Molecular characteristics, fitness, and virulence of high-risk and non-high-risk clones of carbapenemase-producing Klebsiella pneumoniae.

Extensively drug-resistant (XDR) Klebsiella pneumoniae inflict a notable burden on healthcare worldwide. Of specific concern are strains producing carbapenem-hydrolyzing enzymes, as the therapeutic options for these strains are still very limited. Specific sequence types of K. pneumoniae have been noted for their epidemic occurrence globally, but the mechanisms behind the success of specific clones remain unclear. Herein, we have characterized 20 high-risk clones (HiRCs) and 10 non-HiRCs of XDR K. pneumoniae, exploring factors connected to the epidemiological success of some clones. Isolates were subjected to core genome multilocus sequence typing analysis to determine the clonal relationships of the isolates and subsequently characterized with regard to features known to be linked to overall bacterial fitness and virulence. The genomes were analyzed in silico for capsule types, O antigens, virulence factors, antimicrobial resistance genes, prophages, and CRISPR-Cas loci. In vitro growth experiments were conducted to retrieve proxies for absolute and relative fitness for 11 HiRC and 9 non-HiRC isolates selected based on the clonal groups they belonged to, and infections in a Galleria mellonella insect model were used to evaluate the virulence of the isolates in vivo. This study did not find evidence that virulence factors, prophages, CRISPR-Cas loci, or fitness measured in vitro alone would contribute to the global epidemiological success of specific clones of carbapenemase-producing XDR K. pneumoniae. However, this study did find the HiRC group to be more virulent than the non-HiRC group when measured in vivo in a model with G. mellonella. This suggests that the virulence and epidemiological success of certain clones of K. pneumoniae cannot be explained by individual traits investigated in this study and thus warrant further experiments in the future.IMPORTANCEHerein, we explored potential explanations for the successfulness of some epidemic or high-risk clones of carbapenemase-producing Klebsiella pneumoniae. We found differences in mortality in a larva model but found no clear genomic differences in known virulence markers. Most of the research on virulence in K. pneumoniae has been focused on hypervirulent strains, but here, we try to understand differences within the group of highly resistant strains. The results from the larva virulence model could be used to design experiments in higher animals. Moreover, the data could provide further support to a differentiated infection control approach against extensively drug-resistant strains, based on their classification as high-risk clones.

Identification and characterization of plasmids carrying the mobile colistin resistance gene mcr-1 using optical DNA mapping.

Objectives: Colistin is a last-resort antibiotic, but there has been a rapid increase in colistin resistance, threatening its use in the treatment of infections with carbapenem-resistant Enterobacterales (CRE). Plasmid-mediated colistin resistance, in particular the mcr-1 gene, has been identified and WGS is the go-to method in identifying plasmids carrying mcr-1 genes. The goal of this study is to demonstrate the use of optical DNA mapping (ODM), a fast, efficient and amplification-free technique, to characterize plasmids carrying mcr-1. Methods: ODM is a single-molecule technique, which we have demonstrated can be used for identifying plasmids harbouring antibiotic resistance genes. We here applied the technique to plasmids isolated from 12 clinical Enterobacterales isolates from patients at a major hospital in Thailand and verified our results using Nanopore long-read sequencing. Results: We successfully identified plasmids encoding the mcr-1 gene and, for the first time, demonstrated the ability of ODM to identify resistance gene sites in small (∼30 kb) plasmids. We further identified bla CTX-M genes in different plasmids than the ones encoding mcr-1 in three of the isolates studied. Finally, we propose a cut-and-stretch assay, based on similar principles, but performed using surface-functionalized cover slips for DNA immobilization and an inexpensive microscope with basic functionalities, to identify the mcr-1 gene in a plasmid sample. Conclusions: Both ODM and the cut-and-stretch assay developed could be very useful in identifying plasmids encoding antibiotic resistance in hospitals and healthcare facilities. The cut-and-stretch assay is particularly useful in low- and middle-income countries, where existing techniques are limited.

