Current status of antisense RNA-mediated gene regulation in Listeria monocytogenes

Listeria monocytogenes is a Gram-positive human-pathogen bacterium that served as an experimental model for investigating fundamental processes of adaptive immunity and virulence. Recent novel technologies allowed the identification of several hundred non-coding RNAs (ncRNAs) in the Listeria genome and provided insight into an unexpected complex transcriptional machinery. In this review, we discuss ncRNAs that are encoded on the opposite strand of the target gene and are therefore termed antisense RNAs (asRNAs). We highlight mechanistic and functional concepts of asRNAs in L. monocytogenes and put these in context of asRNAs in other bacteria. Understanding asRNAs will further broaden our knowledge of RNA-mediated gene regulation and may provide targets for diagnostic and antimicrobial development

A detailed view of the intracellular transcriptome of Listeria monocytogenes in murine macrophages using RNA-seq

Listeria monocytogenes is a bacterial pathogen and causative agent for the foodborne infection listeriosis, which is mainly a threat for pregnant, elderly or immunocompromised individuals. Due to its ability to invade and colonize diverse eukaryotic cell types including cells from invertebrates, L. monocytogenes has become a well-established model organism for intracellular growth. Almost ten years ago, we and others presented the first whole-genome microarray-based intracellular transcriptome of L. monocytogenes. With the advent of newer technologies addressing transcriptomes in greater detail, we revisit this work, and analyze the intracellular transcriptome of L. monocytogenes during growth in murine macrophages using a deep sequencing based approach.We detected 656 differentially expressed genes of which 367 were upregulated during intracellular growth in macrophages compared to extracellular growth in BHI. This study confirmed ~64% of all regulated genes previously identified by microarray analysis. Many of the regulated genes that were detected in the current study involve transporters for various metals, ions as well as complex sugars such as mannose. We also report changes in antisense transcription, especially upregulations during intracellular bacterial survival. A notable finding was the detection of regulatory changes for a subset of temperate A118-like prophage genes, thereby shedding light on the transcriptional profile of this bacteriophage during intracellular growth. In total, our study provides an updated genome-wide view of the transcriptional landscape of L. monocytogenes during intracellular growth and represents a rich resource for future detailed analysis

Zugriffszahlen auf den SARS-CoV-2-Hygieneplan am Universitätsklinikum Frankfurt am Main im Verlauf der COVID-19-Pandemie sowie Transmissionsraten und Ausbruchsgeschehen

Mit Beginn der COVID-19-Pandemie waren medizinische Einrichtungen in Deutschland vor das Problem gestellt, binnen kürzester Zeit spezifische SARS-CoV-2-Hygienepläne erstellen und in Kraft setzen zu müssen. Der Beitrag setzt den Zeitverlauf der Erstellung und die Frequenz der Änderungen des SARS-CoV-2-Hygieneplans am Universitätsklinikum Frankfurt am Main sowie die Zugriffszahlen der Beschäftigten auf die Hygieneplandokumente in Korrelation mit den an das RKI übermittelten SARS-CoV-2-Fallzahlen in Deutschland. Ebenfalls werden die Transmissionsraten sowie die stattgehabten Ausbruchsgeschehen im Universitätsklinikum im Zeitraum von Februar 2020 bis April 2022 dargestellt.Peer Reviewe

Genomic transmission analysis of multidrug-resistant Gram-negative bacteria within a newborn unit of a Kenyan tertiary hospital: A four-month prospective colonization study

Objective: Multidrug-resistant organisms (MDRO), especially carbapenem-resistant organisms (CRO), represent a threat for newborns. This study investigates the colonization prevalence of these pathogens in a newborn unit at a Kenyan tertiary hospital in an integrated approach combining routine microbiology, whole genome sequencing (WGS) and hospital surveillance data. Methods: The study was performed in the Kenyatta National Hospital (KNH) in 2019 over a four-month period and included 300 mother-baby pairs. A total of 1,097 swabs from newborns (weekly), mothers (once) and the hospital environment were taken. Routine clinical microbiology methods were applied for surveillance. Of the 288 detected MDRO, 160 isolates were analyzed for antimicrobial resistance genes and phylogenetic relatedness using whole genome sequencing (WGS) and bioinformatic analysis. Results: In maternal vaginal swabs, MDRO detection rate was 15% (n=45/300), including 2% CRO (n=7/300). At admission, MDRO detection rate for neonates was 16% (n=48/300), including 3% CRO (n=8/300) with a threefold increase for MDRO (44%, n=97/218) and a fivefold increase for CRO (14%, n=29/218) until discharge. Among CRO, K. pneumoniae harboring blaNDM-1 (n=20) or blaNDM-5 (n=16) were most frequent. WGS analysis revealed 20 phylogenetically related transmission clusters (including five CRO clusters). In environmental samples, the MDRO detection rate was 11% (n=18/164), including 2% CRO (n=3/164). Conclusion: Our study provides a snapshot of MDRO and CRO in a Kenyan NBU. Rather than a large outbreak scenario, data indicate several independent transmission events. The CRO rate among newborns attributed to the spread of NDM-type carbapenemases is worrisome

