothesis or qualitative analysis of possible transmission routes or risk factors and/or contact rates. Risk factors identified include outdoor farms or extensive systems and farms with a low level of biosecurity as well as wildlife behavior; environmental conditions; human activities and movements; fomites, feed (swill feeding), water, carcasses, and bedding materials. We recommend the strengthening of farm biosecurity frameworks with special attention to wildlife-associated parameters, especially in extensive rearing systems and high-risk zones as it was repeatedly found to be an important measure to prevent pathogen transmission to domestic pigs. In addition, there is a need to focus on effective risk-based wildlife surveillance mechanisms and to raise awareness among farmers about existing wildlife-associated risk factors for disease transmission.

Value of simplified lung lesions scoring systems to inform future codes for routine meat inspection in pigs.

BACKGROUND: Across the European Union (EU), efforts are being made to achieve modernisation and harmonisation of meat inspection (MI) code systems. Lung lesions were prioritised as important animal based measures at slaughter, but existing standardized protocols are difficult to implement for routine MI. This study aimed to compare the informative value and feasibility of simplified lung lesion scoring systems to inform future codes for routine post mortem MI. RESULTS: Data on lung lesions in finisher pigs were collected at slaughter targeting 83 Irish pig farms, with 201 batches assessed, comprising 31,655 pairs of lungs. Lungs were scored for cranioventral pulmonary consolidations (CVPC) and pleurisy lesions using detailed scoring systems, which were considered the gold standard. Using the data collected, scenarios for possible simplified scoring systems to record CVPC (n = 4) and pleurisy (n = 4) lesions were defined. The measurable outcomes were the prevalence and (if possible) severity scoring at batch level for CVPC and pleurisy. An arbitrary threshold was set to the upper quartile (i.e., the top 25% of batches with high prevalence/severity of CVPC or pleurisy, n = 50). Each pair of measurable outcomes was compared by calculating Spearman rank correlations and assessing if batches above the threshold for one measurable outcome were also above it for their pairwise comparison. All scenarios showed perfect agreement (k = 1) when compared among themselves and the gold standard for the prevalence of CVPC. The agreement among severity outcomes and the gold standard showed moderate to perfect agreement (k = [0.66, 1]). The changes in ranking were negligible for all measurable outcomes of pleurisy for scenarios 1, 2 and 3 when compared with the gold standard (rs ≥ 0.98), but these changes amounted to 50% for scenario 4. CONCLUSIONS: The best simplified CVPC scoring system is to simply count the number of lung lobes affected excluding the intermediate lobe, which provides the best trade-off between value of information and feasibility, by incorporating information on CVPC prevalence and severity. While for pleurisy evaluation, scenario 3 is recommended. This simplified scoring system provides information on the prevalence of cranial and moderate and severe dorsocaudal pleurisy. Further validation of the scoring systems at slaughter and by private veterinarians and farmers is needed.

PYHIN protein IFI207 regulates cytokine transcription and IRF7 and contributes to the establishment of K. pneumoniae infection.

PYHIN proteins AIM2 and IFI204 sense pathogen DNA, while other PYHINs have been shown to regulate host gene expression through as-yet unclear mechanisms. We characterize mouse PYHIN IFI207, which we find is not involved in DNA sensing but rather is required for cytokine promoter induction in macrophages. IFI207 co-localizes with both active RNA polymerase II (RNA Pol II) and IRF7 in the nucleus and enhances IRF7-dependent gene promoter induction. Generation of Ifi207-/- mice shows no role for IFI207 in autoimmunity. Rather, IFI207 is required for the establishment of a Klebsiella pneumoniae lung infection and for Klebsiella macrophage phagocytosis. These insights into IFI207 function illustrate that PYHINs can have distinct roles in innate immunity independent of DNA sensing and highlight the need to better characterize the whole mouse locus, one gene at a time.

Visualisation of Host-Pathogen Communication.