Four SARS-CoV-2 Genome Sequences from Late April in Stockholm, Sweden, Reveal a Rare Mutation in the Spike Protein.

Here, we report four coding-complete severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome sequences from Stockholm, Sweden, sampled in late April 2020. A rare variant at bp 23463 of the SARS-CoV-2 genome was found, which corresponds to the S1 subunit of the spike protein, changing an arginine (R) residue to histidine (H).

Cathelicidins Modulate TLR-Activation and Inflammation.

Cathelicidins are short cationic peptides that are part of the innate immune system. At first, these peptides were studied mostly for their direct antimicrobial killing capacity, but nowadays they are more and more appreciated for their immunomodulatory functions. In this review, we will provide a comprehensive overview of the various effects cathelicidins have on the detection of damage- and microbe-associated molecular patterns, with a special focus on their effects on Toll-like receptor (TLR) activation. We review the available literature based on TLR ligand types, which can roughly be divided into lipidic ligands, such as LPS and lipoproteins, and nucleic-acid ligands, such as RNA and DNA. For both ligand types, we describe how direct cathelicidin-ligand interactions influence TLR activation, by for instance altering ligand stability, cellular uptake and receptor interaction. In addition, we will review the more indirect mechanisms by which cathelicidins affect downstream TLR-signaling. To place all this information in a broader context, we discuss how these cathelicidin-mediated effects can have an impact on how the host responds to infectious organisms as well as how these effects play a role in the exacerbation of inflammation in auto-immune diseases. Finally, we discuss how these immunomodulatory activities can be exploited in vaccine development and cancer therapies.

Extended-Spectrum-ß-Lactamase- and Plasmid AmpC-Producing Escherichia coli Causing Community-Onset Bloodstream Infection: Association of Bacterial Clones and Virulence Genes with Septic Shock, Source of Infection, and Recurrence.

Invasive infections due to extended-spectrum-ß-lactamase- and pAmpC-producing Escherichia coli (ESBL/pAmpC-EC) are an important cause of morbidity, often caused by the high-risk clone sequence type (ST131) and isolates classified as extraintestinal pathogenic E. coli (ExPEC). The relative influence of host immunocompetence versus microbiological virulence factors in the acquisition and outcome of bloodstream infections (BSI) is poorly understood. Herein, we used whole-genome sequencing on 278 blood culture isolates of ESBL/pAmpC-EC from 260 patients with community-onset BSI collected from 2012 to 2015 in Stockholm to study the association of virulence genes, sequence types, and antimicrobial resistance with severity of disease, infection source, ESBL/pAmpC-EC BSI low-risk patients, and patients with repeated episodes. ST131 subclade C2 comprised 29% of all patients. Factors associated with septic shock in multivariable analysis were patient host factors (hematologic cancer or transplantation and reduced daily living activity), presence of the E. coli virulence factor iss (increased serum survival), absence of phenotypic multidrug resistance, and absence of the genes pap and hsp Adhesins, particularly pap, were associated with urinary tract infection (UTI) source, while isolates from post-prostate biopsy sepsis had a low overall number of virulence operons, including adhesins, and commonly belonged to ST131 clades A, B, and subclade C1, ST1193, and ST648. ST131 was associated with recurrent episodes. In conclusion, the most interesting finding is the association of iss with septic shock. Adhesins are important for UTI pathogenesis, while otherwise low-pathogenic isolates from the microbiota can cause post-prostate biopsy sepsis.

Studies on citrullinated LL-37: detection in human airways, antibacterial effects and biophysical properties.

