Search

Staphylococcus aureus is one of the clinically most relevant pathogens causing infections. Humans are often exposed to S. aureus. In approximately one-third of the healthy population it can be found on the skin either for long or short periods as colonizing “commensals”, without inducing infections or an inflammatory immune response. While tolerating S. aureus seems to be limited to certain individuals and time periods in most cases, Staphylococcus epidermidis is tolerated permanently on the skin of almost all individuals without activating overwhelming skin inflammation. To investigate this, we cocultured a keratinocyte cell line (HaCaT) with viable S. aureus or S. epidermidis to study the differences in the immune activation. S. aureus activated keratinocytes depicted by a profound IL-6 and IL-8 response, whereas S. epidermidis did not. Our data indicate that internalization of S. aureus and the subsequent intracellular sensing of bacterial nucleic acid may be essential for initiating inflammatory response in keratinocytes. Internalized dsRNA activates IL-6 and IL-8 release, but not TNF-α or IFNs by human keratinocytes. This is a non-specific effect of dsRNA, which can be induced using Poly(I:C), as well as RNA from S. aureus and S. epidermidis. However, only viable S. aureus were able to induce this response as these bacteria and not S. epidermidis were actively internalized by HaCaT. The stimulatory effect of S. aureus seems to be independent of the TLR3, -7 and -8 pathways. [ABSTRACT FROM AUTHOR]

Background: Cefiderocol is a novel siderophore cephalosporin active against MDR Gram-negative bacilli, including MBL-harbouring Enterobacterales. The detection of multiple cefiderocol-resistant blaVIM-carrying Enterobacterales isolates (MIC = 4 mg/L) from a single patient suggested an additional, potentially transferable, resistance determinant as blaVIM typically does not elevate cefiderocol MIC above the resistance threshold.Methods: Transfer of a mobile genetic element was performed in liquid mating experiments. All donor isolates and transconjugants were characterized by short-read WGS to identify potential resistance determinants. mRNA expression of siderophore receptors was determined by quantitative RT-PCR. Validation was performed by transformation. Antibiotic susceptibility was determined by broth microdilution.Results: Liquid mating experiments indicated the presence of transferable resistance determinants. Comparative genomic analysis of the clinical isolates and their respective transconjugants revealed the transfer of an accessory fec operon (fecABCDEIR). Transformation of the fec operon-containing vector into a TOP10 Escherichia coli led to an elevation of the cefiderocol MIC by at least 16-fold. Higher expression of fecA as a proxy for the fec operon mRNA expression was associated with phenotypic cefiderocol resistance. Both VIM and the accessory fec operon contribute to the elevation of cefiderocol MIC beyond the resistance threshold. The acquisition of an accessory fec operon via liquid mating confers phenotypic cefiderocol resistance in both E. coli J53 and Pseudomonas aeruginosa PAO1, indicating a broad-host-range nature of this mobile resistance determinant.Conclusions: The emergence of a transferable cefiderocol resistance determinant without prior exposure to the substance is worrisome and should be monitored closely. [ABSTRACT FROM AUTHOR]

Background Infectious complications are a major cause of morbidity and mortality after kidney transplantation. Methods In this transplant cohort study at the German Center of Infectious Diseases (DZIF), we evaluated all infections occurring during the first year after renal transplantation. We assessed microbial etiology, incidence rates, and temporal occurrence of these infections. Results Of 804 renal transplant recipients (65.2% male, 51 ± 14 years), 439 (54.6%) had 972 infections within the first year after transplantation. Almost half of these infections (47.8%) occurred within the first 3 months. Bacteria were responsible for 66.4% (645/972) of all infections, followed by viral (28.9% [281/972]) and fungal (4.7% [46/972]) pathogens. The urinary tract was the most common site of infection (42.4%). Enterococcus was the most frequently isolated bacterium (20.9%), followed by E. coli (17.6%) and Klebsiella (12.5%). E. coli was the leading pathogen in recipients <50 years of age, whereas Enterococcus predominated in older recipients. Resistant bacteria were responsible for at least 1 infection in 9.5% (76/804) of all recipients. Viral infections occurred in 201 recipients (25.0%). Of these, herpes viruses predominated (140/281 [49.8%]), and cytomegalovirus had the highest incidence rate (12.3%). In the 46 fungal infections, Candida albicans (40.8%) was the most commonly isolated. Other fungal opportunistic pathogens, including Aspergillus fumigatus and Pneumocystis , were rare. Conclusions Renal allograft recipients in Germany experience a high burden of infectious complications in the first year after transplantation. Bacteria were the predominating pathogen, followed by opportunistic infections such as cytomegalovirus. Microbial etiology varied between age groups, and resistant bacteria were identified in 10% of recipients. [ABSTRACT FROM AUTHOR]

