Salmonella Typhimurium impairs glycolysis-mediated acidification of phagosomes to evade macrophage defense.

Regulation of cellular metabolism is now recognized as a crucial mechanism for the activation of innate and adaptive immune cells upon diverse extracellular stimuli. Macrophages, for instance, increase glycolysis upon stimulation with pathogen-associated molecular patterns (PAMPs). Conceivably, pathogens also counteract these metabolic changes for their own survival in the host. Despite this dynamic interplay in host-pathogen interactions, the role of immunometabolism in the context of intracellular bacterial infections is still unclear. Here, employing unbiased metabolomic and transcriptomic approaches, we investigated the role of metabolic adaptations of macrophages upon Salmonella enterica serovar Typhimurium (S. Typhimurium) infections. Importantly, our results suggest that S. Typhimurium abrogates glycolysis and its modulators such as insulin-signaling to impair macrophage defense. Mechanistically, glycolysis facilitates glycolytic enzyme aldolase A mediated v-ATPase assembly and the acidification of phagosomes which is critical for lysosomal degradation. Thus, impairment in the glycolytic machinery eventually leads to decreased bacterial clearance and antigen presentation in murine macrophages (BMDM). Collectively, our results highlight a vital molecular link between metabolic adaptation and phagosome maturation in macrophages, which is targeted by S. Typhimurium to evade cell-autonomous defense.

TRIM21 Is Targeted for Chaperone-Mediated Autophagy during Salmonella Typhimurium Infection.

Salmonella enterica serovar Typhimurium (S Typhimurium) is a Gram-negative bacterium that induces cell death of macrophages as a key virulence strategy. We have previously demonstrated that the induction of macrophage death is dependent on the host's type I IFN (IFN-I) response. IFN-I signaling has been shown to induce tripartite motif (TRIM) 21, an E3 ubiquitin ligase with critical functions in autoimmune disease and antiviral immunity. However, the importance and regulation of TRIM21 during bacterial infection remains poorly understood. In this study, we investigated the role of TRIM21 upon S Typhimurium infection of murine bone marrow-derived macrophages. Although Trim21 expression was induced in an IFN-I-dependent manner, we found that TRIM21 levels were mainly regulated posttranscriptionally. Following TLR4 activation, TRIM21 was transiently degraded via the lysosomal pathway by chaperone-mediated autophagy (CMA). However, S Typhimurium-induced mTORC2 signaling led to phosphorylation of Akt at S473, which subsequently impaired TRIM21 degradation by attenuating CMA. Elevated TRIM21 levels promoted macrophage death associated with reduced transcription of NF erythroid 2-related factor 2 (NRF2)-dependent antioxidative genes. Collectively, our results identify IFN-I-inducible TRIM21 as a negative regulator of innate immune responses to S Typhimurium and a previously unrecognized substrate of CMA. To our knowledge, this is the first study reporting that a member of the TRIM family is degraded by the lysosomal pathway.

Leptin signaling impairs macrophage defenses against Salmonella Typhimurium.

The dynamic interplay between metabolism and immune responses in health and disease, by which different immune cells impact on metabolic processes, are being increasingly appreciated. However, the potential of master regulators of metabolism to control innate immunity are less understood. Here, we studied the cross-talk between leptin signaling and macrophage function in the context of bacterial infections. We found that upon infection with Gram-negative pathogens, such as Salmonella Typhimurium, leptin receptor (Lepr) expression increased in both mouse and human macrophages. Unexpectedly, both genetic Lepr ablation in macrophages and global pharmacologic leptin antagonization augmented lysosomal functions, reduced S Typhimurium burden, and diminished inflammation in vitro and in vivo. Mechanistically, we show that leptin induction activates the mTORC2/Akt pathway and subsequently down-regulates Phlpp1 phosphatase, allowing for phosphorylated Akt to impair lysosomal-mediated pathogen clearance. These data highlight a link between leptin signaling, the mTORC2/Phlpp1/Akt axis, and lysosomal activity in macrophages and have important therapeutic implications for modulating innate immunity to combat Gram-negative bacterial infections.

Salmonella Typhimurium disrupts Sirt1/AMPK checkpoint control of mTOR to impair autophagy.