Arginine residues of the antimicrobial peptide LL-37 can be citrullinated by peptidyl arginine deiminases, which reduce the positive charge of the peptide. Notably, citrullinated LL-37 has not yet been detected in human samples. In addition, functional and biophysical properties of citrullinated LL-37 are not fully explored. The aim of this study was to detect citrullinated LL-37 in human bronchoalveolar lavage (BAL) fluid and to determine antibacterial and biophysical properties of citrullinated LL-37. BAL fluid was obtained from healthy human volunteers after intra-bronchial exposure to lipopolysaccharide. Synthetic peptides were used for bacterial killing assays, transmission electron microscopy, isothermal titration calorimetry, mass-spectrometry and circular dichroism. Using targeted proteomics, we were able to detect both native and citrullinated LL-37 in BAL fluid. The citrullinated peptide did not kill Escherichia coli nor lysed human red blood cells. Both peptides had similar α-helical secondary structures but citrullinated LL-37 was more stable at higher temperatures, as shown by circular dichroism. In conclusion, citrullinated LL-37 is present in the human airways and citrullination impaired bacterial killing, indicating that a net positive charge is important for antibacterial and membrane lysing effects. It is possible that citrullination serves as a homeostatic regulator of AMP-function by alteration of key functions.

Klebsiella pneumoniae Expressing VIM-1 Metallo-ß-Lactamase Is Resensitized to Cefotaxime via Thiol-Mediated Zinc Chelation.

Antibiotic-resistant Klebsiella pneumoniae isolates constitute a great clinical challenge. One important resistance mechanism in K. pneumoniae is the metallo-ß-lactamases (MBLs), which require zinc for their function. Thus, zinc chelation could be a strategy to resensitize K. pneumoniae to ß-lactams. However, the potential role for endogenous zinc chelators for this purpose remains to be explored. The aim was to search for endogenous factors that could resensitize MBL-expressing K. pneumoniae to cefotaxime (CTX). Clinical K. pneumoniae isolates expressing different MBLs were screened for sensitivity to CTX in supernatants from human HT-29 colonic epithelial cells. Factors influencing CTX susceptibility were isolated and identified with chromatographic and biochemical methods. Free zinc was measured with a Zinquin assay, the thiol content was assessed with a fluorometric thiol assay, and the reducing ability of the supernatant was measured with a fluorescent l-cystine probe. Urine samples from healthy volunteers were used to validate findings ex vivo VIM-1-expressing K. pneumoniae regained susceptibility to CTX when grown in supernatants from HT-29 cells. This effect was mediated via free thiols in the supernatant, including l-cysteine, and could be prevented by inhibiting thioredoxin reductase activity in the supernatant. Free thiols in urine samples appeared to have a similar function in restoring CTX activity against VIM-1-expressing K. pneumoniae in a zinc-dependent manner. We have identified l-cysteine as an endogenous zinc chelator resulting in the resensitization of VIM-1-expressing K. pneumoniae to CTX. These results suggest that natural zinc chelators in combination with conventional antibiotics could be used to treat infections caused by VIM-1-expressing pathogens.

Innate lymphoid cell type 3-derived interleukin-22 boosts lipocalin-2 production in intestinal epithelial cells via synergy between STAT3 and NF-κB.

Escherichia coli and Klebsiella pneumoniae are opportunistic pathogens that are commonly associated with infections at mucosal surfaces, such as the lung or the gut. The host response against these types of infections includes the release of epithelial-derived antimicrobial factors such as lipocalin-2 (LCN-2), a protein that specifically inhibits the iron acquisition of Enterobacteriaceae by binding and neutralizing the bacterial iron-scavenging molecule enterobactin. Regulation of epithelial antimicrobial responses, including the release of LCN-2, has previously been shown to depend on IL-22, a cytokine produced by innate lymphoid cells type 3 (ILC3) during Enterobacteriaceae infections. However, much remains unknown about the extent to which antimicrobial responses are regulated by IL-22 and how IL-22 regulates the expression and production of LCN-2 in intestinal epithelial cells (IECs). Our study demonstrates how IL-22-induced activation of STAT3 synergizes with NF-κB-activating cytokines to enhance LCN-2 expression in human IECs and elucidates how ILC3 are involved in LCN-2-mediated host defense against Enterobacteriaceae. Together, these results provide new insight into the role of ILC3 in regulating LCN-2 expression in human IECs and could prove useful in future studies aimed at understanding the host response against Enterobacteriaceae as well as for the development of antimicrobial therapies against Enterobacteriaceae-related infections.