The core of biomedical science is the use of laboratory techniques to support the diagnosis and treatment of disease in clinical settings. Despite tremendous advancement in our understanding of medicine in recent years, we are still far from having a complete understanding of human physiology in homeostasis, let alone the pathology of disease states. Indeed medical advances over the last two hundred years would not have been possible without the invention of and continuous development of visualisation techniques available to research scientists and clinicians. As we have all learned from the recent COVID pandemic, despite advances in modern medicine we still have much to learn regarding infection biology. Indeed antimicrobial resistant (AMR) bacteria are a global threat to human health, meaning research into bacterial pathogenesis is vital. In this chapter, we will briefly describe the nature of microbes and host immune responses before delving into some of the visualisation techniques utilised in the field of biomedical research with a focus on host-pathogen interactions. We will give a brief overview of commonly used techniques from gold standard staining methods, in situ hybridisation, microscopy, western blotting, microbial characterisation, to cutting-edge image flow cytometry and mass spectrometry. Specifically, we will focus on techniques utilised to visualise interactions between the host, our own bodies, and invading organisms including bacteria. We will touch on in vitro and ex vivo modelling methodology with examples utilised to delineate pathogenicity in disease. A better understanding of bacterial biology, immunology and how these fields interact (host-pathogen communications) in biomedical research is integral to developing novel therapeutic approaches which circumvent the need for antibiotics, an important issue as we enter a post-antibiotic era.

A trans-kingdom T6SS effector induces the fragmentation of the mitochondrial network and activates innate immune receptor NLRX1 to promote infection.

Bacteria can inhibit the growth of other bacteria by injecting effectors using a type VI secretion system (T6SS). T6SS effectors can also be injected into eukaryotic cells to facilitate bacterial survival, often by targeting the cytoskeleton. Here, we show that the trans-kingdom antimicrobial T6SS effector VgrG4 from Klebsiella pneumoniae triggers the fragmentation of the mitochondrial network. VgrG4 colocalizes with the endoplasmic reticulum (ER) protein mitofusin 2. VgrG4 induces the transfer of Ca2+ from the ER to the mitochondria, activating Drp1 (a regulator of mitochondrial fission) thus leading to mitochondrial network fragmentation. Ca2+ elevation also induces the activation of the innate immunity receptor NLRX1 to produce reactive oxygen species (ROS). NLRX1-induced ROS limits NF-κB activation by modulating the degradation of the NF-κB inhibitor IκBα. The degradation of IκBα is triggered by the ubiquitin ligase SCFß-TrCP, which requires the modification of the cullin-1 subunit by NEDD8. VgrG4 abrogates the NEDDylation of cullin-1 by inactivation of Ubc12, the NEDD8-conjugating enzyme. Our work provides an example of T6SS manipulation of eukaryotic cells via alteration of the mitochondria.

Modelling the Gastrointestinal Carriage of Klebsiella pneumoniae Infections.

Klebsiella pneumoniae is a leading cause of nosocomial and community acquired infections, making K. pneumoniae the pathogen that is associated with the second largest number of deaths attributed to any antibiotic resistant infection. K. pneumoniae colonizes the nasopharynx and the gastrointestinal tract in an asymptomatic manner without dissemination to other tissues. Importantly, gastrointestinal colonization is a requisite for infection. Our understanding of K. pneumoniae colonization is still based on interrogating mouse models in which animals are pretreated with antibiotics to disturb the colonization resistance imposed by the gut microbiome. In these models, infections disseminate to other tissues. Here, we report a murine model to allow for the study of the gastrointestinal colonization of K. pneumoniae without tissue dissemination. Hypervirulent and antibiotic resistant strains stably colonize the gastrointestinal tract of in an inbred mouse population without antibiotic treatment. The small intestine is the primary site of colonization and is followed by a transition to the colon over time, without dissemination to other tissues. Our model recapitulates the disease dynamics of the metastatic K. pneumoniae strains that are able to disseminate from the gastrointestinal tract to other sterile sites. Colonization is associated with mild to moderate histopathology, no significant inflammation, and no effect on the richness of the microbiome. Our model sums up the clinical scenario in which antibiotic treatment disturbs the colonization of K. pneumoniae and results in dissemination to other tissues. Finally, we establish that the capsule polysaccharide is necessary for the colonization of the large intestine, whereas the type VI secretion system contributes to colonization across the gastrointestinal tract. IMPORTANCE Klebsiella pneumoniae is one of the pathogens that is sweeping the world in the antibiotic resistance pandemic. Klebsiella colonizes the nasopharynx and the gut of healthy subjects in an asymptomatic manner, making gut colonization a requisite for infection. This makes it essential to understand the gastrointestinal carriage in preventing Klebsiella infections. Current research models rely on the perturbation of the gut microbiome by antibiotics, resulting in an invasive infection. Here, we report a new model of K. pneumoniae gut colonization that recapitulates key features of the asymptomatic human gastrointestinal tract colonization. In our model, there is no need to disturb the microbiota to achieve stable colonization, and there is no dissemination to other tissues. Our model sums up the clinical scenario in which antibiotic treatment triggers invasive infection. We envision that our model will be an excellent platform upon which to investigate factors enhancing colonization and invasive infections and to test therapeutics to eliminate Klebsiella asymptomatic colonization.