During intracellular infections, autophagy significantly contributes to the elimination of pathogens, regulation of pro-inflammatory signaling, secretion of immune mediators and in coordinating the adaptive immune system. Intracellular pathogens such as S. Typhimurium have evolved mechanisms to circumvent autophagy. However, the regulatory mechanisms targeted by S. Typhimurium to modulate autophagy have not been fully resolved. Here we report that cytosolic energy loss during S. Typhimurium infection triggers transient activation of AMPK, an important checkpoint of mTOR activity and autophagy. The activation of AMPK is regulated by LKB1 in a cytosolic complex containing Sirt1 and LKB1, where Sirt1 is required for deacetylation and subsequent activation of LKB1. S. Typhimurium infection targets Sirt1, LKB1 and AMPK to lysosomes for rapid degradation resulting in the disruption of the AMPK-mediated regulation of mTOR and autophagy. The degradation of cytosolic Sirt1/LKB1/AMPK complex was not observed with two mutant strains of S. Typhimurium, ΔssrB and ΔssaV, both compromising the pathogenicity island 2 (SPI2). The results highlight virulence factor-dependent degradation of host cell proteins as a previously unrecognized strategy of S. Typhimurium to evade autophagy.

The health and economic burden of bloodstream infections caused by antimicrobial-susceptible and non-susceptible Enterobacteriaceae and Staphylococcus aureus in European hospitals, 2010 and 2011: a multicentre retrospective cohort study.

We performed a multicentre retrospective cohort study including 606,649 acute inpatient episodes at 10 European hospitals in 2010 and 2011 to estimate the impact of antimicrobial resistance on hospital mortality, excess length of stay (LOS) and cost. Bloodstream infections (BSI) caused by third-generation cephalosporin-resistant Enterobacteriaceae (3GCRE), meticillin-susceptible (MSSA) and -resistant Staphylococcus aureus (MRSA) increased the daily risk of hospital death (adjusted hazard ratio (HR) = 1.80; 95% confidence interval (CI): 1.34-2.42, HR = 1.81; 95% CI: 1.49-2.20 and HR = 2.42; 95% CI: 1.66-3.51, respectively) and prolonged LOS (9.3 days; 95% CI: 9.2-9.4, 11.5 days; 95% CI: 11.5-11.6 and 13.3 days; 95% CI: 13.2-13.4, respectively). BSI with third-generation cephalosporin-susceptible Enterobacteriaceae (3GCSE) significantly increased LOS (5.9 days; 95% CI: 5.8-5.9) but not hazard of death (1.16; 95% CI: 0.98-1.36). 3GCRE significantly increased the hazard of death (1.63; 95% CI: 1.13-2.35), excess LOS (4.9 days; 95% CI: 1.1-8.7) and cost compared with susceptible strains, whereas meticillin resistance did not. The annual cost of 3GCRE BSI was higher than of MRSA BSI. While BSI with S. aureus had greater impact on mortality, excess LOS and cost than Enterobacteriaceae per infection, the impact of antimicrobial resistance was greater for Enterobacteriaceae.

Investigation of sequential outbreaks of Burkholderia cepacia and multidrug-resistant extended spectrum ß-lactamase producing Klebsiella species in a West African tertiary hospital neonatal unit: a retrospective genomic analysis.

BACKGROUND: Sick newborns admitted to neonatal units in low-resource settings are at an increased risk of developing hospital-acquired infections due to poor clinical care practices. Clusters of infection, due to the same species, with a consistent antibiotic resistance profile, and in the same ward over a short period of time might be indicative of an outbreak. We used whole-genome sequencing (WGS) to define the transmission pathways and characterise two distinct outbreaks of neonatal bacteraemia in a west African neonatal unit. METHODS: We studied two outbreaks of Burkholderia cepacia and multidrug-resistant extended spectrum ß-lactamase (ESBL)-producing Klebsiella pneumoniae in a neonatal unit that provides non-intensive care on the neonatal ward in the Edward Francis Small Teaching Hospital, Banjul, The Gambia. We used WGS to validate and expand findings from the outbreak investigation. We retrospectively sequenced all clinical isolates associated with each outbreak, including isolates obtained from swabs of ward surfaces, environmental fluid cultures, intravenous fluids, and antibiotics administered to newborns. We also sequenced historical B cepacia isolates associated with neonatal sepsis in the same ward. RESULTS: Between March 1 and Dec 31, 2016, 321 blood cultures were done, of which 178 (55%) were positive with a clinically significant isolate. 49 episodes of neonatal B cepacia bacteraemia and 45 episodes of bacteraemia due to ESBL-producing K pneumoniae were reported. WGS revealed the suspected K pneumoniae outbreak to be contemporaneous outbreaks of K pneumoniae (ST39) and previously unreported Klebsiella quasipneumoniae subspecies similipneumoniae (ST1535). Genomic analysis showed near-identical strain clusters for each of the three outbreak pathogens, consistent with transmission within the neonatal ward from extrinsically contaminated in-use intravenous fluids and antibiotics. Time-dated phylogeny, including retrospective analysis of archived bacterial strains, suggest B cepacia has been endemic in the neonatal ward over several years, with the Klebsiella species a more recent introduction. INTERPRETATION: Our study highlights the emerging threat of previously unreported strains of multidrug-resistant Klebsiella species in this neonatal unit. Genome-based surveillance studies can improve identification of circulating pathogen strains, characterisation of antimicrobial resistance, and help understand probable infection acquisition routes during outbreaks in newborn units in low-resource settings. Our data provide evidence for the need to regularly monitor endemic transmission of bacteria within the hospital setting, identify the introduction of resistant strains from the community, and improve clinical practices to reduce or prevent the spread of infection and resistance. FUNDING: Medical Research Council Unit The Gambia at the London School of Hygiene & Tropical Medicine, Fajara, The Gambia.