Imaging the Antistaphylococcal Activity of CATH-2: Mechanism of Attack and Regulation of Inflammatory Response.

Chicken cathelicidin-2 (CATH-2) is a broad-spectrum antimicrobial host defense peptide (HDP) that may serve as a paradigm for the development of new antimicrobial agents. While previous studies have elucidated the mechanism by which CATH-2 kills Escherichia coli, its mode of action against Gram-positive bacteria remains to be determined. In this study, we explored the underlying antibacterial mechanism of CATH-2 against a methicillin-resistant strain of Staphylococcus aureus and the effect of CATH-2-mediated S. aureus killing on immune activation. Visualization of the antimicrobial activity of CATH-2 against S. aureus with live-imaging confocal microscopy demonstrated that CATH-2 directly binds the bacteria, which is followed by membrane permeabilization and cell shrinkage. Transmission electron microscopy (TEM) studies further showed that CATH-2 initiated pronounced morphological changes of the membrane (mesosome formation) and ribosomal structures (clustering) in a dose-dependent manner. Immunolabeling of these sections demonstrated that CATH-2 binds and passes the bacterial membrane at subminimal bactericidal concentrations (sub-MBCs). Furthermore, competition assays and isothermal titration calorimetry (ITC) analysis provided evidence that CATH-2 directly interacts with lipoteichoic acid and cardiolipin. Finally, stimulation of macrophages with S. aureus and CATH-2 showed that CATH-2 not only kills S. aureus but also has the potential to limit S. aureus-induced inflammation at or above the MBC. Taken together, it is concluded that at sub-MBCs, CATH-2 perturbs the bacterial membrane and subsequently enters the cell and binds intracellular S. aureus components, while at or above the MBC, CATH-2 causes disruption of membrane integrity and inhibits S. aureus-induced macrophage activation. IMPORTANCE Due to the high use of antibiotics in both human and veterinary settings, many bacteria have become resistant to those antibiotics that we so heavily rely on. Methicillin-resistant S. aureus (MRSA) is one of these difficult-to-treat resistant pathogens for which novel antimicrobial therapies will be required in the near future. One novel approach could be the utilization of naturally occurring antimicrobial peptides, such as chicken CATH-2, which have been show to act against a wide variety of bacteria. However, before these peptides can be used clinically, more knowledge of their functions and mechanisms of action is required. In this study, we used live imaging and electron microscopy to visualize in detail how CATH-2 kills S. aureus, and we investigated how CATH-2 affects immune activation by S. aureus. Together, these results give a better understanding of how CATH-2 kills S. aureus and what the potential immunological consequences of this killing can be.

Killing of Pseudomonas aeruginosa by Chicken Cathelicidin-2 Is Immunogenically Silent, Preventing Lung Inflammation In Vivo.

The development of antibiotic resistance by Pseudomonas aeruginosa is a major concern in the treatment of bacterial pneumonia. In the search for novel anti-infective therapies, the chicken-derived peptide cathelicidin-2 (CATH-2) has emerged as a potential candidate, with strong broad-spectrum antimicrobial activity and the ability to limit inflammation by inhibiting Toll-like receptor 2 (TLR2) and TLR4 activation. However, as it is unknown how CATH-2 affects inflammation in vivo, we investigated how CATH-2-mediated killing of P. aeruginosa affects lung inflammation in a murine model. First, murine macrophages were used to determine whether CATH-2-mediated killing of P. aeruginosa reduced proinflammatory cytokine production in vitro Next, a murine lung model was used to analyze how CATH-2-mediated killing of P. aeruginosa affects neutrophil and macrophage recruitment as well as cytokine/chemokine production in the lung. Our results show that CATH-2 kills P. aeruginosa in an immunogenically silent manner both in vitro and in vivo Treatment with CATH-2-killed P. aeruginosa showed reduced neutrophil recruitment to the lung as well as inhibition of cytokine and chemokine production, compared to treatment with heat- or gentamicin-killed bacteria. Together, these results show the potential for CATH-2 as a dual-activity antibiotic in bacterial pneumonia, which can both kill P. aeruginosa and prevent excessive inflammation.

