Antibacterial Effect of Human Mesenchymal Stem Cells is Mediated in Part from Secretion of the Antimicrobial Peptide LL-37

Recent in vivo studies indicate that mesenchymal stem cells (MSCs) may have beneficial effects in the treatment of sepsis induced by bacterial infection. Administration of MSCs in these studies improved survival and enhanced bacterial clearance. The primary objective of this study was to test the hypothesis that human MSCs possessed intrinsic antimicrobial properties. We studied the effect of human MSCs derived from bone marrow on the bacterial growth of Gram-negative (Escherichia coli and Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus) bacteria. MSCs as well as their conditioned medium (CM) demonstrated marked inhibition of bacterial growth in comparison with control medium or normal human lung fibroblasts (NHLF). Analysis of expression of major antimicrobial peptides indicated that one of the factors responsible for the antimicrobial activity of MSC CM against Gram-negative bacteria was the human cathelicidin antimicrobial peptide, hCAP-18/LL-37. Both m-RNA and protein expression data showed that the expression of LL-37 in MSCs increased after bacterial challenge. Using an in vivo mouse model of E. coli pneumonia, intratracheal administration of MSCs reduced bacterial growth (in colony-forming unit) in the lung homogenates and in the bronchoalveolar lavage (BAL) fluid, and administration of MSCs simultaneously with a neutralizing antibody to LL-37 resulted in a decrease in bacterial clearance. In addition, the BAL itself from MSC-treated mice had a greater antimicrobial activity in comparison with the BAL of phosphate buffered saline (PBS)-treated mice. Human bone marrow-derived MSCs possess direct antimicrobial activity, which is mediated in part by the secretion of human cathelicidin hCAP-18/ LL-37.

Antimicrobial peptides and proinflammatory cytokines in periprosthetic joint infection

Background: Differentiation between septic and aseptic loosening of joint replacements is essential for successful revision surgery, but reliable markers for the diagnosis of low-grade infection are lacking. The present study was performed to assess intra-articular and systemic levels of antimicrobial peptides and proinflammatory cytokines as diagnostic markers for periprosthetic joint infection. Methods: Fifteen consecutive patients with staphylococcal periprosthetic joint infections and twenty control patients with aseptic loosening of total hip and knee replacements were included in this prospective, single-center, controlled clinical trial. Expression of the antimicrobial peptides human β-defensin-2 (HBD-2), human β-defensin-3 (HBD-3), and cathelicidin LL-37 (LL-37) was determined by ELISA (enzyme-linked immunosorbent assay) in serum and joint aspirates. Proinflammatory cytokines were assessed in serum and joint aspirates with use of cytometric bead arrays. C-reactive protein in serum, microbiology, and histopathology of periprosthetic tissue served as the “gold standard” for the diagnosis of infection. Results: The antimicrobial peptides HBD-3 and LL-37 were significantly elevated in joint aspirates from patients with periprosthetic joint infection compared with patients with aseptic loosening, and the area under the curve (AUC) in a receiver operating characteristic curve analysis was equal to 0.745 and 0.875, respectively. Additionally, significant local increases in the proinflammatory cytokines interleukin (IL)-1β, IL-4, IL-6, IL-17A, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α were observed to be associated with infection. Logistic regression analysis indicated that the combination of an antimicrobial peptide with another synovial fluid biomarker improved diagnostic accuracy; the AUC value was 0.916 for LL-37 and IL-4, 0.895 for LL-37 and IL-6, 0.972 for HBD-3 and IL-4, and 0.849 for HBD-3 and IL-6. In contrast, the only antimicrobial peptides and cytokines in serum that showed a significant systemic increase in association with infection were HBD-2, IL-4, and IL-6 (all of which had an AUC value of <0.75). Conclusions: The present study showed promising results for the use of antimicrobial peptides and other biomarkers in synovial fluid for the diagnosis of periprosthetic joint infection, and analysis of the levels in synovial fluid was more accurate than analysis of serum.

