Rapid Killing of Streptococcus pneumoniae with a Bacteriophage Cell Wall Hydrolase J. M. Loeffler, D. Nelson, V. A. Fischetti

11.

All chemicals were purchased from Sigma (St. Louis, MO) unless stated otherwise. E. coli were harvested by centrifugation, suspended in enzyme buffer (20 mM phosphate buffer (PB), 1 mM EDTA, 10 mM DTT) and broken by sonication for 1.5 min on ice. The crude extracts were ultracentrifuged (75,000g for 1 h at 4°C), the supernatant loaded on a DEAE-cellulose column (volume 20 ml) and washed with 3 volumes of 20 mM PB (pH 7.0), 4 volumes of PB containing 1 M NaCl, and 2 volumes of PB containing 0.1 M NaCl. The enzyme was eluted with PB containing 0.1 M NaCl and 6.5% (w/v) choline. Pooled fractions were dialyzed overnight (1:75) against enzyme buffer. Purification was verified by SDS-PAGE. Protein content was measured with the Bradford method using the dye reagent from Biorad (Hercules, CA).

12.

S. pneumoniae strain DCC 1490 (serotype 14) was grown in brain heart infusion medium (BHI, Difco Laboratories, Detroit, MI.) at 37°C to log-phase, centrifuged at 5000g for 10 min at 4°C, and resuspended in sterile saline to an absorbance at 600 nm of 1.3. Pal was diluted in enzyme buffer in serial 2-fold dilutions. In a 96-well plate 150 l of the bacterial suspension was incubated with 150 l of each Pal dilution (150 l enzyme buffer for the control well). One unit of enzyme was defined as the reciprocal of the highest dilution, which caused a 50% decrease in absorbance after 15 min incubation at 37°C, as compared with the absorbance of the control well.

13.

Pneumococci and viridans streptococci were grown in BHI and Todd-Hewitt broth with 1% yeast extract (Difco Laboratories, Detroit, MI.) to stationary or log-phase, centrifuged and resuspended to an absorbance at 600 nm of 1.0. In a 96-well plate, 150 l of the bacterial suspension were incubated with 150 l of Pal (to a final concentration of 100, 1000 and 10000 U/ml) at 37°C. Samples were assayed in triplicates. At 30 sec, 10 l of each well was serially 10-fold diluted and plated on blood agar for titer determination. A control well contained 150 l of the bacterial solution and 150 l enzyme buffer. The bacterial titer of the control well was determined at time 0 and 10 min. Control titers at an absorbance of 1.0 varied slightly for each strain (Log₁₀ 7.0 - 8.95 cfu/ml). Groups were compared with the Mann-Whitney test. A p value < 0.05 was considered significant.

19.

S. pneumoniae strain DCC 1490 was grown in BHI to log-phase, centrifuged and resuspended in sterile saline to an absorbance at 600nm of 1.0. 500 l of the suspension were incubated at room temperature with 500 l of Pal at a final concentration of 50 U/ml. The lytic reaction was stopped after 10 sec, 1 min and 5 min by addition of glutaraldehyde (final concentration 2.5%). Bacteria and debris were pelleted by centrifugation and overlaid with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4). The samples were then postfixed in 1% osmium tetroxide, block stained with uranyl acetate and processed according to standard procedures by The Rockefeller University Electron Microscopy Service.

21.