In vivo single-cell transcriptomics reveal Klebsiella pneumoniae skews lung macrophages to promote infection.

The strategies deployed by antibiotic-resistant bacteria to counteract host defences are poorly understood. Here, we elucidate a novel host-pathogen interaction resulting in skewing lung macrophage polarisation by the human pathogen Klebsiella pneumoniae. We identify interstitial macrophages (IMs) as the main population of lung macrophages associated with Klebsiella. Single-cell transcriptomics and trajectory analysis of cells reveal type I IFN and IL10 signalling, and macrophage polarisation are characteristic of infected IMs, whereas Toll-like receptor (TLR) and Nod-like receptor signalling are features of infected alveolar macrophages. Klebsiella-induced macrophage polarisation is a singular M2-type we termed M(Kp). To rewire macrophages, Klebsiella hijacks a TLR-type I IFN-IL10-STAT6 axis. Absence of STAT6 limits Klebsiella intracellular survival and facilitates the clearance of the pathogen in vivo. Glycolysis characterises M(Kp) metabolism, and inhibition of glycolysis results in clearance of intracellular Klebsiella. Capsule polysaccharide governs M(Kp). Klebsiella also skews human macrophage polarisation towards M(Kp) in a type I IFN-IL10-STAT6-dependent manner. Klebsiella induction of M(Kp) represents a novel strategy to overcome host restriction, and identifies STAT6 as target to boost defences against Klebsiella.

Meeting report - Cell dynamics: host-pathogen interface.

Two years into the most significant infectious disease event of our generation, infections have populated every conversation and in-depth understanding of host-pathogen interactions has, perhaps, never been more important. In a successful return to in-person conferences, the host-pathogen interface was the focus of the third Cell Dynamics meeting, which took place at the glorious Wotton House in Surrey, UK. The meeting organised by Michaela Gack, Maximiliano Gutierrez, Dominique Soldati-Favre and Michael Way gathered an international group of scientists who shared their recent discoveries and views on numerous aspects, including cell-autonomous defence mechanisms, pathogen interactions with host cytoskeletal or membrane dynamics, and cellular immune regulation. More than 30 years into the beginning of cellular microbiology as a field, the meeting exhibited the unique aspect of the host-pathogen interface in uncovering the fundamentals of both pathogens and their hosts.

Klebsiella pneumoniae hijacks the Toll-IL-1R protein SARM1 in a type I IFN-dependent manner to antagonize host immunity.

Many bacterial pathogens antagonize host defense responses by translocating effector proteins into cells. It remains an open question how those pathogens not encoding effectors counteract anti-bacterial immunity. Here, we show that Klebsiella pneumoniae exploits the evolutionary conserved innate protein SARM1 to regulate negatively MyD88- and TRIF-governed inflammation, and the activation of the MAP kinases ERK and JNK. SARM1 is required for Klebsiella induction of interleukin-10 (IL-10) by fine-tuning the p38-type I interferon (IFN) axis. SARM1 inhibits the activation of Klebsiella-induced absent in melanoma 2 inflammasome to limit IL-1ß production, suppressing further inflammation. Klebsiella exploits type I IFNs to induce SARM1 in a capsule and lipopolysaccharide O-polysaccharide-dependent manner via the TLR4-TRAM-TRIF-IRF3-IFNAR1 pathway. Absence of SARM1 reduces the intracellular survival of K. pneumoniae in macrophages, whereas sarm1-deficient mice control the infection. Altogether, our results illustrate an anti-immunology strategy deployed by a human pathogen. SARM1 inhibition will show a beneficial effect to treat Klebsiella infections.

Environmental Risk Factors Influence the Frequency of Coughing and Sneezing Episodes in Finisher Pigs on a Farm Free of Respiratory Disease.

