The methyltransferase Setdb2 mediates virus-induced susceptibility to bacterial superinfection.

Immune responses are tightly regulated to ensure efficient pathogen clearance while avoiding tissue damage. Here we report that Setdb2 was the only protein lysine methyltransferase induced during infection with influenza virus. Setdb2 expression depended on signaling via type I interferons, and Setdb2 repressed expression of the gene encoding the neutrophil attractant CXCL1 and other genes that are targets of the transcription factor NF-κB. This coincided with occupancy by Setdb2 at the Cxcl1 promoter, which in the absence of Setdb2 displayed diminished trimethylation of histone H3 Lys9 (H3K9me3). Mice with a hypomorphic gene-trap construct of Setdb2 exhibited increased infiltration of neutrophils during sterile lung inflammation and were less sensitive to bacterial superinfection after infection with influenza virus. This suggested that a Setdb2-mediated regulatory crosstalk between the type I interferons and NF-κB pathways represents an important mechanism for virus-induced susceptibility to bacterial superinfection.

Cell-intrinsic regulation of phagocyte function by interferon lambda during pulmonary viral, bacterial super-infection.

Influenza infections result in a significant number of severe illnesses annually, many of which are complicated by secondary bacterial super-infection. Primary influenza infection has been shown to increase susceptibility to secondary methicillin-resistant Staphylococcus aureus (MRSA) infection by altering the host immune response, leading to significant immunopathology. Type III interferons (IFNs), or IFNλs, have gained traction as potential antiviral therapeutics due to their restriction of viral replication without damaging inflammation. The role of IFNλ in regulating epithelial biology in super-infection has recently been established; however, the impact of IFNλ on immune cells is less defined. In this study, we infected wild-type and IFNLR1-/- mice with influenza A/PR/8/34 followed by S. aureus USA300. We demonstrated that global IFNLR1-/- mice have enhanced bacterial clearance through increased uptake by phagocytes, which was shown to be cell-intrinsic specifically in myeloid cells in mixed bone marrow chimeras. We also showed that depletion of IFNLR1 on CX3CR1 expressing myeloid immune cells, but not neutrophils, was sufficient to significantly reduce bacterial burden compared to mice with intact IFNLR1. These findings provide insight into how IFNλ in an influenza-infected lung impedes bacterial clearance during super-infection and show a direct cell intrinsic role for IFNλ signaling on myeloid cells.

Heterotypic Influenza Infections Mitigate Susceptibility to Secondary Bacterial Infection.

Influenza-associated bacterial superinfections have devastating impacts on the lung and can result in increased risk of mortality. New strains of influenza circulate throughout the population yearly, promoting the establishment of immune memory. Nearly all individuals have some degree of influenza memory before adulthood. Due to this, we sought to understand the role of immune memory during bacterial superinfections. An influenza heterotypic immunity model was established using influenza A/Puerto Rico/8/34 and influenza A/X31. We report in this article that influenza-experienced mice are more resistant to secondary bacterial infection with methicillin-resistant Staphylococcus aureus as determined by wasting, bacterial burden, pulmonary inflammation, and lung leak, despite significant ongoing lung remodeling. Multidimensional flow cytometry and lung transcriptomics revealed significant alterations in the lung environment in influenza-experienced mice compared with naive animals. These include changes in the lung monocyte and T cell compartments, characterized by increased expansion of influenza tetramer-specific CD8+ T cells. The protection that was seen in the memory-experienced mouse model is associated with the reduction in inflammatory mechanisms, making the lung less susceptible to damage and subsequent bacterial colonization. These findings provide insight into how influenza heterotypic immunity reshapes the lung environment and the immune response to a rechallenge event, which is highly relevant to the context of human infection.

The Use of the Antigenically Variable Major Surface Protein 2 in the Establishment of Superinfection during Natural Tick Transmission of Anaplasma marginale in Southern Ghana.