S. pneumoniae strain DCC 1490 was grown to log-phase, centrifuged and resuspended to a predefined titer of 10¹⁰ cfu/ml. Swiss CD-1 mice (weight range 22 to 24 g, Charles River Laboratories, Wilmington, MA) were anesthetized with a mixture of ketamine (Fort Dodge Animal Health, Fort Dodge, IA, 1.2 mg/animal) and xylazine (Miles Inc., Shawnee Mission, KS, 0.25 mg/animal), and inoculated in one nostril with 10 l of the bacterial suspension (n = 18) or 10 l of sterile saline (n = 3). Fourty-two hours later, inoculated animals were again anesthetized and 25 l of Pal (350 U, n = 9) or enzyme buffer (n = 9) was instilled in each nostril over several minutes. The mouth of each animal was rinsed with additional 50 l Pal (700 U). Five hours later, all animals were euthanized and the nasal cavity was washed through the dissected trachea with 60 l of sterile saline. The nasal wash was serially 10-fold diluted and plated on blood agar for titer determination. The following day, a-hemolytic colonies were respread on blood agar and incubated with an optochin disk (BBL, Sparks, MD). Bacteria with a zone of inhibition > 14mm were considered to be S. pneumoniae. Groups were compared with the Mann-Whitney test.

22.

Exposure on agar plates: Strain DCC 1490 was grown in BHI to early log- phase, swabbed on blood agar plates and exposed to 10(l of a low concentration of Pal (<10 U/ml). Colonies at the periphery of a clearing zone were picked, grown to log-phase, swabbed on a fresh plate and re-exposed to the same concentration of Pal. Exposure in liquid: Strain DCC 1490, grown to log-phase was harvested and suspended in saline to an OD at 600nm of 1.0. One ml of bacteria was incubated with 1ml of Pal at 1.5 U/ml for 2 min at 37(C (reducing the bacterial titer by Log₁₀ 0.7 cfu/ml). The reaction mixture with the surviving bacteria was then diluted 1:10 in fresh BHI and grown overnight. The next day the culture was again diluted 1:10 in fresh BHI, grown to log-phase as above and mixed with 15 U and 150 U in the following 2 rounds (reducing the titer by Log₁₀ 1.4 and 3.9 cfu/ml, respectively). After 16 rounds of exposure on agar or 3 rounds of exposure in liquid, susceptibility to Pal was tested using the in vitro killing assay (13), compared to the unexposed strain. Results were compared with the Mann-Whitney test. No significant difference could be shown.

Supplemental Figure 1. Observation of colonization status after a single treatment with Pal 1400 U/ml vs. buffer. Buffer-treated mice remained colonized throughout the duration of the experiment, with titers decreasing from day 6. In animals treated with Pal enzyme, S. pneumoniae reappeared discretely from day 2. However, titers did not reach a level necessary for successful recolonization in this model (p < 0.0001 for the comparison of both curves). One control animal was tested on days 0, 4, and 8.

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Supplemental Table 1. Bacterial strains tested for susceptibility to Pal.
Species	Strain	Capsular group type	Susceptibility to Penicillin*	Clonal type	Source
S. pneumoniae	DCC 1355	19F	S		1
S. pneumoniae	DCC 1335	9V	R	Sp9-3	1
S. pneumoniae	DCC 1420	23F	R	Sp23-1	1
S. pneumoniae	DCC 1476	15	I		1
S. pneumoniae	DCC 1490	14	S		1
S. pneumoniae	DCC 1494	14	R	Sp14-1	1
S. pneumoniae	DCC 1714	3	S		1
S. pneumoniae	DCC 1808	24	S		1
S. pneumoniae	DCC 1811	11	S		1
S. pneumoniae	DCC 1850	6B	S		1
S. pneumoniae	AR 314	5	S		1
S. pneumoniae	AR 620	1	S		1
S. pneumoniae	GB 2017	18	S		1
S. pneumoniae	GB 2092	4	S		1
S. pneumoniae	GB 2163	10	S		1
S. pneumoniae	R36A				1
S. pneumoniae	Lyt 4-4				1
S. gordonii	PK 2565				2
S. mitis	J 22				2
S. mutans	OMZ 175				3
S. oralis	H 1				2
S. salivarius	ATCC 27945				2
*R, resistant; I, intermediate; S, susceptible. 1, Alexander Tomasz, The Rockefeller University, New York, NY; 2, Paul Kohlenbrander, National Institute of Dental and Craniofacial Research, Bethesda, MD; 3, Ivo Van de Rijn, Wake Forest University, Winston-Salem, NC.