CHOOSING THE OPTIMAL COMBINATION OF ANTIMICROBIAL PEPTIDES AND ANTIBIOTICS TO TREAT ACUTE SEPSIS Samuel Whitehouse (31573906) Bachelor of Science School of Veterinary and Life Sciences Murdoch University November 2017 Declaration: I declare this thesis is my own account of my research and contains as its main content work which has not been previously submitted for a degree at any tertiary education institution. Signature: Abstract: Sepsis is an infection, usually bacterial, of normally sterile parts of the body and leads to systemic illness and a panoply of non-selective inflammatory responses that are non- specific. Preterm neonates (infants born <37 weeks gestation) are particularly affected by sepsis with a prevalence up to 62%, particularly LOS acquired after 72 hours of life, caused from micro-organisms acquired either perinatally or postnatally usually as a consequence of nosocomial transmission. Increased antibiotic resistant bacteria prevalence has also made treating preterm sepsis more challenging thus new treatments are required to overcome the antibiotic resistance. Synergy between AMPs/antibiotics has been documented before but very few studies at present test various combinations of AMPs/antibiotics in human whole blood. This study set out to determine the optimum combination of AMPs, rBPI , LF, IDR-1018 and LL-37, and antibiotics, 21 Vancomycin and Ceftriaxone, to kill common sensitive and resistant preterm infant clinical isolate sepsis causing bacteria (S. aureus, MRSA, E. coli and ESBL E. coli), inoculated into donated healthy adult whole blood. This study showed that the Vancomycin and IDR-1018 combination was the only combination that had a significantly increased inhibitory effect, against E. coli despite the Vancomycin resistance of E. coli. This study also found that only IDR-1018 had any inhibitory activity against three of the four bacteria used (E. coli, MRSA and ESBL E. coli). We found that the endogenous levels of AMPs and immune cells in donor blood had no effect on the bacteria used in the study at the doses tested. To conclude, this study found a combination of AMPs/antibiotic that can potentially improve sepsis treatment in preterms. But more work is needed to ensure that this combination is a viable new treatment for preterm infant sepsis. iii | P age Acknowledgements I would like to thank my amazing supervisors Dr. Andrew Currie, Dr. Tobias Strunk, Dr. Ed Litton and Dr. Sam Abraham for all you have done for me, your advice and assistance have been invaluable for me throughout the project. I would not have gotten as far as I have without all your help, thank you for putting up with me when I encountered problems in the project. I would like to thank Dr. Kirsty Townsend for all your help with acquiring materials for my experiments and your Microbiology boot camp that gave me the revision I needed for my microbiology techniques. I would like to thank Aaron Roman for being available for as long as you were to take the blood samples from my donors, I hope the lollies I bought for you were a nice payment for having to take samples from all those donors. I would like to thank Terence Lee for helping me by making sure my MIC protocol is correct and showing me how to reconstitute my antibiotics. I would like to thank Caitlyn Granland for all your help with the AMPs and the bacteria in terms of assisting with the calculations that I was unable to understand and any questions about your protocol that I had, you were a magnificent help to me. I would like to thank Tabitha Woodman for allowing me to shadow you and gain experience in the serial dilution plating method, I really learned a lot from you. I would like to thank Sherrianne Ng for all your help in getting materials from Princess Margaret Hospital (PMH) when I could not make it to PMH to pick up the materials I needed. iv | P age I would like to thank all the people at PMH for all the advice you gave me during my practice presentations and for being such a nice and friendly environment for me to work in. I would like to thank my family for all the support you gave me during my Honours project. Without them and the breaks from the thesis they made me take to get a fresh look at the writing, I would not have been able to push through when I encountered difficulties in my project. Finally, I would like to thank all the people, you know who you are, who donated blood samples to my Honours project and by extension of that being available at the times needed to give the samples. v | P age Table of Contents: 1 Chapter 1 Introduction/Literature Review .............................................................................. 1.1 Sepsis Definition: .......................................................................................................... 1 1.2 Sepsis Epidemiology and Pathophysiology: ................................................................. 2 1.2.1 The Pathophysiology of Sepsis: ............................................................................ 2 1.2.2 Epidemiology of Sepsis in Different Age Groups: ............................................... 5 1.2.3 Neonate and Preterm Birth Definition: ................................................................. 6 1.2.4 Sepsis Associated with Preterm Birth: .................................................................. 7 1.2.5 Common Sepsis Causing Bacteria in Preterm EOS/LOS/VLOS: ......................... 