Cathelicidins Inhibit Escherichia coli-Induced TLR2 and TLR4 Activation in a Viability-Dependent Manner.

Activation of the immune system needs to be tightly regulated to provide protection against infections and, at the same time, to prevent excessive inflammation to limit collateral damage to the host. This tight regulation includes regulating the activation of TLRs, which are key players in the recognition of invading microbes. A group of short cationic antimicrobial peptides, called cathelicidins, have previously been shown to modulate TLR activation by synthetic or purified TLR ligands and may play an important role in the regulation of inflammation during infections. However, little is known about how these cathelicidins affect TLR activation in the context of complete and viable bacteria. In this article, we show that chicken cathelicidin-2 kills Escherichia coli in an immunogenically silent fashion. Our results show that chicken cathelicidin-2 kills E. coli by permeabilizing the bacterial inner membrane and subsequently binds the outer membrane-derived lipoproteins and LPS to inhibit TLR2 and TLR4 activation, respectively. In addition, other cathelicidins, including human, mouse, pig, and dog cathelicidins, which lack antimicrobial activity under cell culture conditions, only inhibit macrophage activation by nonviable E. coli In total, this study shows that cathelicidins do not affect immune activation by viable bacteria and only inhibit inflammation when bacterial viability is lost. Therefore, cathelicidins provide a novel mechanism by which the immune system can discriminate between viable and nonviable Gram-negative bacteria to tune the immune response, thereby limiting collateral damage to the host and the risk for sepsis.

Antimicrobial and Immunomodulatory Activity of PMAP-23 Derived Peptides.

INTRODUCTION: The Porcine Myeloid Antibacterial Peptide (PMAP)-23 is a porcine host defence peptide with strong antibacterial activity against Gram-positive and Gram-negative bacteria, and fungi. OBJECTIVE: PMAP-23 and truncated/mutated derivatives were tested for antibacterial and immunomodulatory activities to determine core elements of the peptide required for functionality. METHODS: PMAP-23 and truncated and/or mutated derivatives were synthesized. Antibacterial activity against Gram positive and negative bacteria was determined using colony counting assays. Cytotoxicity was measured against red blood cells and epithelial cells. Peptide induced cytokine production of epithelial cells was determined by ELISA. LPS neutralization was measured using isothermal titration calorimetry and inhibition of LPS induced cytokine production by macrophages. The effect of peptides on phagocytosis was performed by measuring uptake of fluorescently labelled beads by porcine macrophages. RESULTS: Truncation of the peptide did not lead to a strong reduction in antibacterial activity, but interestingly, all C-terminal truncated forms were strongly inhibited by salt addition, unlike the full length peptide or the two N-terminally truncated peptides. None of the peptides were hemolytic or toxic in concentrations up to 40 µM. Full length PMAP-23 induced IL-8 production in porcine epithelial cells, however, this activity was lost in all truncated peptides. None of the peptides bound LPS and subsequently did not inhibit LPS-induced cytokine production of monocytes. Finally, all PMAP-23 derived peptides reduced the uptake of beads by freshly isolated monocytes. CONCLUSION: PMAP-23 is mainly antibacterial with only limited immunomodulating capacity; the full length peptide is required for the full spectrum of activities.

Interspecies cathelicidin comparison reveals divergence in antimicrobial activity, TLR modulation, chemokine induction and regulation of phagocytosis.