The effects of low concentrations of extracellular ATP on cytosolic [Ca.sup.2+], membrane potential, and transcription of IL-6 were studied in monocyte-derived human macrophages. During inflammation or infection many cells secrete ATP. We show here that application of 10 [micro]M ATP or 10 [micro]M UTP induces oscillations in cytosolic [Ca.sup.2+] with a frequency of [approximately equal to] 12 [min.sup.-1] and oscillations in membrane potential. RT-PCR analysis showed expression of P2[Y.sub.1], P2[Y.sub.2], P2[Y.sub.11], P2[X.sub.1], P2[X.sub.4], and P2[X.sub.7] receptors, large-conductance (KCNMA1 and KCNMB1-4), and intermediate-conductance (KCNN4) [Ca.sup.2+]- activated [K.sup.+] channels. The [Ca.sup.2+] oscillations were unchanged after removal of extracellular [Ca.sup.2+], indicating that they were mainly due to movements of [Ca.sup.2+] between intracellular compartments. Comparison of the effects of different nucleotides suggests that the [Ca.sup.2+] oscillations were elicited by activation of P2[Y.sub.2] receptors coupled to phospholipase C. Patch-clamp experiments showed that ATP induced a transient depolarization, probably mediated by activation of P2[X.sub.4] receptors, followed by membrane potential oscillations due to opening of [Ca.sup.2+]- activated [K.sup.+] channels. We also found that 10 [micro]M ATP[gamma]S increased transcription of IL-6 [aproximately equal to] 40-fold within 2 h. This effect was abolished by blockade of P2Y receptors with 100 [micro]M suramin. Our results suggest that ATP released from inflamed, damaged, or metabolically impaired cells represents a 'danger signal' that plays a major role in activating the innate immune system.

The severe course of bloodstream infections with Gram-negative bacilli can lead to organ dysfunctions and compromise the integrity of the vascular barrier, which are the hallmarks of sepsis. This study aimed to investigate the potential effect of cefiderocol on the barrier function of vascular endothelial cells (vECs) in an in vitro experimental set-up. Human umbilical vein cells (HUVECs), co-cultured with erythrocyte-depleted whole blood for up to 48 h, were activated with tumor necrosis factor-alpha (TNF-α) or lipopolysaccharide (LPS) to induce endothelial damage in the absence or presence of cefiderocol (concentrations of 10, 40 and 70 mg/L). The endothelial integrity was quantified using transendothelial electrical resistance (TEER) measurement, performed at 0, 3, 24 and 48 h after stimulation. Stimulation with TNF-α and LPS increased the endothelial permeability assessed by TEER at 24 and 48 h of co-culture. Furthermore, cefiderocol reduces interleukin-6 (IL-6), interleukin-1β (IL-1β) and TNF-α release in peripheral blood mononuclear cells (PBMCs) following LPS stimulation in a dose-dependent manner. Collectively, the data suggest that cefiderocol may have an influence on the cellular immune response and might support the maintenance of vEC integrity during inflammation associated with infection with Gram-negative bacteria, which warrants further investigations. [ABSTRACT FROM AUTHOR]

