Marlene Fredborg

Real-time optical antimicrobial susceptibility testing.

ABSTRACT Rapid antibiotic susceptibility testing is in high demand in health care fields as antimicrobial-resistant bacterial strains emerge and spread. Here, we describe an optical screening system (oCelloScope) which, based on time-lapse imaging of 96 bacteria-antibiotic combinations at a time, introduces real-time detection of bacterial growth and antimicrobial susceptibility with imaging material to support the automatically generated graphs. Automated antibiotic susceptibility tests of a monoculture showed statistically significant antibiotic effects within 6 min and within 30 min in complex samples from pigs suffering from catheter-associated urinary tract infections. The oCelloScope system provides a fast high-throughput screening method for detecting bacterial susceptibility that might entail an earlier diagnosis and introduction of appropriate targeted therapy and thus combat the threat from multidrug-resistant pathogenic bacteria. The oCelloScope system can be employed for a broad range of applications within bacteriology and might present new vistas as a point-of-care instrument in clinical and veterinary settings.

Rational Design of Alpha-Helical Antimicrobial Peptides: Do's and Don'ts.

Antimicrobial peptides (AMPs) are promising candidates for battling multiresistant bacteria. Despite extensive research, structure–activity relationships of AMPs are not fully understood, and there is a lack of structural data relating to AMPs in lipids. Here we present the NMR structure of anoplin (GLLKRIKTLL‐NH2) in a micellar environment. A vast library of substitutions was designed and tested for antimicrobial and hemolytic activity, as well as for changes in structure and lipid interactions. This showed that improvement of antimicrobial activity without concomitant introduction of strong hemolytic activity can be achieved through subtle increases in the hydrophobicity of the hydrophobic face or through subtle increases in the polarity of the hydrophilic face of the helix, or—most efficiently—a combination of both. A set of guidelines based on the results is given, for assistance in how to modify cationic α‐helical AMPs in order to control activity and selectivity. The guidelines are finally tested on a different peptide.

G protein-coupled receptor120 (GPR120) transcription in intestinal epithelial cells is significantly affected by bacteria belonging to the Bacteroides, Proteobacteria, and Firmicutes phyla.

Free fatty acids (FFA) are produced in the intestine by microbial fermentation. Recently, a family of G protein-coupled receptors (GPR) acting as FFA transporters has been reported including GPR120, which is expressed by intestinal epithelial cells. The GPR120 has been reported to affect the expression of glucagon-like peptide (GLP)-1 as well as function as a control point for anti-inflammatory effects. The aim of the present study was to evaluate whether 12 selected intestinal bacteria, representing the 4 major phyla present in the intestine, affect intestinal epithelial cell GPR120 and GLP-1 mRNA abundance. Supernatants of the 12 bacteria were added to differentiated Caco-2 intestinal epithelial cells cultured on filter inserts in concentrations corresponding to a cell:bacteria ratio of 1:200. After 4 h of incubation, changes in cellular mRNA of GLP-1 and GPR120 by bacterial supernatant were examined using real-time reverse transcriptase polymerase chain reaction. The abundance of GLP-1 mRNA decreased when cells were exposed to 4 of the 12 supernatants (P ≤ 0.05) compared with cells without bacteria added. Supernatants from 8 of the 12 bacteria analyzed increased the mRNA level of GPR120 (P ≤ 0.05) compared with cells without bacteria added. The alteration in cellular GPR120 mRNA was observed with bacteria categorized as either probiotics or bacteria capable of inducing an anti-inflammatory effect. The beneficial effect of these bacteria may very well be mediated by regulation of GPR120. The regulation of GPR120 by intestinal microbiota represents a direct signaling pathway for gut bacteria to affect host health and metabolism.