Many vector-borne pathogens, including Anaplasma spp., Borrelia spp., Trypanosoma spp., and Plasmodium spp., establish persistent infection in the mammalian host by using antigenic variation. These pathogens are also able to establish strain superinfection, defined as infection of an infected host with additional strains of the same pathogen despite an adaptive immune response. The ability to establish superinfection results in a population of susceptible hosts even with high pathogen prevalence. It is likely that antigenic variation, responsible for persistent infection, also plays a role in the establishment of superinfection. Anaplasma marginale, an antigenically variable, obligate intracellular, tickborne bacterial pathogen of cattle, is well suited for the study of the role of antigenically variant surface proteins in the establishment of superinfection. Anaplasma marginale establishes persistent infection by variation in major surface protein 2 (msp2), which is encoded by approximately six donor alleles that recombine into a single expression site to produce immune escape variants. Nearly all cattle in regions of high prevalence are superinfected. By tracking the acquisition of strains in calves through time, the complement of donor alleles, and how those donor alleles are expressed, we determined that simple variants derived from a single donor allele, rather than multiple donor alleles, were predominant. Additionally, superinfection is associated with the introduction of new donor alleles, but these new donor alleles are not predominantly used to establish superinfection. These findings highlight the potential for competition among multiple strains of a pathogen for resources within the host and the balance between pathogen fitness and antigenic variation.

Loss of T-bet confers survival advantage to influenza-bacterial superinfection.

The transcription factor, T-bet, regulates type 1 inflammatory responses against a range of infections. Here, we demonstrate a previously unaddressed role of T-bet, to influenza virus and bacterial superinfection. Interestingly, we found that T-bet deficiency did not adversely affect the efficacy of viral clearance or recovery compared to wild-type hosts. Instead, increased infiltration of neutrophils and production of Th17 cytokines (IL-17 and IL-22), in lungs of influenza virus-infected T-bet-/- mice, were correlated with survival advantage against subsequent infection by Streptococcus pneumoniae Neutralization of IL-17, but not IL-22, in T-bet-/- mice increased pulmonary bacterial load, concomitant with decreased neutrophil infiltration and reduced survival of T-bet-/- mice. IL-17 production by CD8+, CD4+ and γδ T cell types was identified to contribute to this protection against bacterial superinfection. We further showed that neutrophil depletion in T-bet-/- lungs increased pulmonary bacterial burden. These results thus indicate that despite the loss of T-bet, immune defences required for influenza viral clearance are fully functional, which in turn enhances protective type 17 immune responses against lethal bacterial superinfections.

Synthetic gene-regulatory networks in the opportunistic human pathogen Streptococcus pneumoniae.

Streptococcus pneumoniae can cause disease in various human tissues and organs, including the ear, the brain, the blood, and the lung, and thus in highly diverse and dynamic environments. It is challenging to study how pneumococci control virulence factor expression, because cues of natural environments and the presence of an immune system are difficult to simulate in vitro. Here, we apply synthetic biology methods to reverse-engineer gene expression control in S. pneumoniae A selection platform is described that allows for straightforward identification of transcriptional regulatory elements out of combinatorial libraries. We present TetR- and LacI-regulated promoters that show expression ranges of four orders of magnitude. Based on these promoters, regulatory networks of higher complexity are assembled, such as logic AND gates and IMPLY gates. We demonstrate single-copy genome-integrated toggle switches that give rise to bimodal population distributions. The tools described here can be used to mimic complex expression patterns, such as the ones found for pneumococcal virulence factors. Indeed, we were able to rewire gene expression of the capsule operon, the main pneumococcal virulence factor, to be externally inducible (YES gate) or to act as an IMPLY gate (only expressed in absence of inducer). Importantly, we demonstrate that these synthetic gene-regulatory networks are functional in an influenza A virus superinfection murine model of pneumonia, paving the way for in vivo investigations of the importance of gene expression control on the pathogenicity of S. pneumoniae.

The prevalence of early- and late-onset bacterial, viral, and fungal respiratory superinfections in invasively ventilated COVID-19 patients.