Effect of subinhibitory antibiotic concentrations on polysaccharide intercellular adhesin expression in biofilm-forming Staphylococcus epidermidis.

Rachid S, Ohlsen K, Witte W, Hacker J, Ziebuhr W. Institut für Molekulare Infektionsbiologie, Röntgenring 11, D-97070 Würzburg, Germany. Biofilm production is an important step in the pathogenesis of Staphylococcus epidermidis polymer-associated infections and depends on the expression of the icaADBC operon leading to the synthesis of a polysaccharide intercellular adhesin. A chromosomally encoded reporter gene fusion between the ica promoter and the beta-galactosidase gene lacZ from Escherichia coli was constructed and used to investigate the influence of both environmental factors and subinhibitory concentrations of different antibiotics on ica expression in S. epidermidis. It was shown that S. epidermidis biofilm formation is induced by external stress (i.e., high temperature and osmolarity). Subinhibitory concentrations of tetracycline and the semisynthetic streptogramin antibiotic quinupristin-dalfopristin were found to enhance ica expression 9- to 11-fold, whereas penicillin, oxacillin, chloramphenicol, clindamycin, gentamicin, ofloxacin, vancomycin, and teicoplanin had no effect on ica expression. A weak (i.e., 2.5-fold) induction of ica expression was observed for subinhibitory concentrations of erythromycin. The results were confirmed by Northern blot analyses of ica transcription and quantitative analyses of biofilm formation in a colorimetric assay.

In vitro bactericidal activity of diterpenoids isolated from Aframomum melegueta K.Schum against strains of Escherichia coli, Listeria monocytogenes and Staphylococcus aureus

Ethnopharmacological relevance: The ethnobotanical use of Aframomum melegueta in the treatment of urinary tract and soft tissue infection suggested that the plant has antimicrobial activity.

Materials and methods: To substantiate the folkloric claims, an acetone, 50:50 acetone:methanol and 2:1 chloroform:methanol extracts were tested against Escherichia coli K12; acetone extract and the fractions of acetone extracts were tested against Listeria monocytogenes. Bioassay-guided fractionation was performed on the extract using L. monocytogenes as the test organism to isolate the bioactive compounds which were then tested against all the other organisms.

Results: Four known labdane diterpenes (G3 and G5) were isolated for the first time from the rhizomes of A. melegueta and purified. These were tested against E. coli, L. monocytogenes, methicillin resistant Staphylococus aureus (MRSA) and S. aureus to determine antibacterial activity. The result showed that two compounds G3 and G5 exhibited more potent antibacterial activity compared to the current clinically used antibiotics ampicillin, gentamicin and vancomycin and can be potential antibacterial lead compounds. The structure of the labdane diterpenes were elucidated using nuclear magnetic resonance (NMR) spectroscopy and Mass spectrometry. A possible mode of action of the isolated compound G3 and its potential cytotoxicity towards mammalian cells were also discussed.

Conclusion: The results confirmed the presence of antibacterial compounds in the rhizomes of A. melegueta with a favourable toxicity profile which could be further optimized as antibacterial lead compounds.

Combined effect of high pressure processing and antimicrobial packaging to control the growth of Listeria monocytogenes on ready-to-eat chicken breast