Synergistic activities of meropenem double and triple combinations against carbapenemase-producing Enterobacteriaceae

The increasing emergence of carbapenemase-producing Enterobacteriaceae poses a considerable threat to global health as only limited treatment options are available and has therefore led to efforts to discover antibiotic combination regimens effective. The aim of this study was to evaluate in vitro synergistic activity of 10 meropenem double and triple combinations against the 5 most frequently encountered carbapenemases-producing Enterobacteriaceae. Broth microdilution assays showed that the meropenem and ertapenem combination was the most efficient regimen of the double combinations tested (>5-log2 fold decrease). The triple combination of meropenem, polymyxin and rifampin exhibited highest synergistic activity of the triple combinations. The divergent reports on synergistic activity of antibiotic combinations suggest that it may not be possible to predict synergy based on carbapenemase type alone. Consequently, we recommend that in vitro evaluation of synergistic activity of antibiotic combinations against carbapenemase-producing Enterobacteriaceae is performed on every isolate to ensure effective treatment regimens.

Apoptotic properties of the type 1 interferon induced family of human mitochondrial membrane ISG12 proteins

Interferons are a family of cytokines with growth inhibitory and antiviral functions, which exert their biological actions through the expression of interferon‐stimulated genes (ISGs). The human ISG12 family of proteins comprises ISG12A, ISG12B, ISG12C and ISG6‐16. Due to differential splicing and a gene variation, the human ISG12A protein exists as a full‐length ISG12A form and three ISG12A variants. ISG12 genes have been found transcriptionally dysregulated in many disorders. High levels of ISG12A mRNA have been found in breast and ovarian cancers. Loss of heterozygosity at the position of the ISG12 genes often occurs in ovarian carcinomas and lymphoblastic leukemias. Both ISG12A and ISG6‐16 are up‐regulated in psoriasis.

Fast Screening of Antibacterial Compounds from Fusaria

Bio-guided screening is an important method to identify bioactive compounds from fungi. In this study we applied a fast digital time-lapse microscopic method for assessment of the antibacterial properties of secondary metabolites from the fungal genus Fusarium. Here antibacterial effects could be detected for antibiotic Y, aurofusarin, beauvericin, enniatins and fusaric acid after six hours of cultivation. The system was then used in a bio-guided screen of extracts from 14 different Fusarium species, which had been fractionated by HPLC. In this screen, fractions containing the red pigments aurofusarin and bikaverin showed effects against strains of Lactobacillus and Bifidobacterium. The IC50 for aurofusarin against Lactobacillus acidophilus was 8 µM, and against Bifidobacterium breve it was 64 µM. Aurofusarin only showed an effect on probiotic bacteria, leading to the speculation that only health-promoting bacteria with a positive effect in the gut system are affected.

Administration of Protein Kinase D1 Induces a Protective Effect on Lipopolysaccharide-Induced Intestinal Inflammation in a Co-Culture Model of Intestinal Epithelial Caco-2 Cells and RAW264.7 Macrophage Cells

Inflammatory bowel diseases (IBD) are chronic inflammatory diseases involving all or part of the gastrointestinal tract. The stress-activated serine-threonine protein kinase D1 (PKD1) protein has previously been implicated in intestinal immune regulation. The objective of this study was to evaluate the effects of human PKD1 in relation to intestinal inflammation, using a co-culture model of intestinal epithelial Caco-2 cells and RAW264.7 macrophages. An inflammatory response was induced in the macrophages by lipopolysaccharide (LPS), upregulating the expression of tumour necrosis factor alpha (TNF-α), interleukin- (IL-) 1β, and IL-6 besides increasing the secretion of TNF-α protein. The effect of administering PKD1 to Caco-2 was evaluated in relation to both amelioration of inflammation and the ability to suppress inflammation initiation. Administration of PKD1 (10–100 ng/ml) following induction of inflammation induced downregulation of TNF-α expression in RAW264.7 cells. In addition, PKD1 administered for 3 h prior to LPS stimulation reduced the subsequent inflammatory response through downregulation of TNF-α, IL-1β, and IL-6 in RAW264.7 cells. These results demonstrate a potential role of PKD1 in the intercellular communication between intestinal epithelial and immune cells, proposing a protective effect of PKD1 on the induction of an inflammatory response in macrophages, an important aspect during the pathogenesis of IBD.