The role of respiratory superinfections in patients with coronavirus disease 2019 (COVID-19) pneumonia remains unclear. We investigated the prevalence of early- and late-onset superinfections in invasively ventilated patients with COVID-19 pneumonia admitted to our department of intensive care medicine between March 2020 and November 2020. Of the 102 cases, 74 (72.5%) received invasive ventilation and were tested for viral, bacterial, and fungal pathogens on Days 0-7, 8-14, and 15-21 after the initiation of mechanical ventilation. Approximately 45% developed one or more respiratory superinfections. There was a clear correlation between the duration of invasive ventilation and the prevalence of coinfecting pathogens. Male patients with obesity and those suffering from chronic obstructive pulmonary disease and/or diabetes mellitus had a significantly higher probability to develop a respiratory superinfection. The prevalence of viral coinfections was high, with a predominance of the herpes simplex virus (HSV), followed by cytomegalovirus. No respiratory viruses or intracellular bacteria were detected in our cohort. We observed a high coincidence between Aspergillus fumigatus and HSV infection. Gram-negative bacteria were the most frequent pathogen group. Klebsiella aerogenes was detected early after intubation, while Klebsiella pneumoniae and Pseudomonas aeruginosa were related to a prolonged respiratory weaning.

Both Coinfection and Superinfection Drive Complex Anaplasma marginale Strain Structure in a Natural Transmission Setting.

Vector-borne pathogens commonly establish multistrain infections, also called complex infections. How complex infections are established, either before or after the development of an adaptive immune response, termed coinfection or superinfection, respectively, has broad implications for the maintenance of genetic diversity, pathogen phenotype, epidemiology, and disease control strategies. Anaplasma marginale, a genetically diverse, obligate, intracellular, tick-borne bacterial pathogen of cattle, commonly establishes complex infections, particularly in regions with high transmission rates. Both coinfection and superinfection can be established experimentally; however, it is unknown how complex infections develop in a natural transmission setting. To address this question, we introduced naive animals into a herd in southern Ghana with a high infection prevalence and high transmission pressure and tracked the strain acquisition of A. marginale through time using multilocus sequence typing. As expected, the genetic diversity among strains was high, and 97% of animals in the herd harbored multiple strains. All the introduced naive animals became infected, and three to four strains were typically detected in an individual animal prior to seroconversion, while one to two new strains were detected in an individual animal following seroconversion. On average, the number of strains acquired via superinfection was 16% lower than the number acquired via coinfection. Thus, while complex infections develop via both coinfection and superinfection, coinfection predominates in this setting. These findings have broad implications for the development of control strategies in high-transmission settings.

Rapid Spread and Control of Multidrug-Resistant Gram-Negative Bacteria in COVID-19 Patient Care Units.

We describe rapid spread of multidrug-resistant gram-negative bacteria among patients in dedicated coronavirus disease care units in a hospital in Maryland, USA, during May-June 2020. Critical illness, high antibiotic use, double occupancy of single rooms, and modified infection prevention practices were key contributing factors. Surveillance culturing aided in outbreak recognition and control.

Predictors of hospital-acquired bacterial and fungal superinfections in COVID-19: a prospective observational study.

BACKGROUND: Bacterial and fungal superinfections may complicate the course of hospitalized patients with COVID-19. OBJECTIVES: To identify predictors of superinfections in COVID-19. METHODS: Prospective, observational study including patients with COVID-19 consecutively admitted to the University Hospital of Pisa, Italy, between 4 March and 30 April 2020. Clinical data and outcomes were registered. Superinfection was defined as a bacterial or fungal infection that occurred ≥48 h after hospital admission. A multivariate analysis was performed to identify factors independently associated with superinfections. RESULTS: Overall, 315 patients with COVID-19 were hospitalized and 109 episodes of superinfections were documented in 69 (21.9%) patients. The median time from admission to superinfection was 19 days (range 11-29.75). Superinfections were caused by Enterobacterales (44.9%), non-fermenting Gram-negative bacilli (15.6%), Gram-positive bacteria (15.6%) and fungi (5.5%). Polymicrobial infections accounted for 18.3%. Predictors of superinfections were: intestinal colonization by carbapenem-resistant Enterobacterales (OR 16.03, 95% CI 6.5-39.5, P < 0.001); invasive mechanical ventilation (OR 5.6, 95% CI 2.4-13.1, P < 0.001); immunomodulatory agents (tocilizumab/baricitinib) (OR 5.09, 95% CI 2.2-11.8, P < 0.001); C-reactive protein on admission >7 mg/dl (OR 3.59, 95% CI 1.7-7.7, P = 0.001); and previous treatment with piperacillin/tazobactam (OR 2.85, 95% CI 1.1-7.2, P = 0.028). Length of hospital stay was longer in patients who developed superinfections ompared with those who did not (30 versus 11 days, P < 0.001), while mortality rates were similar (18.8% versus 23.2%, P = 0.445). CONCLUSIONS: The risk of bacterial and fungal superinfections in COVID-19 is consistent. Patients who need empiric broad-spectrum antibiotics and immunomodulant drugs should be carefully selected. Infection control rules must be reinforced.

