Staphylococcus aureus Mutant Screen Reveals Interaction of the Human Antimicrobial Peptide Dermcidin with Membrane Phospholipids

Antimicrobial peptides (AMPs) form an important part of the innate host defense. In contrast to most AMPs, human dermcidin has an anionic net charge. To investigate whether bacteria have developed specific mechanisms of resistance to dermcidin, we screened for mutants of the leading human pathogen, Staphylococcus aureus, with altered resistance to dermcidin. To that end, we constructed a plasmid for use in mariner-based transposon mutagenesis and developed a high-throughput cell viability screening method based on luminescence. In a large screen, we did not find mutants with strongly increased susceptibility to dermcidin, indicating that S. aureus has no specific mechanism of resistance to this AMP. Furthermore, we detected a mutation in a gene of unknown function that resulted in significantly increased resistance to dermcidin. The mutant strain had an altered membrane phospholipid pattern and showed decreased binding of dermcidin to the bacterial surface, indicating that dermcidin interacts with membrane phospholipids. The mode of this interaction was direct, as shown by assays of dermcidin binding to phospholipid preparations, and specific, as the resistance to other AMPs was not affected. Our findings indicate that dermcidin has an exceptional value for the human innate host defense and lend support to the idea that it evolved to evade bacterial resistance mechanisms targeted at the cationic character of most AMPs. Moreover, they suggest that the antimicrobial activity of dermcidin is dependent on the interaction with the bacterial membrane and might thus assist with the determination of the yet unknown mode of action of this important human AMP.Antimicrobial peptides (AMPs) form an important part of the innate host defense in neutrophil phagosomes, where they contribute to the elimination of ingested bacteria, and on epithelia, where they control the proliferation of commensal and invading microorganisms (9). Many bacteria have developed mechanisms of resistance to AMPs, such as inactivation of AMPs by secreted proteases, the repulsion or sequestration of AMPs by surface polymers, and the expulsion of AMPs from the cytoplasmic membrane (19). Electrostatic forces govern many AMP-bacterium interactions, and frequently, bacterial mechanisms of resistance specifically target the typical cationic character of AMPs (19). For example, the introduction of positive charges in bacterial surface polymers lowers the overall negative charge of the bacterial surface and minimizes the attraction of AMPs to their target.As a result of the long interaction during evolution, hosts have invented ways to circumvent bacterial resistance, such as by stabilizing AMPs via intramolecular bridges or by increasing the density of positive charges to increase the interaction with the negatively charged bacterial surface (23). The active processed forms of the human AMP dermcidin, DCD-1 and DCD-1L, represent peptides with noticeable alternatives to these strategies aimed at circumventing bacterial resistance, inasmuch as in these peptides, the typical cationic charge of the AMPs is replaced by a negative net charge (26). One clear advantage of the anionic character of dermcidin is the ability to evade resistance mechanisms that specifically target cationic AMPs. Possibly for that reason, dermcidin is a main effector of the innate host defense against bacterial pathogens, particularly those on human skin (25). While dermcidin is likely subject to nonspecific resistance mechanisms, such as degradation by secreted proteases (13, 28), it is not known whether specific bacterial resistance mechanisms are targeted at dermcidin. Furthermore, the anionic character comes with the price that the electrostatic attraction to the bacterial surface, which is considered crucial for antimicrobial activity, is mechanistically challenging. Previous research has established that dermcidin binds to the bacterial surface (29), but the molecular basis of binding and the bactericidal mechanism of dermcidin are unknown.In the present study, we aimed to identify the bacterial molecules involved in resistance to dermcidin. To that end, we chose Staphylococcus aureus as a model organism. Staphylococci are part of the normal microflora on the human skin and mucous membranes, where dermcidin is predominantly produced (24, 26). Furthermore, they are important pathogens and thus have a particularly essential need for protection from AMPs in their natural habitat and during infection (15, 31). We developed a luminescence-based assay to screen for mutants with altered resistance to dermcidin. Furthermore, we constructed a plasmid for use in mariner-based transposon mutagenesis to avoid the reported integration bias of the previously used transposon, Tn917 (1). Notably, we did not detect mutants with strongly increased susceptibility to dermcidin and no mutations in genes with a previously established function in antimicrobial resistance. These findings lend support to the idea that dermcidin is an evolutionary adaptation of the human innate immune system formed to evade bacterial resistance mechanisms. In addition, we detected an S. aureus mutant with significantly increased resistance to dermcidin, and characterization of that mutant suggested that the antimicrobial activity of dermcidin is dependent on a specific interaction with bacterial membrane phospholipids.     