This study aimed to assess baseline levels of coughing on a farm free of respiratory disease, and to identify relationships between environmental conditions and coughing frequency in finisher pigs. Six replicates were conducted (690 pigs in total). A cross-correlation analysis was performed and lags of the predictor variables were carried forward for multivariable regression analysis when significant and showing r > 0.25. Results show that coughing frequency was overall low. In the first replicate, coughing was best predicted by exposure to higher ammonia concentrations that occurred with a lag of 1, 7, and 15 days (p = 0.003, p = 0.001, and p < 0.001, respectively), while in the sixth replicate coughing frequency was best predicted by the exposure to lower relative humidity and higher ventilation rates with a lag of 7 and 15 days (p < 0.001 and p = 0.003, respectively). Ammonia concentrations varied according to ventilation rates recorded on the same day (r > −0.70). In conclusion, guidelines on coughing levels in healthy pigs and calibration of the alarm systems of tools that measure coughing frequency can be extrapolated from this study. Environmental risk factors are associated with the respiratory health of finisher pigs.

Effect of Phase Feeding, Space Allowance and Mixing on Productive Performance of Grower-Finisher Pigs.

This study investigates the effects of space allowance (SA), mixing and phase feeding (PF) on performance of grower-finisher pigs. Three trials (T) were conducted. In T1 and T2, 345 pigs/trial were moved to finisher stage at 11 weeks of age and assigned to two SAs: 0.96 (n = 15 pens; 10 pigs/pen) and 0.78 (n = 15; 13 pigs/pen) m2/pig. Mixing was applied to 5 pens of each SA leading to a 2 × 2 factorial arrangement (SA × Mixing). For PF, 2 diets with 0.95 and 0.82 g SID Lys/MJ NE were applied to 5 pens of each SA (not mixed) leading to another 2 × 2 factorial arrangement (SA × PF). In T3, 230 pigs were moved to the grower-finisher stage at 11 weeks of age, mixed, and assigned to 4 treatments (SA × PF; n = 5 pens). Data were analyzed using general linear mixed models. SA did not affect performance (p > 0.05). Non-mixed pigs were 5.40 (T1) and 5.25 (T2) kg heavier than mixed pigs at 21 weeks of age (p < 0.001). PF reduced performance of pigs by 3.45 (T1) and 4.05 (T2) kg at 21 weeks of age (p < 0.001). In conclusion, mixing and reducing SID Lys:NE ratio from 0.95 to 0.82 g/MJ at 15-16 weeks of age, have a more marked impact on performance than reducing SA from 0.96 to 0.78 m2/pig.

Adding value to food chain information: using data on pig welfare and antimicrobial use on-farm to predict meat inspection outcomes.

BACKGROUND: Using Food Chain Information data to objectively identify high-risk animals entering abattoirs can represent an important step forward towards improving on-farm animal welfare. We aimed to develop and evaluate the performance of classification models, using Gradient Boosting Machine algorithms that utilise accurate longitudinal on-farm data on pig health and welfare to predict condemnations, pluck lesions and low cold carcass weight at slaughter. RESULTS: The accuracy of the models was assessed using the area under the receiver operating characteristics (ROC) curve (AUC). The AUC for the prediction models for pneumonia, dorsocaudal pleurisy, cranial pleurisy, pericarditis, partial and total condemnations, and low cold carcass weight varied from 0.54 for pneumonia and 0.67 for low cold carcass weight. For dorsocaudal pleurisy, ear lesions assessed on pigs aged 12 weeks and antimicrobial treatments (AMT) were the most important prediction variables. Similarly, the most important variable for the prediction of cranial pleurisy was the number of AMT. In the case of pericarditis, ear lesions assessed both at week 12 and 14 were the most important variables and accounted for 33% of the Bernoulli loss reduction. For predicting partial and total condemnations, the presence of hernias on week 18 and lameness on week 12 accounted for 27% and 14% of the Bernoulli loss reduction, respectively. Finally, AMT (37%) and ear lesions assessed on week 12 (15%) were the most important variables for predicting pigs with low cold carcass weight. CONCLUSIONS: The findings from our study show that on farm assessments of animal-based welfare outcomes and information on antimicrobial treatments have a modest predictive power in relation to the different meat inspection outcomes assessed. New research following the same group of pigs longitudinally from a larger number of farms supplying different slaughterhouses is required to confirm that on farm assessments can add value to Food Chain Information reports.