Coinfections and their molecular consequences in the porcine respiratory tract.

Understudied, coinfections are more frequent in pig farms than single infections. In pigs, the term "Porcine Respiratory Disease Complex" (PRDC) is often used to describe coinfections involving viruses such as swine Influenza A Virus (swIAV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), and Porcine CircoVirus type 2 (PCV2) as well as bacteria like Actinobacillus pleuropneumoniae, Mycoplasma hyopneumoniae and Bordetella bronchiseptica. The clinical outcome of the various coinfection or superinfection situations is usually assessed in the studies while in most of cases there is no clear elucidation of the fine mechanisms shaping the complex interactions occurring between microorganisms. In this comprehensive review, we aimed at identifying the studies dealing with coinfections or superinfections in the pig respiratory tract and at presenting the interactions between pathogens and, when possible, the mechanisms controlling them. Coinfections and superinfections involving viruses and bacteria were considered while research articles including protozoan and fungi were excluded. We discuss the main limitations complicating the interpretation of coinfection/superinfection studies, and the high potential perspectives in this fascinating research field, which is expecting to gain more and more interest in the next years for the obvious benefit of animal health.

Effects of Influenza on Alveolar Macrophage Viability Are Dependent on Mouse Genetic Strain.

Secondary bacterial coinfections following influenza virus pose a serious threat to human health. Therefore, it is of significant clinical relevance to understand the immunological causes of this increased susceptibility. Influenza-induced alterations in alveolar macrophages (AMs) have been shown to be a major underlying cause of the increased susceptibility to bacterial superinfection. However, the mechanisms responsible for this remain under debate, specifically in terms of whether AMs are depleted in response to influenza infection or are maintained postinfection, but with disrupted phagocytic activity. The data presented in this article resolves this issue by showing that either mechanism can differentially occur in individual mouse strains. BALB/c mice exhibited a dramatic IFN-γ-dependent reduction in levels of AMs following infection with influenza A, whereas AM levels in C57BL/6 mice were maintained throughout the course of influenza infection, although the cells displayed an altered phenotype, namely an upregulation in CD11b expression. These strain differences were observed regardless of whether infection was performed with low or high doses of influenza virus. Furthermore, infection with either the H1N1 A/California/04/2009 (CA04) or H1N1 A/PR8/1934 (PR8) virus strain yielded similar results. Regardless of AM viability, both BALB/c and C57BL/6 mice showed a high level of susceptibility to postinfluenza bacterial infection. These findings resolve the apparent inconsistencies in the literature, identify mouse strain-dependent differences in the AM response to influenza infection, and ultimately may facilitate translation of the mouse model to clinical application.

Strong antimicrobial activity of collinin and isocollinin against periodontal and superinfectant pathogens in vitro.