We report a case of resistance development toward cefiderocol in a patient with intra-abdominal and bloodstream infections caused by carbapenemase-producing Enterobacter cloacae within 21 days of cefiderocol therapy. Whole genome sequencing revealed heterogeneous mutations in the cirA gene, encoding a catecholate siderophore receptor, conferring phenotypic resistance to cefiderocol. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus is one of the clinically most relevant pathogens causing infections. Humans are often exposed to S. aureus. In approximately one-third of the healthy population it can be found on the skin either for long or short periods as colonizing "commensals", without inducing infections or an inflammatory immune response. While tolerating S. aureus seems to be limited to certain individuals and time periods in most cases, Staphylococcus epidermidis is tolerated permanently on the skin of almost all individuals without activating overwhelming skin inflammation. To investigate this, we co-cultured a keratinocyte cell line (HaCaT) with viable S. aureus or S. epidermidis to study the differences in the immune activation. S. aureus activated keratinocytes depicted by a profound IL-6 and IL-8 response, whereas S. epidermidis did not. Our data indicate that internalization of S. aureus and the subsequent intracellular sensing of bacterial nucleic acid may be essential for initiating inflammatory response in keratinocytes. Internalized dsRNA activates IL-6 and IL-8 release, but not TNF-α or IFNs by human keratinocytes. This is a non-specific effect of dsRNA, which can be induced using Poly(I:C), as well as RNA from S. aureus and S. epidermidis. However, only viable S. aureus were able to induce this response as these bacteria and not S. epidermidis were actively internalized by HaCaT. The stimulatory effect of S. aureus seems to be independent of the TLR3, -7 and -8 pathways. [ABSTRACT FROM AUTHOR]

Persistent infections caused by Staphylococcus aureus remain a clinical challenge. Adaptational mechanisms of the pathogen influencing infection persistence, treatment success, and clinical outcome in these types of infections by S. aureus have not been fully elucidated so far. We applied a whole-genome sequencing approach on fifteen isolates retrieved from a persistent S. aureus infection to determine their genetic relatedness, virulome, and resistome. The analysis of the genomic data indicates that all isolates shared a common clonal origin but displayed a heterogenous composition of virulence factors and antimicrobial resistance. This heterogeneity was reflected by different mutations in the rpoB gene that were related to the phenotypic antimicrobial resistance towards rifampicin and different minimal inhibitory concentrations of oxacillin. In addition, one group of isolates had acquired the genes encoding for staphylokinase (sak) and staphylococcal complement inhibitor (scn), leading to the truncation of the hemolysin b (hlb) gene. These features are characteristic for temperate phages of S. aureus that carry genes of the immune evasion cluster and confer triple conversion by integration into the hlb gene. Modulation of immune evasion mechanisms was demonstrated by significant differences in biofilm formation capacity, while invasion and intracellular survival in neutrophils were not uniformly altered by the presence of the immune evasion cluster. Virulence factors carried by temperate phages of S. aureus may contribute to the course of infection at different stages and affect immune evasion and pathogen persistence. In conclusion, the application of comparative genomic demonstrated clonal heterogeneity in persistent S. aureus infection. [ABSTRACT FROM AUTHOR]

Bleeding associated with endothelial damage is a key feature of severe dengue fever. In the current study, we investigated whether Notch ligands were associated with bleeding in 115 patients with confirmed dengue infection in Vietnam. Soluble Notch ligands were determined by means of enzyme-linked immunosorbent assay. Seventeen of 115 patients (14.8%) experienced bleeding manifestations. High soluble delta-like ligand 1 (sDLL1) plasma levels was associated with bleeding (median, 15 674 vs 7117 pg/mL; P < .001). Receiver operating characteristic (ROC) curve analysis demonstrated that sDLL1 had the best test performance (area under the ROC curve, 0.852), with 88% sensitivity and 84% specificity. The combination with alanine aminotransferase and aspartate aminotransferase slightly increased sDLL1 performance. sDLL1 may be useful to guide clinical management of patients with patients in endemic settings. [ABSTRACT FROM AUTHOR]