8 1.2.6 Antibiotic Resistant Sepsis Causing Bacteria on the Rise: ................................... 9 1.3 Preterm Compared to Term/Adult Immune Response to Sepsis Infection: .................. 9 1.3.1 Preterm Immune System when Challenged by Sepsis Infection: ......................... 9 1.3.2 Preterm Immunoglobulins and Other Soluble Molecules Involved in Fighting Sepsis Infection: .................................................................................................................. 10 1.3.3 Preterm Cells Involved in Fighting Sepsis Infection: ......................................... 12 1.3.4 Preterm Cytokines Involved in The Immune Response to Sepsis Infection: ...... 15 1.4 Antibiotics Treat Bacteria and Regulate Inflammatory Cytokines: ............................ 15 1.4.1 Antibiotics: .......................................................................................................... 15 1.4.2 Ceftriaxone: ........................................................................................................ 16 1.4.3 Vancomycin: ....................................................................................................... 17 1.5 Bacteria are Developing Resistance to the Antibiotic Treatment: .............................. 18 1.5.1 Bacterial Antibiotic Resistance Mechanisms: ..................................................... 18 1.6 Alternative Treatments for Sepsis: .............................................................................. 20 1.6.1 Potential Adjunct Treatments with/without Antibiotics: .................................... 20 1.7 Antimicrobial Peptides Effect on Bacteria: ................................................................ 21 1.7.1 Antimicrobial Peptides (AMPs): ......................................................................... 21 1.7.2 Bactericidal/Permeability Increasing Protein: .................................................... 22 1.7.3 Lactoferrin: ......................................................................................................... 23 1.7.4 LL-37: ................................................................................................................. 24 1.7.5 Innate Defence Regulator peptide-1018: ............................................................ 24 1.7.6 The Problems stopping AMPs being used as therapeutic treatments: ................ 25 1.8 Bacterial Resistance to AMPs:.................................................................................... 25 1.8.1 Methods Used by Bacteria to Resist AMPs: ....................................................... 25 1.9 AMPs May Help Antibiotics to Kill the Bacteria: ...................................................... 28 1.9.1 The Synergy Between AMPs and Antibiotics: ................................................... 28 1.9.2 Project Aims and Hypotheses: ............................................................................ 30 vi | P age 2 Chapter 2 Materials and Methods: .......................................................................................... 2.1 Table 7. Bacteria used in this project: ......................................................................... 33 2.2 Table 8. AMPs used in this project: ............................................................................ 34 2.3 Table 9. Antibiotics used in this project: .................................................................... 34 2.4 Donors for the Project: ................................................................................................ 34 2.5 Reconstitution of Antibiotics: ..................................................................................... 35 2.6 Reconstitution of Lyophilised LL-37: ........................................................................ 36 2.7 Broth Used for Bacterial Culture: ............................................................................... 36 2.8 RPMI + 5% TSB Used for Bacterial Culture: ............................................................ 36 2.9 Sub-culturing of Bacterial Stocks: .............................................................................. 37 2.10 Growth Curves and Mid-Log Stocks: ......................................................................... 37 2.11 Viability Checks for the Mid-Log Bacterial Stocks: .................................................. 37 2.12 Minimum Inhibitory Concentration of Antibiotics: .................................................... 38 2.13 Bacteriostasis Experiments: ........................................................................................ 39 2.14 Combination of Antibiotics and Antimicrobial Peptides Experiment: ....................... 40 2.15 Statistical Test Conducted on the Resulting Data: ...................................................... 40 3 Chapter 3 Results: ................................................................................................................... 3.1 Growth Curves of Sensitive and Resistant Sepsis Causing Bacteria: ......................... 43 3.2 Minimum Inhibitory Concentrations of Sensitive and Resistant Sepsis Causing Bacteria: .................................................................................................................................. 