Student Theses and Dissertations

Date of Award

2014

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Fischetti Laboratory

Abstract

Streptococcus suis infects pigs worldwide and may be zoonotically transmitted to humans with a mortality rate of up to 20%. Methicillin-resistant Staphylococcus aureus (MRSA) and Streptococcus pyogenes (group A streptococci – GrAS) cause potentially fatal human diseases. These are just three of the many Gram-positive pathogens for which resistance to leading antibiotics has emerged. The goal of this work was to develop a novel antimicrobial treatment to combat these and other antibiotic-resistant pathogens. We identified a novel bacteriophage lysin, derived from an S. suis phage termed PlySs2 (phage lysin from S. suis 2). This thesis is divided into four main sections detailing PlySs2: characterization (chapter 2); activity against S. suis (chapter 3); broad lytic activity (chapter 4); and efficacy in vivo (chapter 5). PlySs2 has an N-terminal CHAP catalytic domain and a C-terminal SH3b binding domain. It is stable at 50°C for 30 min, 37°C for >24 h, 4°C for 15 days, and -80°C for >7 months; it maintained full activity after 10 freeze-thaw cycles. PlySs2 displays potent lytic activity against most strains of S. suis including the type strain S735, the pathogenic serotype 2, strain 10, and the pathogenic serotype 9 strain 7997. At 64 μg/ml, PlySs2 reduced multiple strains of S. suis by 6-logs within 1 hour in vitro. PlySs2 exhibited a minimum inhibitory concentration (MIC) of 32 μg/ml for S. suis strain S735 and 64 μg/ml for strain 7997. While resistance to gentamicin was observed after systematically increasing levels of gentamicin in an S. suis culture, the same protocol resulted in no observable resistance to PlySs2. The bacteriophage lysin PySs2 also exhibits broad lytic activity against MRSA, vancomycin-intermediate S. aureus (VISA), Streptococcus suis, Listeria, Staphylococcus simulans, Staphylococcus epidermidis, Streptococcus equi, Streptococcus agalactiae (group B streptococci – GBS), S. pyogenes, Streptococcus sanguinis, group G streptococci (GGS), group E streptococci (GES), and Streptococcus pneumoniae. PlySs2 at 128 μg/ml in vitro reduced MRSA and S. pyogenes by 5-logs and 3-logs within 1 hour respectively, and exhibited a minimum inhibitory concentration (MIC) of 16 μg/ml for MRSA. Serially increasing exposure of MRSA and S. pyogenes to PlySs2 or mupirocin resulted in no observed resistance to PlySs2 and resistance to mupirocin. The relevance of our in vitro work was confirmed with multiple in vivo experiments. Using a single 0.1-mg dose of PlySs2, the colonizing S. suis strain 7997 was reduced from the murine intranasal mucosa by >4 logs; a 0.1-mg dose of gentamicin reduced S. suis by <3-logs. A combination of 0.05 mg PlySs2 + 0.05 mg gentamicin reduced S. suis by >5-logs. In protecting against mixed infections, a single, 2-mg dose of PlySs2 protected 92% (22/24) of the mice in a bacteremia model of dual MRSA and S. pyogenes infection. This is the first known lysin with broad activity against multiple serotypes and strains of S. suis, making it a vital tool in the treatment and prevention of S. suis infections in pigs and humans. To date, no other lysin has shown such notable broad lytic activity, stability, and efficacy against multiple, leading, human bacterial pathogens; PlySs2 has all the characteristics to be an effective therapeutic.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

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