Development of banana (Musa balbisiana) pseudo stem fiber as a surgical bio-tool to avert post-operative wound infections

The search to develop an ideal suture material encourages us to explore novel suture biomaterials with superior characteristics to the current commercially available products. Surgical sutures play a crucial role in the development of post-operative wound infection by acting as a substrate for biofilm formation which leads to dehisced wounds. In this context, the present invention meets this need by fabricating banana (Musa balbisiana) fibre into an advanced antimicrobials releasing suture biomaterial (BSc) for the prevention of post-operative wound infection. Suture material developed from banana pseudo stem fiber was impregnated with chloramphenicol, clotrimazole and growth factors with the aid of a hydro-gel system. The fabricated suture material was found to be biocompatible towards human erythrocytes and L929 mouse fibroblast cells. BSc exhibited promising physico-chemical characteristics which were comparable to the commercially available Bombyx mori silk fibroin (BMSF) suture. BSc displayed a biphasic release pattern with sustained release of chloramphenicol for up to 140 h. Apart from being environment friendly and having a facile fabrication method, this advanced suture biomaterial showed broad spectrum in vitro antimicrobial activity against bacterial and fungal pathogens. BSc successfully impeded biofilm formation on its surface, as is evident from the confocal microscopy analysis. This contributes to superior wound healing efficacy in terms of reduced microbial burden and a subsequent decrease in the inflammatory cytokine levels. Histopathological observations further supported the pronounced healing efficacy of BSc sutured wounds. The findings of this study establish the banana pseudo stem fiber as a novel advanced suture biomaterial to prevent post-operative wound infections.

A novel antimicrobial suture biomaterial developed from banana waste fibers to avert post operative wound infections.     

A novel method to prepare tussah/Bombyx mori silk fibroin-based films

The possibility of using silk fibroin in biomaterials for tissue engineering is a subject of broad interest. In this study, Bombyx mori/tussah silk fibroin (BSF/TSF) blend films were prepared by solution casting using CaCl₂/formic acid as a co-solvent and water as a rinse solvent. The morphology, crystallinity, thermal resistance, mechanical properties and water contact angle of the blend films as well as the biocompatibility were investigated. The BSF/TSF blend films displayed a smooth surface and specific nanostructure in their cross-section, originating from the nanofibril-preservation during fibroin dissolution. The water rinse process induced the formation of a stable β-sheet structure. The BSF film showed superior mechanical properties to the TSF film, and the blending with TSF led to a significant reduction in the strength and elasticity of blend films. However, adding the TSF component could regulate the hydrophilic properties and enhance cell growth on the blend films. The BSF/TSF blend films with specific nanostructure, stable secondary structure, appropriate mechanical properties, and good biocompatibility, are promising candidates for application in regenerative medicine.

A novel method is reported to prepare tussah/Bombyx mori silk fibroin blend films featured transparent, flexible and biocompatible.     

Prospects for antimicrobial peptide-based immunotherapy approaches in Leishmania control

Introduction: Leishmaniasis is one of the neglected tropical diseases and is highly endemic in many countries. Currently, there is no adequate human vaccine and treatment to control the disease.

Areas covered: As a result of the failure of chemotherapy and toxicity, it is necessary to find another approach for the treatment of leishmaniasis. Recently, antimicrobial peptides (AMPs), originating from natural resources, have attracted much attention for their use as a new antibiotics for many infectious and noninfectious diseases. Natural AMPs are named interchangeably as host defense peptides. They are naturally active in the innate immune system as a primary defense mechanism in most species all over the world. Several AMPs have been tested in in vitro and in vivo experiments against leishmaniasis.