In the European Union, food is considered safe with regard to Listeria monocytogenes if its numbers do not exceed 100 cfu/g throughout the shelf-life of the food. Therefore, it is important to determine if a food supports growth of L. monocytogenes. Challenge tests are laboratory-based studies that measure the growth of L. monocytogenes on artificially contaminated food stored under foreseeable conditions of transportation, distribution and storage. The aim of this study was to elaborate and optimize a user-friendly protocol to perform challenge tests on food and to apply it to determine whether growth of L. monocytogenes is supported during the production and distribution of a potentially risky food i.e. mushrooms. A three-strain mixture of L. monocytogenes was inoculated onto three independent batches of whole mushrooms, sliced mushrooms, mushroom casing and mushroom substrate at a concentration of about 100 -1000 cfu/g. The batches were incubated at potential abuse temperatures, as a worst case scenario, and at intervals during storage L. monocytogenes numbers, % moisture and pH were determined. The results showed that the sliced and whole mushrooms supported growth of L. monocytogenes while mushroom casing allowed survival but did not support growth. Mushroom substrate showed a rich background microflora able of growing in Listeria selective media which hindered enumeration of L. monocytogenes. Combase predictions were not always accurate, indicating that challenge tests are a necessary part of growth determination of L. monocytogenes.

Detection of the icaADBC gene cluster and biofilm formation in Staphylococcus epidermidis isolates from catheter-related urinary tract infections.

Cho SH, Naber K, Hacker J, Ziebuhr W. Institut für Molekulare Infektionsbiologie, Röntgenring 11, D-97070 Würzburg, Germany. Biofilm production in Staphylococcus epidermidis is an important virulence factor that is mediated by the expression of the icaADBC operon. In this study 41 S. epidermidis isolates obtained from catheter-related urinary tract infections were analyzed for the presence of the icaADBC operon and biofilm formation. Eighteen of 41 isolates (44%) were shown to carry ica-specific DNA, but only 11 isolates (27%) produced biofilms spontaneously under normal growth conditions. Upon induction by external stress or antibiotics, biofilm formation could be stimulated in five of seven ica-positive, biofilm-negative isolates, indicating that the icaADBC expression was down-regulated in these strains. Genetic analyses of the ica gene clusters of the remaining two ica-positive, biofilm-negative strains revealed a spontaneous ICAC::IS256 insertion in one strain. Insertion of the element caused a target site duplication of seven base pairs and a biofilm-negative phenotype. After repeated passages the insertion mutant was able to revert to a biofilm-forming phenotype which was due to the precise excision of IS256 from the icaC gene. The data show that icaC::IS256 integrations occur during S. epidermidis polymer-related infections and the results highlight the biological relevance of the IS256-mediated phase variation of biofilm production in S. epidermidis during an infection.

Alternative transcription factor sigma(B) is involved in regulation of biofilm expression in a Staphylococcus aureus mucosal isolate.

Rachid S, Ohlsen K, Wallner U, Hacker J, Hecker M, Ziebuhr W. Institut für Molekulare Infektionsbiologie, D-97070 Würzburg, Germany. Osmotic stress was found to induce biofilm formation in a Staphylococcus aureus mucosal isolate. Inactivation of a global regulator of the bacterial stress response, the alternative transcription factor sigma(B), resulted in a biofilm-negative phenotype and loss of salt-induced biofilm production. Complementation of the mutant strain with an expression plasmid encoding sigma(B) completely restored the wild-type phenotype. The combined data suggest a critical role of sigma(B) in S. aureus biofilm regulation under environmental stress conditions.

Antimicrobial activity of neuropeptides against a range of micro-organisms from skin, oral, respiratory and gastrointestinal tract sites

Many neuropeptides are similar in size, amino acid composition and charge to antimicrobial peptides. This study aimed to determine whether the neuropeptides substance P (SP), neurokinin A (NKA), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP), displayed antimicrobial activity against Streptococcus mutans, Lactobacillus acidophilus, Enterococcus faecalis, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. SP, NPY, VIP and CGRP displayed variable degrees of antimicrobial activity against all the pathogens tested with the exception of S. aureus. These antimicrobial activities add a further dimension to the immunomodulatory roles for neuropeptides in the inflammatory and immune responses. (c) 2008 Elsevier B.V. All rights reserved.