Objectives: Increasing spread of resistance could jeopardize the use of antifolates against MRSA infections.Methods: We compared the prevalence of phenotypic trimethoprim/sulfamethoxazole resistance in 20 534 clinical Staphylococcus aureus isolates (19 096 MSSA and 1438 MRSA) of non-redundant patients at Heidelberg University Hospital over 8 years and performed WGS on trimethoprim/sulfamethoxazole-resistant MRSA.Results: From 2012 to 2019, trimethoprim/sulfamethoxazole resistance in MSSA (674/19 096; 3.5%) ranged between 1.5% and 7.2% and in MRSA (135/1438; 9.4%) between 0.5% and 20.2%, reaching a peak in 2016 and 2018, respectively (Ptrend < 0.001). Trimethoprim/sulfamethoxazole resistance was more likely in outpatients than inpatients (P = 0.005), younger patients (P < 0.001), skin and soft tissue infections (SSTIs) (MRSA only, P = 0.05), submissions from pulmonology (MRSA only, P = 0.001), the upper respiratory tract (MSSA only, P < 0.001) and general surgery (MSSA only, P = 0.001). WGS of 76 trimethoprim/sulfamethoxazole-resistant MRSA revealed that 59% belonged to major pandemic CA-MRSA clones (ST22, ST8, ST398, ST772, ST30), 47% harboured Panton-Valentine leucocidin (PVL), 97% SCCmec IV/V, 71% dfrG and 28% dfrA. SNP-based phylogeny of trimethoprim/sulfamethoxazole-resistant MRSA core genomes favoured independent introduction over clonal expansion as the source, most prominently of dfrA+ trimethoprim/sulfamethoxazole-resistant ST22 MRSA from the Gaza Strip.Conclusions: The presented results support that trimethoprim/sulfamethoxazole-resistant S. aureus, formerly associated with SSTI from outpatients and S. aureus in the (sub)tropics, is on the rise in the temperate zone, potentially due to migration. Closer monitoring of trimethoprim/sulfamethoxazole resistance in S. aureus is recommended to safeguard the effectiveness of antifolate compounds. [ABSTRACT FROM AUTHOR]

This document is a corrigendum published in the journal Frontiers in Immunology. It corrects an error in the published Figure 5 of the article titled "Inflammatory response against Staphylococcus aureus via intracellular sensing of nucleic acids in keratinocytes." The original figure was misleading due to the duplication of the negative control in the Western blot. The corrected figure has been provided, along with the original blots and raw data as supplementary files. The authors apologize for the error and state that it does not affect the scientific conclusions of the article. [Extracted from the article]

Vascular leakage associated with vascular endothelial cell (vEC) dysfunction is a hallmark of sepsis. Causative for the decreased integrity of the vascular endothelium (vE) is a complex concurrence of pathogen components, inflammation-associated host factors, and the interaction of vECs and activated circulating immune cells. One signaling pathway that regulates the integrity of the vE is the Notch cascade, which is activated through the binding of a Notch ligand to its respective Notch receptor. Recently, we showed that the soluble form of the Notch ligand Delta-like1 (sDLL1) is highly abundant in the blood of patients with sepsis. However, a direct connection between DLL1-activated Notch signaling and loss of vEC barrier function has not been addressed so far. To study the impact of infection-associated sDLL1, we used human umbilical vein cells (HUVEC) grown in a transwell system and cocultured with blood. Stimulation with sDLL1 induced activation as well as loss of endothelial tight structure and barrier function. Moreover, LPS-stimulated HUVEC activation and increase in endothelial cell permeability could be significantly decreased by blocking DLL1-receptor binding and Notch signaling, confirming the involvement of the cascade in LPS-mediated endothelial dysfunction. In conclusion, our results suggest that during bacterial infection and LPS recognition, DLL1-activated Notch signaling is associated with vascular permeability. This finding might be of clinical relevance in terms of preventing vascular leakage and the severity of sepsis. [ABSTRACT FROM AUTHOR]

Background: There is a growing interest in the rapid genotypic identification of antimicrobial resistance (AMR). In routine diagnostics, we detected multiple KPC-positive Escherichia coli (KPC-Ec) with discordant phenotypic meropenem susceptibility from a single patient's blood cultures, which prompted a more thorough investigation.Objectives: We investigated the potential clinical relevance of, and the mechanism behind, discordant phenotypic and genotypic meropenem susceptibility in KPC-Ec.Methods: WGS was used to perform a comparative analysis of the isolates' genetic characteristics and their blaKPC-2 locus. Expression of blaKPC-2 was determined by quantitative PCR and the potency of meropenem hydrolysis was determined using a semi-quantitative carbapenem inactivation method. An in vivo infection assay using Galleria mellonella was performed to assess the potential clinical relevance of KPC expression in E. coli.Results: Despite the presence of blaKPC-2, three of five isolates were susceptible to meropenem (MICVITEK2 ≤ 0.25 mg/L), while two isolates were resistant (MICVITEK2 ≥ 16 mg/L). The isolates with high MICs had significantly higher blaKPC-2 expression, which corresponds to phenotypic meropenem inactivation. The genetic environment of blaKPC-2, which may impact KPC production, was identical in all isolates. In vivo infection assay with G. mellonella suggested that meropenem was effective in reducing mortality following infection with low-expressing KPC-Ec.Conclusions: Our findings clearly highlight a limitation of genotypic AMR prediction for blaKPC. For the time being, genotypic AMR prediction requires additional analysis for accurate antibiotic therapy decision-making. [ABSTRACT FROM AUTHOR]