Surgical Implications for Diagnosis and Treatment of Intestinal Aspergillosis in Pediatric Patients with ALL.

BACKGROUND: The incidence of invasive aspergillosis (IA) in children with hematooncological malignancies is increasing as a result of intensive treatment, immunosuppression, and extended use of broad-spectrum antibiotics. Infection of the GI tract by Aspergillus spp. is a rare and fatal complication, which often requires surgical diagnostic and therapeutic exploration. OBJECTIVE: The aim of this study was to determine the characteristics of symptomatic intestinal aspergillosis, diagnosis, treatment, and outcome of pediatric patients with an underlying hemato-oncologic disease. PATIENTS AND METHODS: We analyzed 2,307 German patients with acute lymphoblastic leukemia (ALL) from age 1 to 17 years registered in the AIEOP-BFM ALL 2000 study from 2000 to 2006. All reported adverse events were assessed for symptoms of IA and retrospectively reviewed for any sign or proof of intestinal involvement of IA. RESULTS: In this cohort, IA was reported in 30 of 2,307 patients while intestinal involvement was documented in five patients. Four of these patients had intestinal symptoms and three patients underwent explorative laparotomy. Among clinical cases with IA, gastrointestinal manifestation of IA mostly occurred in adolescent patients (10-16 years). Symptoms varied from abdominal tenderness and pain to constipation. Intestinal aspergillosis was proven by microbiological and histopathological examination and fungal infection was observed macroscopically in the jejunal lumen during surgery. Despite the extended surgery and antifungal therapy, outcome of disseminated IA with intestinal involvement remains poor. CONCLUSION: Surgeons should be aware of surgical complications of intestinal aspergillosis in children with hematooncological diseases requiring exploration and resection. IA is a rare event and still difficult to diagnose due to unspecific abdominal symptoms. Thus, biopsy sampling is of utmost importance to ensure diagnosis, and resection of necrotic or perforated tissue should be attempted early.

Type I interferon enhances necroptosis of Salmonella Typhimurium-infected macrophages by impairing antioxidative stress responses.

Salmonella enterica serovar Typhimurium exploits the host's type I interferon (IFN-I) response to induce receptor-interacting protein (RIP) kinase-mediated necroptosis in macrophages. However, the events that drive necroptosis execution downstream of IFN-I and RIP signaling remain elusive. In this study, we demonstrate that S Typhimurium infection causes IFN-I-mediated up-regulation of the mitochondrial phosphatase Pgam5 through RIP3. Pgam5 subsequently interacts with Nrf2, which sequesters Nrf2 in the cytosol, thereby repressing the transcription of Nrf2-dependent antioxidative genes. The impaired ability to respond to S Typhimurium-induced oxidative stress results in reactive oxygen species-mediated mitochondrial damage, energy depletion, transient induction of autophagy, and autophagic degradation of p62. Reduced p62 levels impair interaction of p62 with Keap1, which further decreases Nrf2 function and antioxidative responses to S Typhimurium infection, eventually leading to cell death. Collectively, we identify impaired Nrf2-dependent redox homeostasis as an important mechanism that promotes cell death downstream of IFN-I and RIP3 signaling in S Typhimurium-infected macrophages.

Determining vancomycin Etest MICs in patients with MRSA bloodstream infection does not support switching antimicrobials.