Expert commentary: Most AMPs require proper conformation to be active. Leishmania (L.) tarentolae as a nonpathogenic strain, is an effective tool not only for vaccine development but also for therapy. Recombinant L. tarentolae expressing selective or combined AMPs is a suggestive approach for leishmaniasis or any other infectious disease treatment.     

Phosphorus Solubilization and Plant Growth Promotion Ability of Rhizobacteria of <Emphasis Type="Italic">R. communis</Emphasis> L Growing in Assam,India

Castor (Ricinus communis L) is the primary host plant of eri silkworm and its rhizosphere harbours diverse group of microbial community with biofertilizer potentiality. Phosphate solubilizing bacteria (PSB) render available phosphate (P) in agricultural soil by P mineralization process through enzyme mediated reaction. In search for PSB strains, 15 castor rhizobacteria were isolated and characterized for morphological and biochemical properties. The isolates were screened in vitro for P solubilization efficiency both qualitatively and quantitatively. Isolate MAJ PSB12 produced highest soluble P concentration (322.20 µmol/l) in National Botanical Research Institute Phosphate medium after 96 h of incubation with a maximum drop in pH to 5.4 from 7.0. Among the isolates, maximum content of IAA (24.6 mg/l) and GA₃ (3.921 mg/l) was also found to be produced by the same strain. The most potential isolate was identified as Bacillus firmus MAJ PSB12 by 16S rRNA gene homology analysis and the sequence was submitted to National Centre for Biotechnology Information GenBank. Although many species belonging to the genus Bacillus are efficient P solubilizer, application of native rhizobacteria is easier for adaptation and succession during biofertilization process. B. firmus MAJ PSB12 can be utilized as potential biofertilizer to promote sustainable castor cultivation in sericulture for upliftment of rural livelihood.     

The bitter taste of infection

The human innate immune response to pathogens is complex, and it has been difficult to establish the contribution of epithelial signaling in the prevention of upper respiratory tract infection. The prevalence of chronic sinusitis in the absence of systemic immune defects indicates that there may be local defects in innate immunity associated with such mucosal infections. In this issue of the JCI, Cohen and colleagues investigate the role of the bitter taste receptors in airway epithelial cells, and find that these are critical to sensing the presence of invading pathogens. The participation of respiratory mucosal epithelial cells in innate immune defense has been increasingly appreciated. Not only do airway cells express the full complement of pattern recognition receptors and corresponding adaptor proteins to signal the recruitment of professional immune cells in response to perceived infection, they also participate directly in pathogen eradication. Mucociliary clearance is activated in response to bacterial components, and bacterial killing is mediated through epithelial production of NO and antimicrobial peptides. Although major defects in ciliary function (e.g., Kartagener syndrome) are clearly associated with increased respiratory infection rates, more subtle epithelial abnormalities that might be important in susceptibility to common conditions such as chronic sinus infection have not been fully characterized. Mutations in cystic fibrosis transmembrane conductance regulator (CFTR) that do not cause cystic fibrosis have been associated with chronic rhinosinusitis, although the specific pathogenetic mechanisms involved have not been determined (1). Given the complexity of the human innate immune response to pathogens, it has been difficult to establish the contribution of epithelial signaling in the prevention of upper respiratory tract infection. Nonetheless, given the large number of patients with chronic sinusitis, in the absence of any clinically apparent systemic immune defect, it seems likely that there must be local defects in innate immunity associated with such mucosal infections. In this issue of the JCI, Cohen and coworkers explore unexpected players in innate immune defense: the bitter taste receptors (2).     

Antimicrobial Activity of High-Mobility-Group Box 2: a New Function to a Well-Known Protein