A Systematic Review on the Effectiveness of Pre-Harvest Meat Safety Interventions in Pig Herds to Control Salmonella and Other Foodborne Pathogens.

This systematic review aimed to assess the effectiveness of pre-harvest interventions to control the main foodborne pathogens in pork in the European Union. A total of 1180 studies were retrieved from PubMed® and Web of Science for 15 pathogens identified as relevant in EFSA's scientific opinion on the public health hazards related to pork (2011). The study selection focused on controlled studies where a cause-effect could be attributed to the interventions tested, and their effectiveness could be inferred. Altogether, 52 studies published from 1983 to 2020 regarding Campylobacter spp., Clostridium perfringens, Methicillin-resistant Staphylococcus aureus, Mycobacterium avium, and Salmonella spp. were retained and analysed. Research was mostly focused on Salmonella (n = 43 studies). In-feed and/or water treatments, and vaccination were the most tested interventions and were, overall, successful. However, the previously agreed criteria for this systematic review excluded other effective interventions to control Salmonella and other pathogens, like Yersinia enterocolitica, which is one of the most relevant biological hazards in pork. Examples of such successful interventions are the Specific Pathogen Free herd principle, stamping out and repopulating with disease-free animals. Research on other pathogens (i.e., Hepatitis E, Trichinella spiralis and Toxoplasma gondii) was scarce, with publications focusing on epidemiology, risk factors and/or observational studies. Overall, high herd health coupled with good management and biosecurity were effective to control or prevent most foodborne pathogens in pork at the pre-harvest level.

Laparoscopic colorectal surgery and discharge within 24 h-who is at risk for readmission?

AIM: The aim was to describe risk factors for hospital readmission in patients undergoing laparoscopic colorectal procedures and being discharged in ≤24 h. METHOD: All consecutive patients undergoing minimally invasive colorectal surgery between 2010 and 2019 from a single institution were retrospectively reviewed. All patients were included in an enhanced recovery programme. Patients who met criteria for hospital discharge were compared according to the need for readmission in a 45-day follow-up. RESULTS: In all, 664 patients underwent minimally invasive colorectal surgery during the study period and 237 (35.7%) were discharged in ≤24 h. Readmission was required in 16 (6.8%) patients discharged in ≤24 h and no postoperative mortality was observed in this group. Patients discharged in ≤24 h were more likely to have benign disease (P < 0.001), fewer associated procedures (P < 0.025) and intracorporeal anastomoses (P < 0.001). The type of surgical procedure (abdominoperineal resection), low rectal tumour, malignant disease, older age and longer operating time were associated with readmission. Age (OR 1.06; P = 0.037), malignant disease (OR 4.39; P = 0.05) and operating time (OR 1.03; P < 0.001) were identified as independent predictive factors for readmission amongst patients being discharged in ≤24 h. CONCLUSION: Highly selected patients undergoing minimally invasive procedures in colorectal surgery may be safely discharged within 24 h following the procedure. High-risk features for readmission include older age, malignant disease and longer operating time.

Targeting Candida albicans in dual-species biofilms with antifungal treatment reduces Staphylococcus aureus and MRSA in vitro.

Polymicrobial biofilms consisting of fungi and bacteria are frequently formed on endotracheal tubes and may contribute to development of ventilator associated pneumonia (VAP) in critically ill patients. This study aimed to determine the role of early Candida albicans biofilms in supporting dual-species (dual-kingdom) biofilm formation with respiratory pathogens in vitro, and investigated the effect of targeted antifungal treatment on bacterial cells within the biofilms. Dual-species biofilm formation between C. albicans and three respiratory pathogens commonly associated with VAP (Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus) was studied using quantitative PCR. It was shown that early C. albicans biofilms enhanced the numbers of E. coli and S. aureus (including methicillin resistant S. aureus; MRSA) but not P. aeruginosa within dual-species biofilms. Transwell assays demonstrated that contact with C. albicans was required for the increased bacterial cell numbers observed. Total Internal Reflection Fluorescence microscopy showed that both wild type and hyphal-deficient C. albicans provided a scaffold for initial bacterial adhesion in dual species biofilms. qPCR results suggested that further maturation of the dual-species biofilm significantly increased bacterial cell numbers, except in the case of E.coli with hyphal-deficient C. albicans (Ca_gcn5Δ/Δ). A targeted preventative approach with liposomal amphotericin (AmBisome®) resulted in significantly decreased numbers of S. aureus in dual-species biofilms, as determined by propidium monoazide-modified qPCR. Similar results were observed when dual-species biofilms consisting of clinical isolates of C. albicans and MRSA were treated with liposomal amphotericin. However, reductions in E. coli numbers were not observed following liposomal amphotericin treatment. We conclude that early C. albicans biofilms have a key supporting role in dual-species biofilms by enhancing bacterial cell numbers during biofilm maturation. In the setting of increasing antibiotic resistance, an important and unexpected consequence of antifungal treatment of dual-species biofilms, is the additional benefit of decreased growth of multi-drug resistant bacteria such as MRSA, which could represent a novel future preventive strategy.