OBJECTIVES: Elevated vancomycin minimum inhibitory concentrations (MIC) have been reported to adversely affect clinical outcome in methicillin-resistant Staphylococcus aureus (MRSA) bloodstream infection (BSI). We therefore examined the association between vancomycin MIC and outcome considering various potential confounders. METHODS: Clinical data and bacterial isolates were prospectively collected from patients with MRSA BSI from 2006 to 2012 as part of the Invasive Staphylococcus aureus Infection Cohort (INSTINCT) study. Antimicrobial susceptibility was assessed by Etest, broth microdilution (BMD) and VITEK 2. Bacterial genotypes were determined by spa typing. Using univariate and Cox regression analyses, we investigated the impact of low (≤1.0 mg/L) and high (≥1.5 mg/L) vancomycin Etest MIC on clinical outcomes. RESULTS: Ninety-one MRSA BSI episodes were included, of which 79 (86.8%) were caused by spa types t003, t032 and t045. High vancomycin MICs were seen only if using Etest but not confirmed using standard reference BMD. When episodes were stratified into low and high vancomycin Etest MIC groups, 30-day overall mortality was 34.5% and 27.3%, respectively (P = 0.64, OR 0.71; 95% confidence interval [CI] 0.27-1.79). Variables significantly associated with all-cause mortality in the Cox model were age (P = 0.003), acute physiology score (P = 0.0006), and Charlson comorbidity index (P = 0.018). CONCLUSIONS: Vancomycin MICs may vary dependent on testing methodologies and local MRSA epidemiology. The patients' underlying disease and individual comorbidities rather than elevated vancomycin MICs determine adverse clinical outcomes in MRSA BSI. Routine Etest MIC testing of MRSA isolates is of limited value for treatment decisions.

Amino acid alterations in fibronectin binding protein A (FnBPA) and bacterial genotype are associated with cardiac device related infection in Staphylococcus aureus bacteraemia.

OBJECTIVES: Staphylococcus aureus initiates cardiac device-related infection (CDI) by binding of fibronectin binding protein A (FnBPA) to the device's surface. In FnBPA, specific "binding enhancing" amino acid alterations are associated with CDI. However, no study has investigated whether these mutations also occur in geographically different regions and whether they arise during infection or are inherent properties of the infecting isolate. METHODS: We analysed bacterial isolates from 34 patients with S. aureus bacteraemia and implanted cardiac devices for association with CDI, FnBPA sequence, classification into a clonal complex (CC), and binding to fibronectin (Fn). RESULTS: We confirmed that amino acid alterations at positions 652, 782, and 786 in FnBPA were associated with CDI (p = 0.005). Furthermore, CC15 and CC45 isolates were associated with CDI (p = 0.004). All isolates within a CC exhibited a characteristic mutation pattern, with major changes occurring in CC45 including a duplication of D1 and an altered immunogenic epitope in the D3 repeat. Isolates harbouring the "binding enhancing" mutations showed a slightly increased Fn binding capability, whereas Fn binding was decreased in CC45 isolates, according to a microtiter plate assay. CONCLUSIONS: FnBPA sequence variations are lineage specific and display inherent properties of the infecting isolate. Sequence analysis of FnBPA, as well as the bacterial genotype, may be used to predict the risk for device-related infection.

Polymorphisms in Fibronectin Binding Proteins A and B among Staphylococcus aureus Bloodstream Isolates Are Not Associated with Arthroplasty Infection.

BACKGROUND: Nonsynonymous single nucleotide polymorphisms (SNPs) in fibronectin binding protein A (fnbA) of Staphylococcus aureus are associated with cardiac device infections. However, the role of fnbA SNPs in S. aureus arthroplasty infection is unknown. METHODS: Bloodstream S. aureus isolates from a derivation cohort of patients at a single U.S. medical center with S. aureus bacteremia (SAB) and prosthetic hip or knee arthroplasties that were infected (PJI, n = 27) or uninfected (PJU, n = 43) underwent sequencing of fnbA and fnbB. A validation cohort of S. aureus bloodstream PJI (n = 12) and PJU (n = 58) isolates from Germany also underwent fnbA and fnbB sequencing. RESULTS: Overall, none of the individual fnbA or fnbB SNPs were significantly associated with the PJI or PJU clinical groups within the derivation cohort. Similarly, none of the individual fnbA or fnbB SNPs were associated with PJI or PJU when the analysis was restricted to patients with either early SAB (i.e., bacteremia occurring <1 year after placement or manipulation of prostheses) or late SAB (i.e., bacteremia >1 year after placement or manipulation of prostheses). CONCLUSIONS: In contrast to cardiac device infections, there is no association between nonsynonymous SNPs in fnbA or fnbB of bloodstream S. aureus isolates and arthroplasty infection. These results suggest that initial steps leading to S. aureus infection of cardiovascular and orthopedic prostheses may arise by distinct processes.