Studies of a Novel Phage Lytic Enzyme, PlySs2

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 \u3e24 h, 4°C for 15 days, and -80°C for \u3e7 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 \u3e4 logs; a 0.1-mg dose of gentamicin reduced S. suis by \u3c3-logs. A combination of 0.05 mg PlySs2 + 0.05 mg gentamicin reduced S. suis by \u3e5-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

Impact of opioid-free analgesia on pain severity and patient satisfaction after discharge from surgery: multispecialty, prospective cohort study in 25 countries

Background: Balancing opioid stewardship and the need for adequate analgesia following discharge after surgery is challenging. This study aimed to compare the outcomes for patients discharged with opioid versus opioid-free analgesia after common surgical procedures.Methods: This international, multicentre, prospective cohort study collected data from patients undergoing common acute and elective general surgical, urological, gynaecological, and orthopaedic procedures. The primary outcomes were patient-reported time in severe pain measured on a numerical analogue scale from 0 to 100% and patient-reported satisfaction with pain relief during the first week following discharge. Data were collected by in-hospital chart review and patient telephone interview 1 week after discharge.Results: The study recruited 4273 patients from 144 centres in 25 countries; 1311 patients (30.7%) were prescribed opioid analgesia at discharge. Patients reported being in severe pain for 10 (i.q.r. 1-30)% of the first week after discharge and rated satisfaction with analgesia as 90 (i.q.r. 80-100) of 100. After adjustment for confounders, opioid analgesia on discharge was independently associated with increased pain severity (risk ratio 1.52, 95% c.i. 1.31 to 1.76; P < 0.001) and re-presentation to healthcare providers owing to side-effects of medication (OR 2.38, 95% c.i. 1.36 to 4.17; P = 0.004), but not with satisfaction with analgesia (beta coefficient 0.92, 95% c.i. -1.52 to 3.36; P = 0.468) compared with opioid-free analgesia. Although opioid prescribing varied greatly between high-income and low- and middle-income countries, patient-reported outcomes did not.Conclusion: Opioid analgesia prescription on surgical discharge is associated with a higher risk of re-presentation owing to side-effects of medication and increased patient-reported pain, but not with changes in patient-reported satisfaction. Opioid-free discharge analgesia should be adopted routinely

Best Practices for Foundations in Molecular Simulations [Article v1.0].

This document provides a starting point for approaching molecular simulations, guiding beginning practitioners to what issues they need to know about before and while starting their first simulations, and why those issues are so critical. This document makes no claims to provide an adequate introduction to the subject on its own. Instead, our goal is to help people know what issues are critical before beginning, and to provide references to good resources on those topics. We also provide a checklist of key issues to consider before and while setting up molecular simulations which may serve as a foundation for other best practices documents

The Phage Lysin PlySs2 Decolonizes <i>Streptococcus suis</i> from Murine Intranasal Mucosa

<div><p><i>Streptococcus suis</i> infects pigs worldwide and may be zoonotically transmitted to humans with a mortality rate of up to 20%. <i>S</i>. <i>suis</i> has been shown to develop <i>in vitro</i> resistance to the two leading drugs of choice, penicillin and gentamicin. Because of this, we have pursued an alternative therapy to treat these pathogens using bacteriophage lysins. The bacteriophage lysin PlySs2 is derived from an <i>S</i>. <i>suis</i> phage and displays potent lytic activity against most strains of that species including serotypes 2 and 9. At 64 μg/ml, PlySs2 reduced multiple serotypes of <i>S</i>. <i>suis</i> by 5 to 6-logs within 1 hour <i>in vitro</i> and exhibited a minimum inhibitory concentration (MIC) of 32 μg/ml for a <i>S</i>. <i>suis</i> serotype 2 strain and 64 μg/ml for a serotype 9 strain. Using a single 0.1-mg dose, the colonizing <i>S</i>. <i>suis</i> serotype 9 strain was reduced from the murine intranasal mucosa by >4 logs; a 0.1-mg dose of gentamicin reduced <i>S</i>. <i>suis</i> by <3-logs. A combination of 0.05 mg PlySs2 + 0.05 mg gentamicin reduced <i>S</i>. <i>suis</i> by >5-logs. While resistance to gentamicin was induced after systematically increasing levels of gentamicin in an <i>S</i>. <i>suis</i> culture, the same protocol resulted in no observable resistance to PlySs2. Thus, PlySs2 has both broad and high killing activity against multiple serotypes and strains of <i>S</i>. <i>suis</i>, making it a possible tool in the control and prevention of <i>S</i>. <i>suis</i> infections in pigs and humans.</p></div

PlySs2 and gentamicin may act additively to reduce <i>S</i>. <i>suis in vivo</i>.

<p>PlySs2 removed <i>S</i>. <i>suis</i> from the murine intranasal mucosa. CD-1^® mice were nasally colonized with the pathogenic <i>S</i>. <i>suis</i> strain 7997. Twenty-four hours after colonization, in each nostril, mice received 10 μl of either 50 mM PB, pH 7.4 (buffer C), 5 mg/ml PlySs2 in buffer C, 5 mg/ml gentamicin in buffer C, or a combination of 2.5 mg/ml PlySs2 and 2.5 mg/ml gentamicin in buffer C. <i>S</i>. <i>suis</i> CFU counts were calculated for the nasal passage of each mouse.</p

<i>S</i>. <i>suis</i> 7997 and S735 did not develop resistance to PlySs2 <i>in vitro</i>.

<p><i>S</i>. <i>suis</i> strain S735 or <i>S</i>. <i>suis</i> strain 7997 grew in media containing 1/32× (3.13%) to 4× (400%) the MIC of PlySs2 or gentamicin over 8 days. Comparing the MICs of PlySs2 after each day to the initial MIC of PlySs2 for each strain determined resistance. Neither developed resistance to PlySs2. Both <i>S</i>. <i>suis</i> strain S735 and <i>S</i>. <i>suis</i> strain 7997 developed resistance to the positive control, gentamicin. The fluctuation observed in this assay was +/- 1x MIC.</p

MIC of PlySs2 against <i>S</i>. <i>suis</i> strains^a.

<p>MIC of PlySs2 against <i>S</i>. <i>suis</i> strains<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169180#t001fn001" target="_blank">^a</a>.</p

PlySs2 displayed activity against almost all strains of <i>S</i>. <i>suis</i>.

<p>Bacteria in logarithmic growth were exposed to 32 μg/ml PlySs2 for 30 minutes in PB (for 60-minute readings, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169180#pone.0169180.s004" target="_blank">S3 Fig</a>). The activity was measured by OD₆₀₀ reduction. To normalize and combine values from multiple tests, the final OD₆₀₀ of the treated samples was divided by the final OD₆₀₀ of the untreated samples. An OD₆₀₀ ratio of 1.0 indicates no lysis, while an OD₆₀₀ ratio of ~0.02 indicates complete lysis.</p

PlySs2 was bactericidal to nearly all strains of <i>S</i>. <i>suis</i>.

<p>Bacteria were grown to log-phase. After exposure to 64 μg/ml PlySs2 in buffer A for 60 min in 96-well plates, bacteria were serially diluted and plated to BHI agar for CFU enumeration. The CFU numbers of most <i>S</i>. <i>suis</i> strains dropped by 5 to 6 logs after PlySs2 treatment including the type strain S735 and the pathogenic strains 10 and 7997. Death (log fold kill) was calculated as -log[(CFUs in the test condition) ÷ (CFUs in the control condition)].</p