Managing respiratory disease in finisher pigs: Combining quantitative assessments of clinical signs and the prevalence of lung lesions at slaughter.

This study aimed to assess the relationship between quantitative assessments of clinical signs of respiratory disease (recorded manually and automatically) and the prevalence of lung lesions at slaughter to validate the use of both in the management of respiratory disease on farm. This was an observational study where pigs (n = 1573) were monitored from 25 ± 5.3 kg (week 12) to slaughter at 114 ± 15.4 kg (week 24). Pigs were housed in eight rooms divided into six pens on a wean-to-finish farm. A manual pen-based coughing (CF) and sneezing (SF) frequency was recorded weekly, for ten consecutive weeks, and a SOMO box (SoundTalks®) was installed in each room, issuing a daily respiratory distress index (RDI) for 13 weeks. Lungs were individually scored for pneumonia, scarring and dorsocaudal (DC) and cranial (CP) pleurisy lesions at slaughter. Relationship between prevalence of lung lesions and weekly RDI and CF and SF was assessed using Spearman's rank correlations and multivariable linear and logit-normal models. Both coughing and lung lesions were largely pen-specific, which fit the disease presentation of Mycoplasma hyopneumoniae. Results showed agreement between RDI and CF (rs = 0.5, P < 0.001), measuring higher levels of coughing at the beginning (weeks 13-14) and end (weeks 21-24, and weeks 21-22, respectively) of the finisher period. Positive associations were found between the prevalence of pneumonia and CF on week 21 and 22 (P < 0.001 and P = 0.011, respectively) and RDI on week 21-24 (rs > 0.70; P < 0.050); the prevalence of DC and CP, and CF on week 22 (P < 0.001); and prevalence of scar lesions and CF on week 17 and 21 (P = 0.013 and P = 0.004, respectively), and RDI on week 21-24 (rs > 0.70; P < 0.050). In the earlier weeks of the finisher stage, coughing was recorded but was not reflected in a higher prevalence of lung lesions at slaughter. These findings highlight the benefit of including measurements of coughing frequency to complement post mortem findings, to improve the management of respiratory disease on farm.

Klebsiella pneumoniae Reduces SUMOylation To Limit Host Defense Responses.

Klebsiella pneumoniae is an important cause of multidrug-resistant infections worldwide. Understanding the virulence mechanisms of K. pneumoniae is a priority and timely to design new therapeutics. Here, we demonstrate that K. pneumoniae limits the SUMOylation of host proteins in epithelial cells and macrophages (mouse and human) to subvert cell innate immunity. Mechanistically, in lung epithelial cells, Klebsiella increases the levels of the deSUMOylase SENP2 in the cytosol by affecting its K48 ubiquitylation and its subsequent degradation by the ubiquitin proteasome. This is dependent on Klebsiella preventing the NEDDylation of the Cullin-1 subunit of the ubiquitin ligase complex E3-SCF-ßTrCP by exploiting the CSN5 deNEDDylase. Klebsiella induces the expression of CSN5 in an epidermal growth factor receptor (EGFR)-phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT)-extracellular signal-regulated kinase (ERK)-glycogen synthase kinase 3 beta (GSK3ß) signaling pathway-dependent manner. In macrophages, Toll-like receptor 4 (TLR4)-TRAM-TRIF-induced type I interferon (IFN) via IFN receptor 1 (IFNAR1)-controlled signaling mediates Klebsiella-triggered decrease in the levels of SUMOylation via let-7 microRNAs (miRNAs). Our results revealed the crucial role played by Klebsiella polysaccharides, the capsule, and the lipopolysaccharide (LPS) O-polysaccharide, to decrease the levels of SUMO-conjugated proteins in epithelial cells and macrophages. A Klebsiella-induced decrease in SUMOylation promotes infection by limiting the activation of inflammatory responses and increasing intracellular survival in macrophages.IMPORTANCEKlebsiella pneumoniae has been singled out as an urgent threat to human health due to the increasing isolation of strains resistant to "last-line" antimicrobials, narrowing the treatment options against Klebsiella infections. Unfortunately, at present, we cannot identify candidate compounds in late-stage development for treatment of multidrug-resistant Klebsiella infections; this pathogen is exemplary of the mismatch between unmet medical needs and the current antimicrobial research and development pipeline. Furthermore, there is still limited evidence on K. pneumoniae pathogenesis at the molecular and cellular levels in the context of the interactions between bacterial pathogens and their hosts. In this research, we have uncovered a sophisticated strategy employed by Klebsiella to subvert the activation of immune defenses by controlling the modification of proteins. Our research may open opportunities to develop new therapeutics based on counteracting this Klebsiella-controlled immune evasion strategy.