The human intestinal tract is highly colonized by a vast number of microorganisms. Despite this permanent challenge, infections remain rare, due to a very effective barrier defense system. Essential effectors of this system are antimicrobial peptides and proteins (AMPs), which are secreted by intestinal epithelial and lymphoid cells, balance the gut microbial community, and prevent the translocation of microorganisms. Several antimicrobial proteins have already been identified in the gut. Nonetheless, we hypothesized that additional AMPs are yet to be discovered in this setting. Using biological screening based on antimicrobial function, here we identified competent antibacterial activity of high-mobility-group box 2 (HMGB2) against Escherichia coli. By recombinant expression, we confirmed this biologically new antimicrobial activity against different commensal and pathogenic bacteria. In addition, we demonstrated that the two DNA-binding domains (HMG boxes A and B) are crucial for the antibiotic function. We detected HMGB2 in several gastrointestinal tissues by mRNA analysis and immunohistochemical staining. In addition to the nuclei, we also observed HMGB2 in the cytoplasm of intestinal epithelial cells. Furthermore, HMGB2 was detectable in vitro in the supernatants of two different cell types, supporting an extracellular function. HMGB2 expression was not changed in inflammatory bowel disease but was detected in certain stool samples of patients, whereas it was absent from control individuals. Taken together, we characterized HMGB2 as an antimicrobial protein in intestinal tissue, complementing the diverse repertoire of gut mucosal defense molecules.     

Combination Effects of Antimicrobial Peptides

Antimicrobial peptides (AMPs) are ancient and conserved across the tree of life. Their efficacy over evolutionary time has been largely attributed to their mechanisms of killing. Yet, the understanding of their pharmacodynamics both in vivo and in vitro is very limited. This is, however, crucial for applications of AMPs as drugs and also informs the understanding of the action of AMPs in natural immune systems. Here, we selected six different AMPs from different organisms to test their individual and combined effects in vitro. We analyzed their pharmacodynamics based on the Hill function and evaluated the interaction of combinations of two and three AMPs. Interactions of AMPs in our study were mostly synergistic, and three-AMP combinations displayed stronger synergism than two-AMP combinations. This suggests synergism to be a common phenomenon in AMP interaction. Additionally, AMPs displayed a sharp increase in killing within a narrow dose range, contrasting with those of antibiotics. We suggest that our results could lead a way toward better evaluation of AMP application in practice and shed some light on the evolutionary consequences of antimicrobial peptide interactions within the immune system of organisms.     

Molecular cloning and characterization of a short peptidoglycan recognition protein from silkworm Bombyx mori

Peptidoglycan is the major bacterial component recognized by the insect immune system. Peptidoglycan recognition proteins (PGRPs) are a family of pattern‐recognition receptors that recognize peptidoglycans and modulate innate immune responses. Some PGRPs retain N‐acetylmuramoyl‐L‐alanine amidase (Enzyme Commission number: 3.5.1.28) activity to hydrolyse bacterial peptidoglycans. Others have lost the enzymatic activity and work only as immune receptors. They are all important modulators for innate immunity. Here, we report the cloning and functional analysis of PGRP‐S4, a short‐form PGRP from the domesticated silkworm, Bombyx mori. The PGRP‐S4 gene encodes a protein of 199 amino acids with a signal peptide and a PGRP domain. PGRP‐S4 was expressed in the fat body, haemocytes and midgut. Its expression level was significantly induced by bacterial challenges in the midgut. The recombinant PGRP‐S4 bound bacteria and different peptidoglycans. In addition, it inhibited bacterial growth and hydrolysed an Escherichia coli peptidoglycan in the presence of Zn²⁺. Scanning electron microscopy showed that PGRP‐S4 disrupted the bacterial cell surface. PGRP‐S4 further increased prophenoloxidase activation caused by peptidoglycans. Taken together, our data suggest that B. mori PGRP‐S4 has multiple functions in immunity.     

Dermcidin-Derived Peptides Show a Different Mode of Action than the Cathelicidin LL-37 against Staphylococcus aureus

