Isolation and Characterization of a Bacteriophage Specific for Neisseria perfiava

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, JUly 1976, p Copyright 1976 American Society for Microbiology Vol. 4, No. 1 Printed in U.S.A. Isolation and Characterization of a Bacteriophage Specific for Neisseria perfiava V. I. STEINBERG,* E. J. HART, J. HANDLEY, AND I. D. GOLDBERG Department of Microbiology, University of Kansas Medical Center, Kansas City, Kansas 66103,* and Department of Microbiology, The University of Kansas, Lawrence, Kansas Received for publication 24 March 1976 Six isolates from normal throat samples have been shown to contain phage active against Neisseria perflava. The phage isolates were similar in terms of host range, latent period, burst size, antigenic properties, morphology, and nucleic acid content. Neutralization studies with antisera demonstrated that the isolates exhibited a very high degree of serological relatedness. These results taken together suggested that the isolates represented a single strain of bacteriophage. This phage, which we have designated NP-1, exhibited a high degree of host specificity, attacking only one of the several strains ofn. perfiava tested and none of the other species tested. One-step growth experiments yielded minimum latent periods of approximately 35 min; average burst sizes varied from 34 to 63 plaque-forming units per cell. Electron micrographs revealed particles with heads averaging 75 nm in diameter and tails averaging 300 nm in length and 18 nm in diameter. The phage contained double-stranded DNA with a guanine plus cytosine content of 38%. In the absence of adequate serological methods for following the epidemiology of Neisseria gonorrhoeae, the availability of a phage typing system would be of great value for strain differentiation. Although several laboratories have searched intensively for phages able to attack the pathogenic species of Neisseria, no reproducible systems have been established. One indirect approach to the problem is the isolation and characterization of phages against a nonpathogenic species ofneisseria. From the information gained, one would be in a better position to predict the probable nature of a gonococcal phage in terms of filterability, stability, degree of host specificity, and perhaps other characteristics. Bacteriophages able to attack nonpathogenic species of Neisseria have been described previously. A bacteriophage specific for N. perflava was first isolated by Stone et al. (8). They demonstrated propagation of the phage and studied its host range and morphological characteristics by electron microscopy. The phage exhibited extreme specificity for N. perflava and did not plaque on the other related species tested: N. subflava, N. sicca, N. flavescens, Branhamella catarrhalis, or N. meningitidis. Phelps (6) has isolated 16 bacteriophages specific for nonpathogenic Neisseria species. These phages have been divided into five groups on the basis of serology and host preference. Because of the absence of a detailed study of a Neisseria phage, we have investigated a phage specific for N. perfiava. This communication will describe the isolation and characterization of this phage in terms of host range, latent period, burst size, antigenic properties, particle morphology, and nucleic acid content. MATERIALS AND METHODS Isolation of bacteriophages. Six separate isolates were obtained from normal throat specimens. Throat swabs were incubated overnight with actively growing cells of N. perflava LS. The enrichment cultures were centrifuged at 8,000 rpm for 15 min (Sorvall RC-2B, rotor SS34) and filtered through membrane filters (0.45,tm, Millipore Corp.), and dilutions of the supernatant fluid were spotted onto lawns of N. perfiava LS indicator cells. Plates were checked for the appearance of plaques. Isolates were purified by at least three successive single-plaque transfers. These were tentatively designated LK1, LK2, LK3, LK4, UK1, and UK2. Propagation of host bacteria. Bacterial strains and their sources are listed in Table 1. N. perflava was routinely grown in broth containing Trypticase, phytone, and yeast extract (TPY) (6) and on Trypticase soy agar (BBL) plates. For host range studies, N. perfiava, N. flava, N. flavescens, and B. catarrhalis were grown in GC broth plus defined supplements (GCBB-DS) (3). N. lactamica and N. gonorrhoeae were grown in the biphasic medium of Sparling (7). All strains were plated on GC medium base (Difco) plus defined supplements (GCBA-DS). Phage assay. Phages were assayed by the soft agar overlay procedure (1). Trypticase soy agar plates were overlaid with 3 ml of TPY soft agar 87