Listeria monocytogenes Induces a Virulence-Dependent microRNA Signature That Regulates the Immune Response in Galleria mellonella

microRNAs (miRNAs) coordinate several physiological and pathological processes by regulating the fate of mRNAs. Studies conducted in vitro indicate a role of microRNAs in the control of host-microbe interactions. However, there is limited understanding of miRNA functions in in vivo models of bacterial infections. In this study, we systematically explored changes in miRNA expression levels of Galleria mellonella larvae (greater-wax moth), a model system that recapitulates the vertebrate innate immunity, following infection with L. monocytogenes. Using an insect-specific miRNA microarray with more than 2000 probes, we found differential expression of 90 miRNAs (39 upregulated and 51 downregulated) in response to infection with L. monocytogenes. We validated the expression of a subset of miRNAs which have mammalian homologs of known or predicted function. In contrast, non-pathogenic L. innocua failed to induce these miRNAs, indicating a virulence-dependent miRNA deregulation. To predict miRNA targets using established algorithms, we generated a publically available G. mellonella transcriptome database. We identified miRNA targets involved in innate immunity, signal transduction and autophagy, including spätzle, MAP kinase, and optineurin, respectively, which exhibited a virulence-specific differential expression. Finally, in silico estimation of minimum free energy of miRNA-mRNA duplexes of validated microRNAs and target transcripts revealed a regulatory network of the host immune response to L. monocytogenes. In conclusion, this study provides evidence for a role of miRNAs in the regulation of the innate immune response following bacterial infection in a simple, rapid and scalable in vivo model that may predict host-microbe interactions in higher vertebrates

Streptobacillus moniliformis and IgM and IgG Immune Response in Patient with Endocarditis

We describe a case of endocarditis caused by Streptobacillus moniliformis bacteria, a known cause of rat-bite fever, in a 32-year-old woman with pet rats in Germany. The patient had a strong serologic response, with high IgM and IgG titers. Serologic analysis is a promising tool to identify S. moniliformis bacterial infection

Molecular surveillance of carbapenem-resistant gram-negative bacteria in liver transplant candidates

Background: Carbapenem-resistant Gram-negative bacteria (CRGN) cause life-threatening infections due to limited antimicrobial treatment options. The occurrence of CRGN is often linked to hospitalization and antimicrobial treatment but remains incompletely understood. CRGN are common in patients with severe illness (e.g., liver transplantation patients). Using whole-genome sequencing (WGS), we aimed to elucidate the evolution of CRGN in this vulnerable cohort and to reconstruct potential transmission routes. Methods: From 351 patients evaluated for liver transplantation, 18 CRGN isolates (from 17 patients) were analyzed. Using WGS and bioinformatic analysis, genotypes and phylogenetic relationships were explored. Potential epidemiological links were assessed by analysis of patient charts. Results: Carbapenem-resistant (CR) Klebsiella pneumoniae (n=9) and CR Pseudomonas aeruginosa (n=7) were the predominating pathogens. In silico analysis revealed that 14/18 CRGN did not harbor carbapenemase-coding genes, whereas in 4/18 CRGN, carbapenemases (VIM-1, VIM-2, OXA-232, and OXA-72) were detected. Among all isolates, there was no evidence of plasmid transfer-mediated carbapenem resistance. A close phylogenetic relatedness was found for three K. pneumoniae isolates. Although no epidemiological context was comprehensible for the CRGN isolates, evidence was found that the isolates resulted of a transmission of a carbapenem-susceptible ancestor before individual radiation into CRGN. Conclusion: The integrative epidemiological study reveals a high diversity of CRGN in liver cirrhosis patients. Mutation of carbapenem-susceptible ancestors appears to be the dominant way of CR acquisition rather than in-hospital transmission of CRGN or carbapenemase-encoding genetic elements. This study underlines the need to avoid transmission of carbapenem-susceptible ancestors in vulnerable patient cohorts

The secRNome of Listeria monocytogenes Harbors Small Noncoding RNAs That Are Potent Inducers of Beta Interferon.