Dermcidin (DCD) is an antimicrobial peptide which is constitutively expressed in eccrine sweat glands. By postsecretory proteolytic processing in sweat, the DCD protein gives rise to anionic and cationic DCD peptides with a broad spectrum of antimicrobial activity. Many antimicrobial peptides induce membrane permeabilization as part of their killing mechanism, which is accompanied by a loss of the bacterial membrane potential. In this study we show that there is a time-dependent bactericidal activity of anionic and cationic DCD-derived peptides which is followed by bacterial membrane depolarization. However, DCD-derived peptides do not induce pore formation in the membranes of gram-negative and gram-positive bacteria. This is in contrast to the mode of action of the cathelicidin LL-37. Interestingly, LL-37 as well as DCD-derived peptides inhibit bacterial macromolecular synthesis, especially RNA and protein synthesis, without binding to microbial DNA or RNA. Binding studies with components of the cell envelope of gram-positive and gram-negative bacteria and with model membranes indicated that DCD-derived peptides bind to the bacterial envelope but show only a weak binding to lipopolysaccharide (LPS) from gram-negative bacteria or to peptidoglycan, lipoteichoic acid, and wall teichoic acid, isolated from Staphylococcus aureus. In contrast, LL-37 binds strongly in a dose-dependent fashion to these components. Altogether, these data indicate that the mode of action of DCD-derived peptides is different from that of the cathelicidin LL-37 and that components of the bacterial cell envelope play a role in the antimicrobial activity of DCD.Antimicrobial peptides (AMPs) serve as a first line of innate host defense in many species such as plants, amphibians, insects, and mammals. AMPs show a broad-spectrum antimicrobial activity against a wide range of pathogens including bacteria, fungi, and enveloped viruses (51). Most gene-encoded AMPs are synthesized as proforms, which are subsequently processed into mature peptides of various lengths. A common feature of most of these peptides is that they are cationic and form amphipathic structures (3). The mode of action of most AMPs is incompletely understood. It is believed that most AMPs kill microorganisms by membrane permeation either via pore formation or via membrane disintegration like that induced by the human cathelicidin LL-37. However, membrane disruption may not reflect the complex processes involved in the killing of microorganisms (5, 48). In addition, several AMPs clearly act differently and intracellular target sites have been identified (15). The mode of action has been unraveled for only a few AMPs which act via defined targets, such as the lantibiotic nisin, which specifically binds to bacterial lipid II, a membrane-bound component involved in peptidoglycan (PG) synthesis (7, 46). Similarly, the lantibiotic mersacidin interferes with transglycosylation and PG synthesis in gram-positive bacteria by direct targeting of lipid II (6). Furthermore, buforin II kills microorganisms by disruption of critical intracellular processes such as the inhibition of macromolecular biosynthesis or by interacting with nucleic acids inside the microorganisms (33). For several AMPs it has been demonstrated that the charge and the composition of the bacterial cell envelope determine sensitivity to AMPs (37). Staphylococcus aureus mutants lacking specific modifications in the bacterial envelope are highly susceptible to a variety of cationic AMPs. For example, incorporation of d-alanine into S. aureus teichoic acids by the dltA enzymes or the lysinylation of phosphatidylglycerol by mprF confers resistance to defensins, protegrins, and other AMPs by repulsion of the cationic peptides (36).Dermcidin (DCD) was identified by our group as a human AMP which is constitutively expressed in eccrine sweat glands and secreted into sweat (40). By postsecretory proteolytic processing in human sweat, the precursor protein gives rise to several truncated DCD peptides varying in length from 25 to 48 amino acids and with net charges between −2 and +2 (2, 10, 38). Several DCD peptides show antimicrobial activity against pathogenic microorganisms such as S. aureus, Escherichia coli, Enterococcus faecalis, Candida albicans, Staphylococcus epidermidis, Pseudomonas putida, and methicillin-resistant S. aureus as well as rifampin- and isoniazid-resistant Mycobacterium tuberculosis (9, 25, 40, 41, 45). We were able to show that DCD-derived peptides are also active under high-salt conditions and in a buffer resembling human sweat (40). Antimicrobially active DCD peptides, namely, the anionic peptides DCD-1L (48-mer) and DCD-1 (47-mer) and the cationic peptides SSL-25 (25-mer) and SSL-23 (23-mer), are derived from the C-terminal region of the precursor protein. Interestingly, these peptides have diverse and overlapping spectra of activity which are independent of the net peptide charge (41). In previous studies we showed that DCD peptides interact with the bacterial cell envelope and kill gram-negative bacteria without forming pores in membranes (41). In this study we investigated the mode of antimicrobial activity of DCD-derived peptides in more detail and studied bacterial factors that govern sensitivity or tolerance to DCD in the model microorganism S. aureus. In our first approach, we tried to identify the bacterial surface molecules to which DCD peptides bind. Second, we analyzed the bacterial response to DCD peptide challenge. Finally, we analyzed bacterial mutants to elucidate the mechanisms determining DCD sensitivity.     