Periodontitis pathogenesis involves activation of host immune responses triggered by microbial dysbiosis. Therefore, controlling periodontal pathogens in-vivo is a main goal of periodontal therapy. New antimicrobials might help to control periodontal infection and improve treatment outcomes at "the dark times" of increasing antibiotic resistance. Here, we determined the biological activity of collinin and isocollinin against 8 bacterial strains. Antimicrobial activity of collinin and isocollinin, chlorhexidine digluconate (CHX) and sodium hypochlorite (NaClO) was evaluated against clinically relevant periodontal bacteria, like Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Fusobacterium nucleatum, Prevotella intermedia, Dialister pneumosintes strains and superinfectants like Escherichia coli, Staphylococcusaureus, and Pseudomonasaeruginosa strains. A broth microdilution test was carried out to determine the minimum inhibitory concentration of collinin and isocollinin against those strains, and bacterial viability was determined by resazurin assay at diverse concentration and exposure times. P. gingivalis was the most susceptible strain to collinin and isocollinin (MIC 2.1 µg/mL and 4.2 µg/mL respectively). Other periodontal pathogens showed MICs <17 µg/mL for collinin and MICs between 20 and 42 µg/mL for isocollinin, whereas CHX and NaClO showed MICs of 62 and 326 µg/mL, respectively. Collinin and isocollinin also exhibited antimicrobial activity against superinfectant bacteria (MIC < 21 and < 42 µg/mL, respectively). Overall, collinin and isocollinin showed a remarkable antibacterial activity against relevant periodontal and superinfective bacteria, especially against P. gingivalis (MIC 2.1 µg/mL and 4.2 µg/mL respectively) and the highly virulent P. aeruginosa (MIC 5.2 and 20.8 µg/mL, respectively).

A self-perpetuating repressive state of a viral replication protein blocks superinfection by the same virus.

Diverse animal and plant viruses block the re-infection of host cells by the same or highly similar viruses through superinfection exclusion (SIE), a widely observed, yet poorly understood phenomenon. Here we demonstrate that SIE of turnip crinkle virus (TCV) is exclusively determined by p28, one of the two replication proteins encoded by this virus. p28 expressed from a TCV replicon exerts strong SIE to a different TCV replicon. Transiently expressed p28, delivered simultaneously with, or ahead of, a TCV replicon, largely recapitulates this repressive activity. Interestingly, p28-mediated SIE is dramatically enhanced by C-terminally fused epitope tags or fluorescent proteins, but weakened by N-terminal modifications, and it inversely correlates with the ability of p28 to complement the replication of a p28-defective TCV replicon. Strikingly, p28 in SIE-positive cells forms large, mobile punctate inclusions that trans-aggregate a non-coalescing, SIE-defective, yet replication-competent p28 mutant. These results support a model postulating that TCV SIE is caused by the formation of multimeric p28 complexes capable of intercepting fresh p28 monomers translated from superinfector genomes, thereby abolishing superinfector replication. This model could prove to be applicable to other RNA viruses, and offer novel targets for antiviral therapy.

Microorganisms associated with respiratory syncytial virus pneumonia in the adult population.

Respiratory syncytial virus (RSV) has been recognized as responsible for severe respiratory illness in adults, especially in the elderly. While pneumonia is commonly observed during RSV infection, the burden and epidemiology of bacterial superinfection is poorly understood. The aim of this study was to identify microorganisms associated with RSV-positive pneumonia in adults. A retrospective study was conducted during three consecutive winters (October to April 2013-2016) in the University Hospital of Lyon, France. During RSV circulation periods, a systematic RSV screening was performed by reverse-transcription PCR on all respiratory samples collected from adults. Records of RSV-positive patients were subsequently analyzed to identify radiologically confirmed pneumonia cases. Bacteria were identified by standard bacteriology cultures or urinary antigen screening and classified as potentially causative of pneumonia if quantification was above the specific threshold as defined by the European Manual of Clinical Microbiology. Overall, 14,792 adult respiratory samples were screened for RSV detection by PCR. In total, 292 had a positive RSV detection (2.0%) among which 89 presented with pneumonia including 27 bacterial superinfections (9.3%) with Streptococcus pneumonia, Haemophilus influenza, Staphylococcus aureus, Pseudomonas aeruginosa, and Moraxella catarrhalis. Most patients were elderly (55.6%) and patients with comorbidities (77.8%). A more severe outcome was observed for RSV-bacteria-associated pneumonia compared with RSV pneumonia: length of stay was significantly longer (16 days vs 10 days) and ICU hospitalization more frequent (66.7% vs 21.0%) (p < 0.05). In conclusion, we did not observe major differences in the epidemiology of bacterial superinfections in RSV-positive pneumonia compared to reports on post-influenza pneumonia.