Cellular sensing of bacterial RNA is increasingly recognized as a determinant of host-pathogen interactions. The intracellular pathogen Listeria monocytogenes induces high levels of type I interferons (alpha/beta interferons [IFN-α/β]) to create a growth-permissive microenvironment during infection. We previously demonstrated that RNAs secreted by L. monocytogenes (comprising the secRNome) are potent inducers of IFN-β. We determined the composition and diversity of the members of the secRNome and found that they are uniquely enriched for noncoding small RNAs (sRNAs). Testing of individual sRNAs for their ability to induce IFN revealed several sRNAs with this property. We examined ril32, an intracellularly expressed sRNA that is highly conserved for the species L. monocytogenes and that was the most potent inducer of IFN-β expression of all the sRNAs tested in this study, in more detail. The rli32-induced IFN-β response is RIG-I (retinoic acid inducible gene I) dependent, and cells primed with rli32 inhibit influenza virus replication. We determined the rli32 motif required for IFN induction. rli32 overproduction promotes intracellular bacterial growth, and a mutant lacking rli32 is restricted for intracellular growth in macrophages. rli32-overproducing bacteria are resistant to H2O2 and exhibit both increased catalase activity and changes in the cell envelope. Comparative transcriptome sequencing (RNA-Seq) analysis indicated that ril32 regulates expression of the lhrC locus, previously shown to be involved in cell envelope stress. Inhibition of IFN-β signaling by ruxolitinib reduced rli32-dependent intracellular bacterial growth, indicating a link between induction of the interferon system and bacterial physiology. rli32 is, to the best of our knowledge, the first secreted individual bacterial sRNA known to trigger the induction of the type I IFN response.IMPORTANCE Interferons are potent and broadly acting cytokines that stimulate cellular responses to nucleic acids of unusual structures or locations. While protective when induced following viral infections, the induction of interferons is detrimental to the host during L. monocytogenes infection. Here, we identify specific sRNAs, secreted by the bacterium, with the capacity to induce type I IFN. Further analysis of the most potent sRNA, rli32, links the ability to induce RIG-I-dependent induction of the type I IFN response to the intracellular growth properties of the bacterium. Our findings emphasize the significance of released RNA for Listeria infection and shed light on a compartmental strategy used by an intracellular pathogen to modulate host responses to its advantage

Comparative analysis of phenotypic and genotypic antibiotic susceptibility patterns in Mycobacterium avium complex

Objective: Phenotypic (Sensititre Myco, pDST) and genotypic drug susceptibility testing (GenoType NTM DR, gDST) in M. avium complex (MAC) have become available as standardized assays, but comparable data is needed. This study aimed to investigate the phenotypic and genotypic drug susceptibility patterns in MAC clinical isolates. Methods: Overall, 98 isolates from 85 patients were included. pDST and gDST were performed on all isolates and results compared regarding specificity and sensitivity using pDST as a reference method. The impact of drug instability on pDST results was studied using a biological assay over 14 days. In addition, the evolution of antimicrobial resistance was investigated in sequential isolates of 13 patients. Results: Macrolide resistance was rare, 1.2% (95% CI 0.7–7.3) of isolates in the base cohort. No aminoglycoside resistances were found, but 14.1% of the studied isolates (95% CI 7.8–23.8) showed intermediate susceptibility. The GenoType NTM DR identified two out of four macrolide-resistant isolates. Antibiotic stability was demonstrated to be poor in rifampicin, rifabutin, and doxycycylin. Conclusions: pDST results in NTM for unstable antibiotics must be interpreted with care. A combination of pDST and gDST will be useful for the guidance of antimicrobial therapy in MAC-disease

Colonization with multidrug-resistant organisms is associated with in increased mortality in liver transplant candidates.

ObjectivesRising prevalence of multidrug-resistant organisms (MDRO) is a major health problem in patients with liver cirrhosis. The impact of MDRO colonization in liver transplantation (LT) candidates and recipients on mortality has not been determined in detail.MethodsPatients consecutively evaluated and listed for LT in a tertiary German liver transplant center from 2008 to 2018 underwent screening for MDRO colonization including methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistant gram-negative bacteria (MDRGN), and vancomycin-resistant enterococci (VRE). MDRO colonization and infection status were obtained at LT evaluation, planned and unplanned hospitalization, three months upon graft allocation, or at last follow-up on the waiting list.ResultsIn total, 351 patients were listed for LT, of whom 164 (47%) underwent LT after a median of 249 (range 0-1662) days. Incidence of MDRO colonization increased during waiting time for LT, and MRDO colonization was associated with increased mortality on the waiting list (HR = 2.57, pConclusionsColonization with MDRO is associated with increased mortality on the waiting list, but not in short-term follow-up after LT. Moreover, colonization with CRGN seems associated with high mortality in liver transplant candidates and recipients