Studies on Staphylococcus epidermidis biofilm formation and the bacterial interaction with the human cathelicidin antimicrobial peptide LL-37

The long-term use of central venous catheters for delivering nutrients and drugs in preterm neonates has been related to nosocomial infections. The majority of late-onset sepsis in very preterm infants (<28 gestational weeks) are caused by Gram positive bacteria. Coagulasenegative staphylococci (CoNS) are responsible for almost the half of these cases. Staphylococcus epidermidis is the most prevalent bacteria identified from CoNS bacteraemia and biofilm production is found to be the main determinant of persistent infection. The major host defense peptide LL-37 is the only cathelicidin antimicrobial peptide that exists in humans. The peptide is broadly distributed in the human body and possesses several additional functions related to host defense. As a cathionic peptide, it interacts with the negatively charged bacterial surface. LL-37 is shown to inhibit biofilm formation and regulates biofilm-associated gene expression in Pseudomonas aeruginosa in vitro. In Paper I, we showed that S. epidermidis strains obtained from bloodstream infection in preterm infants had different characteristics than the skin strains isolated from healthy term neonates. The blood isolates were equipped with an invasive genetic element IS256 and showed higher antimicrobial resistance compared with the skin isolates. However, vancomycin resistance was not detected among any of the strains. We also observed short and long filamentlike structures on the cell surface of S. epidermidis. These filaments were involved in the attachment to the catheter surface and also in cell to cell attachment and/or communication. Our in vitro studies in Paper II and Paper III, revealed that physiological LL-37 peptide concentrations, below those that kill or inhibit growth of the free-floating bacteria, inhibited S. epidermidis attachment and biofilm formation on abiotic surfaces. In Paper III, we observed that the peptide regulates genes involved in the biofilm formation. In Paper IV, we found that the circulating serum level of hCAP18/LL-37 was similar in preterm and term neonates at birth and both the inactive protein and the active peptide were detectable independent of the gestational time. We observed positive correlation between maternal and infant peptide concentration. This may indicate that the peptide passes over early during pregnancy. In summary, our work revealed that S. epidermidis strains that cause bloodstream infection in preterm infants are more virulent compared with skin strains in term neonates. Physiological concentration of the human cathionic peptide LL-37 had inhibitory effect on S. epidermidis biofilm formation by regulating biofilm genes. The similar LL-37 peptide concentration in preterm and term infants’ blood might suggest that these neonate’s vulnerability is not connected to the lower antimicrobial peptide level at birth. LIST OF PUBLICATIONS I. Hell, E. , Giske, CG. , Hultenby, K. , Marchini, G. , Gemzell Danielsson, K. Attachment and biofilm forming capabilities of Staphylococcus epidermidis strains isolated from preterm infants. Current Microbiology (2013)67(6):712-7 II. Hell, E. , Giske, CG. , Nelson, A. , Romling, U. , Marchini, G. Human cathelicidin peptide LL-37 inhibits both attachment capability and biofilm formation of Staphylococcus epidermidis. Letters in Appl Microbiol (2010), 50:211-215 III. Hell, E. , Ujvari, D. , Hultenby, K. , Rohde, H. , Gemzell Danielsson, K. , Marchini, G. Biofilm-associated gene regulation by the human cathelicidin LL-37 in Staphylococcus epidermidis. Manuscript IV. Hell, E. , Dubicke, A. , Fransson, E. , Gemzell Danielsson, K. , Marchini, G. Circulating cathelicidin antimicrobial peptide LL-37 levels in newborn infants in relation to gestational time. Manuscript CONTENTS 1 Background . 7 1.1 Staphylococcus epidermidis . 7 1.1.1 The first colonizer after birth and the nosocomial pathogen . 7 1.1.2 Virulence determinants . 8 1.2 S. epidermidis Biofilm formation . 8 1.2. 1 Adhesion, initial attachment . 9 1.2. 2 Biofilm maturation . 9 1.2. 3 Biofilm detachment and regulation . 11 1.3 Antimicrobial susceptibility of S. epidermidis . 12 1.4 the neonatal innate immunity . 13 1.5 Antimicrobial peptides . 13 1.5. 1 The human cathelicidin peptide LL-37 . 13 2 Aims of the thesis . 16 3 Materials and Methods . 17 3.1 PATIENT MATERIALS . 17 3.2 BACTERIA, PEPTIDE, GROWTH MEDIA AND BLOOD SAMPLES . 17 3.2. 1 Bacterial strains . 17 3.2. 2 Human cathelicidin peptide LL-37 . 17 3.2. 3 Growth media and bacterial culture conditions . 17 3.2. 4 Blood samples . 18 3.3 GROWTH EXPERIMENT (PAPER II) . 18 3.4 CULTURE PLATE ASSAYS . 18 3.4.1 Initial attachment and biofilm formation in polystyrene microtiter plates (Paper I) . 18 3.4.2 Initial attachment and biofilm formation in the presence of peptide LL-37 in polystyrene microtiter plates (Paper II) . 18 3.4. 3 Biofilm formation in the presence of peptide LL-37 in 6-well culture plate (Paper III) . 18 3.5 ELECTRON MICROSCOPY. 19 3.5. 1 Transmission electron microscopy (TEM) (Paper I) . 19 3.5. 2 Scanning electron microscopy (SEM) (Paper III) . 19 3.6 DNAAND RNA BASED ANALYSIS . 19 3.6. 1 PCR (Paper I) . 19 3.6. 2 PFGE (Paper I) . 19 3.6. 3 RNA extraction, reverse transcription and qRT-PCR (Paper III) . 20 3.7 PROTEIN ANALYSIS . 20 3.7. 1 Immuno dot-blot analysis (Paper III) . 20 3.7. 2 ELISA (Paper IV) . 21 3.7. 3 Immunoprecipitation and western blot analysis (Paper IV) . 21 3.8 ANTIMICROBIAL SUSCEPTIBILITY TESTING (PAPER I). 21 4 Results and Discussion . 22 4.1 CHARACTERISTICS OF STAPHYLOCOCCUS EPIDERMIDIS STAINS ISOLATED FROM NEONATES (PAPER I) . 22 4.1.1 Primary attachment and biofilm formation . 22 4.1.2 Prevalence of the biofilm-associated genes . 22 4.1.3 Antimicrobial susceptibility, IS256 and clonal relationship . 22 4.2 INTERACTION BETWEEN S. EPIDERMIDIS AND THE HUMAN CATHELICIDIN PEPTIDE LL-37, IN VITRO STUDIES (PAPER II AND III) . 23 4.2. 1 Effect of the peptide on primary attachment and biofilm formation . 23 4.2. 2 Effect of the peptide on biofilm-associated gene expression . 23 4.2. 3 Circulating cathelicidin antimicrobial peptide LL-37 levels in newborn infants in relation to gestational time (Paper IV) . 25 5 General Conclusions . 26 6 Future perspectives . 27 8 Acknowledgements . 28 9 REFERENCES . 32 LIST OF ABBREVIATIONS NICUs neonatal intensive care units CVC central venous catheter VLWB very low birth weight