2 88 STEINBERG ET AL. J. CLIN. MICROBIOL. (TPY broth plus 0.4% agar) containing 0.1 ml of N. perflava cells (3 x 108 to 5 x 108 colony-forming units [CFU]/ml) and 0.1 ml of phage. The plates were incubated for 16 to 20 h at 370C in the presence of 10% CO2 and humidity; CO2 was not essential for production of plaques but plaques were larger in its presence. Plaques were counted, and phage counts were expressed as plaque-forming units (PFU) per ml. Phage propagation. Phages were propagated on plates using the soft agar mascerate procedure (1). Cells (0.1 ml of culture containing 108 CFU/ml) and phage (0.1 ml of lysate containing 106 PFU/ml) were added to 3 ml of soft agar to give confluent lysis on the plates. The soft agar layers were harvested in 5 ml oftpy broth after incubation as described above. The suspensions were centrifuged at 8,000 rpm (Sorvall RC-2B, rotor SS34) for 15 min to remove agar and cells. The supernatant fluids were sterilized by filtration through 0.45-,um membrane filters (Gelman) on a Millipore filter apparatus with a stainless-steel screen filter base. Gelman filters were chosen because significant phage loss occurred with most of the other membranes tested (Table 2). Phages propagated by this method yielded titers of 1 x 10"' to 5 x 1010 PFU/ml. Phage lysates were stored either at 4 C or frozen at -20 C after the addition of 10% dimethylsulfoxide (10). The phage was very stable and no loss of viability was noted. Host range studies. GCBA-DS plates were overlaid with 3 ml of TPY soft agar containing cells (approximately 5 x 107 CFU). Tenfold dilutions (undiluted through 10-7) of phage were made. A portion (0.01 ml) of each phage dilution was spotted onto the preseeded plates, and the plates were incubated as described for phage assays. Plates were scored for lysis and formation of plaques. One-step growth experiments. One-step experiments were performed as described by Adams (1) at a multiplicity of infection of Phage was allowed to adsorb for 8 min. Samples were withdrawn at 8- min intervals for a total of 72 min and assayed for PFU. Concentration of phage. Phage particles were precipitated by the polyethylene glycol (PEG) method (9). A stock solution of PEG was made by adding PEG 4000 (Matheson Scientific, Inc.) and NaCl to TPY broth. The mixture was autoclaved and then added to phage lysates so that the final concentration of PEG was 5% and the final concentration of NaCl was 0.5 M. The mixture was incubated for 1 h at 40C with occasional shaking and then was centrifuged at 8,000 rpm (Sorvall RC-2B, rotor SS34) for 10 min. Recovery of phage particles TABLE 1. Host range studies -ability ofphages to form plaques on strains of Neisseria Isolates" Indicator strain Source LK1 LK2 LK3 LK4 UK1 UK2 N. perflava LS D. S. Kellogg N. perflava N7 D. S. Kellogg N. perflava ATCC N. flava ATCC N. flavescens ATCC N. lactamica ATCC B. catarrhalis ATCC - NT - NT NT N. gonorrhoeae ATCC L L L L L L N. gonorrhoeae F62 type T4 D. S. Kellogg - NT NT NT NT NT " +, Lysis and formation of plaques at dilutions undiluted through 10-7; -, no lysis or no plaques; NT, not tested; L, lysis not due to plaques. TABLE 2. Effect of filtration with various types of membranes on phage titer" PFU/ml Membrane Filter apparatus Millipore Gelman Sartorius Selas 0.45,um 0.65,um 0.8 /Am 0.45 Am 0.2,um (0.45 Mm) (0.45 Mum) Control (before filtration) 2.2 x 10"' Millipore with fritted glass base 1.5 x x x x x 109 <1.0 X x 10'0 Millipore with stainless-steel 3.0 x x 10" 4.8 x x x x x 1010 screen filter base Nalgene filter unit (0.45,um) <1.0 x 107 Sintered glass (ultrafine) filter 2.9 x 1010 " Isolate LK4 was propagated and centrifuged as described in Materials and Methods. The supernatant was assayed before and after filtration.

3 VOL. 4, 1976 using this procedure was 100%. The pellet was resuspended in fresh TPY broth. Phage antiserum. Antiserum to each of the phages was prepared in male New Zealand white rabbits weighing 5 to 5.5 pounds (23 to 25 kg). Preimmune sera were collected from all animals before injection. Concentrated phage (1 ml) varying in titer from 1 x 1010 to 5 x 1011 PFU/ml was injected subcutaneously twice a week for 3 weeks. Animals were bled 5 days later, and antisera were tested for the ability to neutralize phage. If the antiserum was not of a sufficient titer, the animals were given 2-ml booster injections. Sera were sterilized by filtration and heated at 56 C for 30 min to inactivate complement. Neutralization of phage. Antiserum was tested for its ability to neutralize phage using a modification of the method of Adams (1). Serum (0.1 ml) was added to 0.9 ml of phage containing approximately 107 PFU. The mixture was incubated, dilutions were made in TPY broth, and samples were assayed for the number of PFU remaining. Incubation for the preliminary testing of serum was for 10 min at ambient temperature (29 C). Antiserum that would neutralize 90 to 100% of phage when diluted 1:10 was considered to be of sufficiently high titer for the neutralization K studies. Incubation for neutralization K studies was at 37 C. Samples were withdrawn at 5, 10, 15, and 20 min, diluted, and assayed for the number of PFU. K values were calculated as described by Adams (1). DNA extraction. DNA was isolated from concentrated phage by means of phenol extraction (4). Equal volumes of redistilled phenol and phage were added to a tube and mixed for 10 min by gentle rolling of the tube. The mixture was centrifuged at 10,000 rpm (Sorvall RC-2B, rotor SS34) for 30 min. The aqueous layer was removed and reextracted with phenol. After the second extraction the aqueous layer was removed and dialyzed against sterile SSC (SSC=0.15 M NaCl M sodium citrate, ph 8.0). DNA was quantitated