Psychological stress downregulates epidermal antimicrobial peptide expression and increases severity of cutaneous infections in mice

The skin is the first line of defense against microbial infection, and psychological stress (PS) has been shown to have adverse effects on cutaneous barrier function. Here we show that PS increased the severity of group A Streptococcus pyogenes (GAS) cutaneous skin infection in mice; this was accompanied by increased production of endogenous glucocorticoids (GCs), which inhibited epidermal lipid synthesis and decreased lamellar body (LB) secretion. LBs encapsulate antimicrobial peptides (AMPs), and PS or systemic or topical GC administration downregulated epidermal expression of murine AMPs cathelin-related AMP and beta-defensin 3. Pharmacological blockade of the stress hormone corticotrophin-releasing factor or of peripheral GC action, as well as topical administration of physiologic lipids, normalized epidermal AMP levels and delivery to LBs and decreased the severity of GAS infection during PS. Our results show that PS decreases the levels of 2 key AMPs in the epidermis and their delivery into LBs and that this is attributable to increased endogenous GC production. These data suggest that GC blockade and/or topical lipid administration could normalize cutaneous antimicrobial defense during PS or GC increase. We believe this to be the first mechanistic link between PS and increased susceptibility to infection by microbial pathogens.     

Antimicrobial peptides and the skin

In recent years, hundreds of naturally occurring peptide antibiotics have been discovered based on their ability to inhibit the growth of microbial pathogens. These antimicrobial peptides (AMPs) participate in the innate immune response by providing a rapid first-line defence against infection. This review discusses the biology and clinical relevance of the two major families of AMPs, cathelicidins and defensins, with emphasis on their function in mammalian skin and their association with skin pathology. Current evidence shows that cathelicidins and defensins act as both natural antibiotics and as signalling molecules that activate host cell processes involved in immune defence and tissue repair. Alterations in the expression pattern of AMPs have been associated with a variety of pathological processes. Ongoing and future studies are likely to implicate AMPs in several unexplained human inflammatory disorders and to provide novel therapeutic approaches for the treatment of these diseases.     

Inventing a facile method to construct Bombyx mori (B. mori) silk fibroin nanocapsules for drug delivery

Bombyx mori (B. mori) silk fibroin (SF) microcapsules have acted as a great candidate in delivering drugs. However, it is difficult to fabricate SF nanocapsules using the present layer-by-layer (LBL) technique. In addition, the current SF microcapsules have limits in loading negatively charged drugs. Here, we invent a novel LBL method by introducing silane (APTES) as a structure indicator to produce SF nanocapsules that can load drugs with negative or positive charge. LBL assembly was completed by alternately coating SF and APTES on the template of polystyrene (PS) nanospheres by electrostatic attraction. SF nanocapsules were obtained after removal of the PS templates. Zeta potential analysis proved LBL assembly was indeed driven by the interaction between negative charge of SF and positive charge of APTES. Fluorescence images and electric microscope images indicated that SF nanocapsules had a hollow and stable structure with diameter at nearly 250 nm. The highest encapsulation rate of DOX or Ce6 were up to 80% and 90%, respectively, indicating SF nanocapsules have a high loading capability for both cationic and anionic drugs. In vitro cell experiments proved the biocompatibility of SF nanocapsules and their burst drug release in response to acidic environment. Furthermore, chemotherapy and photodynamic therapy proved SF nanocapsules loaded with DOX or Ce6 had significant inhibition on tumor cells. Our results suggested that this LBL technique is a facile method for polymers with negative charge to fabricate nanocapsules for antitumor drug carrier.

A novel LBL method was proposed here by introducing silane to produce stable SF nanocapsules for better drug delivery.     