Cathelicidins are short cationic peptides initially described as antimicrobial peptides, which can also modulate the immune system. Because most findings have been described in the context of human LL-37 or murine CRAMP, or have been investigated under varying conditions, it is unclear which functions are cathelicidin specific and which functions are general cathelicidin properties. This study compares 12 cathelicidins from 6 species under standardized conditions to better understand the conservation of cathelicidin functions. Most tested cathelicidins had strong antimicrobial activity against E. coli and/or MRSA. Interestingly, while more physiological culture conditions limit the antimicrobial activity of almost all cathelicidins against E. coli, activity against MRSA is enhanced. Seven out of 12 cathelicidins were able to neutralize LPS and another 7 cathelicidins were able to neutralize LTA; however, there was no correlation found with LPS neutralization. In contrast, only 4 cathelicidins enhanced DNA-induced TLR9 activation. In conclusion, these results provide new insight in the functional differences of cathelicidins both within and between species. In addition, these results underline the importance not to generalize cathelicidin functions and indicates that caution should be taken in extrapolating results from LL-37- or CRAMP-related studies to other animal settings.

Imaging the antimicrobial mechanism(s) of cathelicidin-2.

Host defence peptides (HDPs) have the potential to become alternatives to conventional antibiotics in human and veterinary medicine. The HDP chicken cathelicidin-2 (CATH-2) has immunomodulatory and direct killing activities at micromolar concentrations. In this study the mechanism of action of CATH-2 against Escherichia coli (E. coli) was investigated in great detail using a unique combination of imaging and biophysical techniques. Live-imaging with confocal fluorescence microscopy demonstrated that FITC-labelled CATH-2 mainly localized at the membrane of E. coli. Upon binding, the bacterial membrane was readily permeabilized as was shown by propidium iodide influx into the cell. Concentration- and time-dependent effects of the peptide on E. coli cells were examined by transmission electron microscopy (TEM). CATH-2 treatment was found to induce dose-dependent morphological changes in E. coli. At sub-minimal inhibitory concentrations (sub-MIC), intracellular granulation, enhanced vesicle release and wrinkled membranes were observed, while membrane breakage and cell lysis occurred at MIC values. These effects were visible within 1-5 minute of peptide exposure. Immuno-gold TEM showed CATH-2 binding to bacterial membranes. At sub-MIC values the peptide rapidly localized intracellularly without visible membrane permeabilization. It is concluded that CATH-2 has detrimental effects on E. coli at concentrations that do not immediately kill the bacteria.

Importance of Endosomal Cathelicidin Degradation To Enhance DNA-Induced Chicken Macrophage Activation.

Cathelicidins are essential in the protection against invading pathogens through both their direct antimicrobial activity and their immunomodulatory functions. Although cathelicidins are known to modulate activation by several TLR ligands, little is known about their influence on DNA-induced macrophage activation. In this study, we explored the effects of cathelicidins on DNA-induced activation of chicken macrophages and elucidated the intracellular processes underlying these effects. Our results show that chicken cathelicidin (CATH)-2 strongly enhances DNA-induced activation of both chicken and mammalian macrophages because of enhanced endocytosis of DNA-CATH-2 complexes. After endocytosis, DNA is liberated from the complex because of proteolytic breakdown of CATH-2, after which TLR21 is activated. This leads to increased cytokine expression and NO production. Through the interaction with DNA, CATH-2 can play an important role in modulating the immune response at sites of infection. These observations underline the importance of cathelicidins in sensing bacterial products and regulating immune responses.

Avian host defense peptides.

Host defense peptides (HDPs) are important effector molecules of the innate immune system of vertebrates. These antimicrobial peptides are also present in invertebrates, plants and fungi. HDPs display broad-spectrum antimicrobial activities and fulfill an important role in the first line of defense of many organisms. It is becoming increasingly clear that in the animal kingdom the functions of HDPs are not confined to direct antimicrobial actions. Research in mammals has indicated that HDPs have many immunomodulatory functions and are also involved in other physiological processes ranging from development to wound healing. During the past five years our knowledge about avian HDPs has increased considerably. This review addresses our current knowledge on the evolution, regulation and biological functions of HDPs of birds.