Burden of Disease Estimates of Seven Pathogens Commonly Transmitted Through Foods in Denmark, 2017.

Burden of disease metrics are increasingly established to prioritize food safety interventions. We estimated the burden of disease caused by seven foodborne pathogens in Denmark in 2017: Campylobacter, Salmonella, Shiga toxin-producing Escherichia coli, norovirus, Yersinia enterocolitica, Listeria monocytogenes, and Toxoplasma gondii. We used public health surveillance data and scientific literature to estimate incidence, mortality, and total disability-adjusted life year (DALY) of each, and linked results with estimates of the proportion of disease burden that is attributable to foods. Our estimates showed that Campylobacter caused the highest burden of disease, leading to a total burden of 1709 DALYs (95% uncertainty interval [UI] 1665-1755), more than threefold higher than the second highest ranked pathogen (Salmonella: 492 DALYs; 95% UI 481-504). Campylobacter still led the ranking when excluding DALYs attributable to nonfoodborne routes of exposure. The total estimated incidence was highest for norovirus, but this agent ranked sixth when focusing on foodborne burden. Salmonella ranked second in terms of foodborne burden of disease, followed by Listeria and Yersinia. Foodborne congenital toxoplasmosis was estimated to cause the loss of ∼100 years of healthy life, a burden that was borne by a low number of cases in the population. The ranking of foodborne pathogens varied substantially when based on reported cases, estimated incidence, and burden of disease estimates. Our results reinforce the need to continue food safety efforts throughout the food chain in Denmark, with a particular focus on reducing the incidence of Campylobacter infections.

2-Hydroxylation of Acinetobacter baumannii Lipid A Contributes to Virulence.

Acinetobacter baumannii causes a wide range of nosocomial infections. This pathogen is considered a threat to human health due to the increasingly frequent isolation of multidrug-resistant strains. There is a major gap in knowledge on the infection biology of A. baumannii, and only a few virulence factors have been characterized, including lipopolysaccharide. The lipid A expressed by A. baumannii is hepta-acylated and contains 2-hydroxylaurate. The late acyltransferases controlling the acylation of lipid A have been already characterized. Here, we report the characterization of A. baumannii LpxO, which encodes the enzyme responsible for the 2-hydroxylation of lipid A. By genetic methods and mass spectrometry, we demonstrate that LpxO catalyzes the 2-hydroxylation of the laurate transferred by A. baumannii LpxL. LpxO-dependent lipid A 2-hydroxylation protects A. baumannii from polymyxin B, colistin, and human ß-defensin 3. LpxO contributes to the survival of A. baumannii in human whole blood and is required for pathogen survival in the waxmoth Galleria mellonella LpxO also protects Acinetobacter from G. mellonella antimicrobial peptides and limits their expression. Further demonstrating the importance of LpxO-dependent modification in immune evasion, 2-hydroxylation of lipid A limits the activation of the mitogen-activated protein kinase Jun N-terminal protein kinase to attenuate inflammatory responses. In addition, LpxO-controlled lipid A modification mediates the production of the anti-inflammatory cytokine interleukin-10 (IL-10) via the activation of the transcriptional factor CREB. IL-10 in turn limits the production of inflammatory cytokines following A. baumannii infection. Altogether, our studies suggest that LpxO is a candidate for the development of anti-A. baumannii drugs.