Antimicrobial peptides and the skin

In recent years, hundreds of naturally occurring peptide antibiotics have been discovered based on their ability to inhibit the growth of microbial pathogens. These antimicrobial peptides (AMPs) participate in the innate immune response by providing a rapid first-line defence against infection. This review discusses the biology and clinical relevance of the two major families of AMPs, cathelicidins and defensins, with emphasis on their function in mammalian skin and their association with skin pathology. Current evidence shows that cathelicidins and defensins act as both natural antibiotics and as signalling molecules that activate host cell processes involved in immune defence and tissue repair. Alterations in the expression pattern of AMPs have been associated with a variety of pathological processes. Ongoing and future studies are likely to implicate AMPs in several unexplained human inflammatory disorders and to provide novel therapeutic approaches for the treatment of these diseases.     

Epidemiology,antibiotic therapy and outcomes of bacteremia caused by drug-resistant ESKAPE pathogens in cancer patients

Purpose

Infection due to the six ESKAPE pathogens has recently been identified as a serious emerging problem. However, there is still a lack of information on bacteremia caused by these organisms in cancer patients. We aimed to assess the epidemiology, antibiotic therapy and outcomes of bacteremia due to drug-resistant ESKAPE pathogens (rESKAPE) in patients with cancer.

Methods

All episodes of bacteremia prospectively documented in hospitalized adults with cancer from 2006 to 2011 were analyzed.

Results

Of 1,148 episodes of bacteremia, 392 (34 %) were caused by ESKAPE pathogens. Fifty-four episodes (4.7 %) were due to rESKAPE strains (vancomycin-resistant Enterococcus faecium 0, methicillin-resistant Staphylococcus aureus (MRSA) 13, extended-spectrum beta-lactamase (ESLB)-producing Klebsiella pneumoniae 7, carbapenem-resistant Acinetobacter baumannii 4, carbapenem- and quinolone-resistant Pseudomonas aeruginosa 18 and derepression chromosomic ß-lactam and ESBL-producing Enterobacter species 12. Risk factors independently associated with rESKAPE bacteremia were comorbidities, prior antibiotic therapy, urinary catheter and urinary tract source. Inappropriate empirical antibiotic therapy was more frequent in patients with rESKAPE bacteremia than in the other cases (55.6 % vs. 21.5 %, p?<?0.001). Persistence of bacteremia (25 % vs. 9.7 %), septic metastasis (8 % vs. 4 %) and early case-fatality rate (23 % vs. 11 %) were more frequent in patients with rESKAPE bacteremia than in patients with other etiologies (p?<?0.05).

Conclusions

Bacteremia due to rESKAPE pathogens in cancer patients occurs mainly among those with comorbidities who have received prior antibiotic therapy and have a urinary tract source. These patients often receive inappropriate empirical antibiotic therapy and have a poor outcome.     

Temporins A and B Stimulate Migration of HaCaT Keratinocytes and Kill Intracellular Staphylococcus aureus

The growing number of microbial pathogens resistant to available antibiotics is a serious threat to human life. Among them is the bacterium Staphylococcus aureus, which colonizes keratinocytes, the most abundant cell type in the epidermis. Its intracellular accumulation complicates treatments against resulting infections, mainly due to the limited diffusion of conventional drugs into the cells. Temporins A (Ta) and B (Tb) are short frog skin antimicrobial peptides (AMPs). Despite extensive studies regarding their antimicrobial activity, very little is known about their activity on infected cells or involvement in various immunomodulatory functions. Here we show that Tb kills both ATCC-derived and multidrug-resistant clinical isolates of S. aureus within infected HaCaT keratinocytes (80% and 40% bacterial mortality, respectively) at a nontoxic concentration, i.e., 16 μM, whereas a weaker effect is displayed by Ta. Furthermore, the peptides prevent killing of keratinocytes by the invading bacteria. Further studies revealed that both temporins promote wound healing in a monolayer of HaCaT cells, with front speed migrations of 19 μm/h and 12 μm/h for Ta and Tb, respectively. Migration is inhibited by mitomycin C and involves the epidermal growth factor receptor (EGFR) signaling pathway. Finally, confocal fluorescence microscopy indicated that the peptides diffuse into the cells. By combining antibacterial and wound-healing activities, Ta and Tb may act as multifunctional mediators of innate immunity in humans. Particularly, their nonendogenous origin may reduce microbial resistance to them as well as the risk of autoimmune diseases in mammals.