Strongyloides stercoralis larvae or egg: Which came first?

Strongyloides stercoralis (SS) hyperinfection is a well-documented condition. However, SS eggs in stool samples are not commonly observed during routine analysis. Here, we report a case on SS hyperinfection where both larvae and eggs were observed in the stool sample of an immunossupressed liver allograft transplanted patient.

A unique presentation of superinfected pseudomyxoma peritonei secondary to a low-grade appendiceal mucinous neoplasm.

BACKGROUND: Pseudomyxoma peritonei (PMP) is an uncommon condition characterized by diffuse mucinous material in the abdomen and pelvis, generally arising from a perforated epithelial neoplasm. Typically, the disease presents as suspected acute appendicitis, ovarian mass, abdominal distension, or ventral hernia. Our case represents a very rare presentation of superinfected PMP. CASE PRESENTATION: A 46-year-old female with a past medical history notable for depression, asthma, and uterine leiomyomas presented to an urgent care with 5 days of progressive abdominal pain, bloating, nausea, and subjective fevers. The patient had a diffusely tender abdomen, without peritonitis, was mildly tachycardic, and had a white blood cell count of 15 K. A CT of the abdomen/pelvis was consistent with PMP with a ruptured appendiceal mucocele versus PMP secondary to an adnexal ovarian neoplastic pathology with an infectious component. The patient initially improved on antibiotics but ultimately required two surgeries, the first of which controlled intraabdominal sepsis while the second permitted definitive management of PMP with cytoreductive surgery (CRS) and HIPEC. CONCLUSION: Superinfected PMP is a rare entity with very few documented cases. A staged approach that incorporates clearing the peritoneal infection, with or without resection of the primary tumor, followed by rehabilitation and definitive surgery appears to be a safe and effective management strategy.

Keratocyst Capnocytophaga superinfection.

INTRODUCTION: Capnocytophaga spp are Gram-negative bacteria that cause severe infections in immunosuppressed patients. This situation is extremely rare in immunocompetent patients. CASE REPORT: This clinical report describes the unusual infection of an immunocompetent patient with Capnocytophaga spp. The imaging studies showed the existence of a cyst in the left jawbone. After treatment and a microbiological study of the content, it was found to be an outbreak of septicaemia. DISCUSSION: Capnocytophaga spp, commensal bacteria of the oral cavity, can lead to serious illness and that is why an empirical treatment is needed until a diagnostic confirmation can be obtained.

Establishment of a Wolbachia Superinfection in Aedes aegypti Mosquitoes as a Potential Approach for Future Resistance Management.

Wolbachia pipientis is an endosymbiotic bacterium estimated to chronically infect between 40-75% of all arthropod species. Aedes aegypti, the principle mosquito vector of dengue virus (DENV), is not a natural host of Wolbachia. The transinfection of Wolbachia strains such as wAlbB, wMel and wMelPop-CLA into Ae. aegypti has been shown to significantly reduce the vector competence of this mosquito for a range of human pathogens in the laboratory. This has led to wMel-transinfected Ae. aegypti currently being released in five countries to evaluate its effectiveness to control dengue disease in human populations. Here we describe the generation of a superinfected Ae. aegypti mosquito line simultaneously infected with two avirulent Wolbachia strains, wMel and wAlbB. The line carries a high overall Wolbachia density and tissue localisation of the individual strains is very similar to each respective single infected parental line. The superinfected line induces unidirectional cytoplasmic incompatibility (CI) when crossed to each single infected parental line, suggesting that the superinfection would have the capacity to replace either of the single constituent infections already present in a mosquito population. No significant differences in fitness parameters were observed between the superinfected line and the parental lines under the experimental conditions tested. Finally, the superinfected line blocks DENV replication more efficiently than the single wMel strain when challenged with blood meals from viremic dengue patients. These results suggest that the deployment of superinfections could be used to replace single infections and may represent an effective strategy to help manage potential resistance by DENV to field deployments of single infected strains.

Influenza Casts a Lung Shadow.

This commentary highlights the article by Pociask et al that provides new insights into the lingering effects of influenza infection.