Abstract Background Lipopolysaccharide (LPS)-triggered Toll-like receptor (TLR) 4-signalling belongs to the key innate defence mechanisms upon infection with Gram-negative bacteria and triggers the subsequent activation of adaptive immunity. There is an active crosstalk between TLR4-mediated and other signalling cascades to secure an effective immune response, but also to prevent excessive inflammation. Many pathogens induce signalling cascades via secreted factors that interfere with TLR signalling to modify and presumably escape the host response. In this context heterotrimeric G proteins and their coupled receptors have been recognized as major cellular targets. Toxigenic strains of Gram-negative Pasteurella multocida produce a toxin (PMT) that constitutively activates the heterotrimeric G proteins Gαq, Gα13 and Gαi independently of G protein-coupled receptors through deamidation. PMT is known to induce signalling events involved in cell proliferation, cell survival and cytoskeleton rearrangement. Results Here we show that the activation of heterotrimeric G proteins through PMT suppresses LPS-stimulated IL-12p40 production and eventually impairs the T cell-activating ability of LPS-treated monocytes. This inhibition of TLR4-induced IL-12p40 expression is mediated by Gαi-triggered signalling as well as by Gβγ-dependent activation of PI3kinase and JNK. Taken together we propose the following model: LPS stimulates TLR4-mediated activation of the NFĸB-pathway and thereby the production of TNF-α, IL-6 and IL-12p40. PMT inhibits the production of IL-12p40 by Gαi-mediated inhibition of adenylate cyclase and cAMP accumulation and by Gβγ-mediated activation of PI3kinase and JNK activation. Conclusions On the basis of the experiments with PMT this study gives an example of a pathogen-induced interaction between G protein-mediated and TLR4-triggered signalling and illustrates how a bacterial toxin is able to interfere with the host’s immune response.

Abstract Background RhoH is a constitutively active member of the family of Rho GTPases. Its expression is restricted to the haematopoietic lineage, where it serves as a positive regulator for T cell selection and mast cell function and as a negative regulator for growth-related functions in other lineages. Here, we examined the activation of signal transducer and activator of transcription (STAT) proteins in response to stimulation with interleukin 3 (IL3). Results Using the murine IL3-dependent cell line BaF3 we investigated the influence of RhoH protein expression levels on IL3-mediated cellular responses. RhoH overexpressing cells showed lower sensitivity to IL3 and decreased STAT5 activation. SiRNA-mediated repression of RhoH gene expression led to an increase in proliferation and STAT5 activity which correlated with an increased number of IL3 receptor α chain molecules, also known as CD123, expressed at the cell surface. Interestingly, these findings could be reproduced using human THP-1 cells as a model system for acute myeloid leukaemia, where low RhoH levels are known to be an unfavourable prognostic marker. Overexpression of RhoH on the other hand caused an induction of STAT1 activity and western blot analysis revealed that activated STAT1 is phosphorylated on Tyr701. STAT1 is known to induce apoptosis or cell cycle arrest and we detected an upregulation of cyclin-dependent kinase inhibitors (CDKI) p21Cip1 and p27Kip1 in RhoH overexpressing BaF3 cells. Conclusions We propose that RhoH functions as a negative regulator for IL3-induced signals through modulation of the JAK-STAT pathway. High levels of RhoH allow the IL3-dependent activation of STAT1 causing decreased proliferation through upregulation of p21Cip1 and p27Kip1. Low RhoH levels on the other hand led to an upregulation of IL3-dependent cell growth, STAT5 activity and an increase of CD123 surface expression, linking RhoH to a CD123/STAT5 phenotype that has been described in AML patients.