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

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 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

<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

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

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