43 3.3 Determining Bacteriostasis Using Different Blood Volumes and Bacterial Concentrations: ....................................................................................................................... 49 3.4 Determining Bacteriostasis Using Different Bacterial Concentrations in a Fixed Blood Volume: .................................................................................................................................. 54 3.5 Determining Bacteriostasis Using Different Bacterial Concentrations, Fixed Blood Volume and Reduced Incubation Time: ................................................................................. 61 3.6 Determining the Optimum AMP and Antibiotic Combination that Inhibits Common Sensitive and Resistant Sepsis Causing Bacteria: ................................................................... 64 4 Chapter 4 Discussion: ............................................................................................................. 4.1 Discussion of Previous Research: ............................................................................... 71 4.1.1 AMP Monotherapy Activity Against the Bacteria Used in the Study: ............... 71 4.1.2 AMP and Antibiotic Combination Effect on the Bacteria Used in the Study: .... 76 4.1.3 The Donor Blood Sample’s Effect on the Bacteria Used in the Study ............... 82 4.2 Conclusion: ................................................................................................................. 84 4.3 Limitations: ................................................................................................................. 86 5 References:.......................................................................................................................... 88 vii | P age CHAPTER 1: INTRODUCTION/ LITERATURE REVIEW 1 Chapter 1 Introduction/Literature Review 1.1 Sepsis Definition: Sepsis is an infection, usually bacterial, of normally sterile parts of the body and leads to systemic illness and a panoply of non-selective inflammatory responses that are non- specific (1). Common sites include the respiratory tract (the most common site), skin, urinary tract, the peritoneal cavity and other places, the origin can be from indwelling catheters or any devices put into the patient, trauma can also allow bacterial entry such as burns and major surgery (2, 3). The respiratory tract, is associated with the highest sepsis mortality, in men and alcoholics (3). Women are more prone to urogenital site infection (3). Other common sites are endocarditis, device related, Central Nervous System, soft tissues, abdomen and skin (3). The presence of infection differentiates sepsis from identical clinical syndromes that can occur from several non-microbial conditions, pancreatitis being the archetype (1). Sepsis is also a multi-step process involving an uncontrolled inflammatory response by the patient’s cells, the result leads to multi-organ failure and death (4). Finally, sepsis can be seen as the final common pathway of many different infectious pathways such as parasitic, viral, fungal and bacterial infections in high risk groups (5). The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3) states that sepsis should be defined as “life-threatening organ dysfunction caused by a dysregulated host response to infection. For clinical operationalization, organ dysfunction can be represented by an increase in the Sequential [Sepsis-related] Organ Failure Assessment (SOFA) score of 2 points or more, which is associated with an in- hospital mortality greater than 10%. Septic shock should be defined as a subset of sepsis in which particularly profound circulatory, cellular, and metabolic abnormalities are 1 | P age associated with a greater risk of mortality than with sepsis alone” as seen in Table 1 from (6). Table 1. SOFA scores representing increasing mortality probability of the patient (6). Score System 0 1 2 3 4 Respiration ≥400 (53.3) <400 (53.3) <300 (40) <200 (26.7) <100 (13.3) PaO /FIO , mm without with 2 2 Hg (kPa) respiratory respiratory support support Coagulation ≥150 <150 <100 <50 <20 Platelets, x103/µL Liver <1.2 (20) 1.2-1.9 2.0-5.9 (33- 6.0-11.9 (102- >12.0 (204) Bilirubin, mg/dL (20-32) 101) 204) (µmol/L) Cardiovascular MAP ≥70 MAP <70 Dopamine <5 Dopamine 5.1- Dopamine >15 mm Hg mm Hg or dobutamine 1.5 or or epinephrine (any dose)1 epinephrine or >0.1 or norepinephrine norepinephrine ≤0.11 >0.11 Central Nervous 15 13-14 10-12 6-9 <6 System Glasgow Coma Score2 Renal <1.2 (110) 1.2-1.9 2.0-3.4 (171- 3.5-4.9 (300- >5.0 (440) Creatinine, mg/dL (110-170) 299) 440) (µmol/L) Urine output, <500 <200 mL/d 1. The dose of catecholamine are given for at least an hour as µg/kg/min. 2. The range of the Glasgow Coma Score are 3-15; a higher score means a better neurological function 1.2 Sepsis Epidemiology and Pathophysiology: 1.2.1 The Pathophysiology of Sepsis: When bacteria replicate within a patient they release antigens and other active factors, such as endotoxins from the Gram-negative (GN) bacteria and exotoxins from the Gram-positive (GP) bacteria, activating an immune response to clear the invading bacteria (2). The activated immune system releases cytokines including, Tumor Necrosis Factor (TNF)-α, IL-1β, IL-2, IL-6, IL-8, and Interferon IFN-γ, which further activate neutrophils, monocytes, and endothelial cells (2, 7). As well as the cellular and cytokine involvement in bacterial sepsis, there is also involvement from the 2 | P age