Resistance of Staphylococcus aureus to the cationic antimicrobial agent poly(2-(dimethylamino ethyl)methacrylate) (pDMAEMA) is influenced by cell-surface charge and hydrophobicity

Cationic antimicrobial agents may prevent device-associated infections caused by Staphylococcus epidermidis and Staphylococcus aureus. This study reports that the cationic antimicrobial polymer poly(2-(dimethylamino ethyl)methacrylate) (pDMAEMA) was more effective at antagonizing growth of clinical isolates of S. epidermidis than of S. aureus. Importantly, mature S. epidermidis biofilms were significantly inactivated by pDMAEMA. The S. aureus isolates tested were generally more hydrophobic than the S. epidermidis isolates and had a less negative charge, although a number of individual S. aureus and S. epidermidis clinical isolates had similar surface hydrophobicity and charge values. Fluorescence spectroscopy and flow cytometry revealed that fluorescently labelled pDMAEMA interacted strongly with S. epidermidis compared with S. aureus. S. aureus Delta dltA and Delta mprF mutants were less hydrophobic and therefore more susceptible to pDMAEMA than wild-type S. aureus. Although the different susceptibility of S. epidermidis and S. aureus isolates to pDMAEMA is complex, influenced in part by surface hydrophobicity and charge, these findings nevertheless reveal the potential of pDMAEMA to treat S. epidermidis infections.

Antimicrobial peptide incorporated poly(2-hydroxyethyl methacrylate) hydrogels for the prevention of Staphylococcus epidermidis-associated biomaterial infections

The effectiveness of the antimicrobial peptide maximin-4, the ultrashort peptide H-Orn-Orn-Trp-Trp-NH(2) , and the lipopeptide C(12) -Orn-Orn-Trp-Trp-NH(2) in preventing adherence of pathogens to a candidate biomaterial were tested utilizing both matrix- and immersion-loaded poly(2-hydroxyethyl methacrylate) (poly(HEMA)) hydrogels. Antiadherent properties correlated to both the concentration released and the relative antimicrobial concentrations of each compound against Staphylococcus epidermidis ATCC 35984, at each time point. Immersion-loaded samples containing C(12) -Orn-Orn-Trp-Trp-NH(2) exhibited the lowest adherence profile for all peptides studied over 1, 4, and 24 h. The results outlined in this article show that antimicrobial peptides have the potential to serve as an important weapon against biomaterial associated infections. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012.

Efficacy of Biocides Used in the Modern Food Industry To Control Salmonella enterica, and Links between Biocide Tolerance and Resistance to Clinically Relevant Antimicrobial Compounds

Biocides play an essential role in limiting the spread of infectious disease. The food industry is dependent on these agents, and their increasing use is a matter for concern. Specifically, the emergence of bacteria demonstrating increased tolerance to biocides, coupled with the potential for the development of a phenotype of cross-resistance to clinically important antimicrobial compounds, needs to be assessed. In this study, we investigated the tolerance of a collection of susceptible and multidrug-resistant (MDR) Salmonella enterica strains to a panel of seven commercially available food-grade biocide formulations. We explored their abilities to adapt to these formulations and their active biocidal agents, i.e., triclosan, chlorhexidine, hydrogen peroxide, and benzalkonium chloride, after sequential rounds of in vitro selection. Finally, cross-tolerance of different categories of biocidal formulations, their active agents, and the potential for coselection of resistance to clinically important antibiotics were investigated. Six of seven food-grade biocide formulations were bactericidal at their recommended working concentrations. All showed a reduced activity against both surface-dried and biofilm cultures. A stable phenotype of tolerance to biocide formulations could not be selected. Upon exposure of Salmonella strains to an active biocidal compound, a high-level of tolerance was selected for a number of Salmonella serotypes. No cross-tolerance to the different biocidal agents or food-grade biocide formulations was observed. Most tolerant isolates displayed changes in their patterns of susceptibility to antimicrobial compounds. Food industry biocides are effective against planktonic Salmonella. When exposed to sublethal concentrations of individual active biocidal agents, tolerant isolates may emerge. This emergence was associated with changes in antimicrobial susceptibilities.