by the diphenylamine method of Burton (2). Determination of TM. The DNA sample was precipitated in 95% ethanol and the precipitate was dissolved in 0.01 M potassium phosphate plus M EDTA (ethylenediaminetetraacetic acid), ph 7.0. T. values were determined by the method of Marmur and Doty (5) using a spectrophotometer (Beckman DB-G) with jacketed quartz cuvettes. The temperature of the sample was measured using a telethermometer probe placed directly into the sample cuvette. The temperature of the sample and the blank was raised slowly by circulating heated water through the jackets of the cuvettes using a circulating water pump. Electron microscopy. Phage was concentrated and suspended in 2% ammonium acetate to a titer of approximately 1012 PFU/ml. This preparation was put on a grid, dried, and negatively stained with 2% sodium silicotungstate, ph 7.3, for about 10 s. The grids were examined in an electron microscope (RCA EMU-3F2) at 50 kv, and electron micrographs were made. N. PERFLAVA BACTERIOPHAGE 89 RESULTS AND DISCUSSION The six phage isolates came from two different sources and formed plaques that differed in size, definition of edge, and degree of clarity. The isolates were treated separately in the following experiments, which were designed to characterize them in terms of latent period, burst size, host range, serological relatedness, and particle morphology. One-step growth experiments. One-step growth experiments were done for several of the phage isolates. The isolates exhibited minimum latent periods of 35 min and average burst sizes that varied from 35 to 63 PFU per infective center. Host range studies. The plaque-forming ability of all six isolates was tested on N. per- /lava LS and other related strains and species (Table 1). None of the isolates was able to form plaques on N. perflava N7, N. perflava 14799, N. flava, N. flavescens, N. lactamica, or N. gonorrhoeae No plaques were seen on N. gonorrhoeae F62 or on B. catarrhalis with the isolates tested. Lysis was visible when each of the undiluted and diluted (dilutions 10-' and 10-2) isolates was spotted on N. gonorrhoeae However, plaques were never seen. The lytic ability of the isolates could not be reduced by ultraviolet treatment sufficient to decrease the number of PFUs per milliliter by 107-fold. Using antiserum, isolates containing 10'0 PFU/ml were neutralized to 104 PFU/ml. This treatment was also insufficient to reduce the lytic ability of the isolates on N. gonorrhoeae We concluded that the lysis seen on this particular strain of N. gonorrhoeae was not caused by phage particles but was due to an unknown component in the lysates. It is not unusual to find that a phage, although unable to form plaques on a given strain of bacteria, may still exhibit an extended host range as measured by adsorption. Experiments were done to compare adsorption of phage to N. perflava LS (99.9%) with adsorption to N. gonorrhoeae and other Neisseria species. However, the maximum level of adsorption with these nonhomologous species was only 30%. Adsorption was determined based on unadsorbed (free) phage because of the absence of infective centers. As a result, the sensitivity of the system was very low and it could not be determined if a level of 30% represented true adsorption. Phage neutralization. The results of the burst size, latent period, and host range studies suggested that all of the isolates could possibly

4 90 STEINBERG ET AL. represent the same phage. To test whether or not the phages were closely related serologically, antiserum against LK4 and antiserum against UK1 were tested against all six isolates. The rates of inactivation of all six isolates with these antisera were very similar (Table 3, Fig. 1). Though the possibility exists that minor antigenic differences are present, these results suggest strongly that the isolates are serologically identical. Electron microscopy. Phages UK1 and LK1 were examined by electron microscopy. The heads were polyhedral, averaging 75 nm in diameter, and the tails averaged 300 nm in length and 18 nm in diameter (Fig. 2). The size differs from that reported for other Neisseria phages (6, 8). Stone's phage had a head measuring 50 by 75 nm and a tail measuring 160 by 20 nm. Phelps reported two sizes of phage particles. One had a head measuring 70 nm in diameter and a tail 150 by 15 nm. The other phage was 10% larger. Nucleic acid. Nucleic acid was isolated from the phage isolates and shown to be deoxyribonucleic acid (DNA) by the diphenylamine test (2). DNA from LK2, LK4, LK1, and UK1 was thermally denatured. A sharp hyperchromatic shift was seen at 260 nm, and it was concluded that the DNA was double stranded. A T,,, of 65 C was obtained in potassium-edta buffer, which corresponds to an average guanine plus cytosine content of 38%. This value is lower than that given in the literature (5) for the T,,, TABLE 3. Neutralization ofphages with antiserum prepared against isolate LK4' Phage titer (PFU/ml) Neutralization Isolate Control in In antiserum preimmune se- to LK4 % K rum LK1 1.7 x x 10' LK2 3.1 x x LK3 1.6 x x LK4 1.2 x x 10" UK1 1.4 x x UK2 2.3 x x 10" 89 -b UK2c 2.3 x x a Antiserum was diluted at 1:200 and added to phage isolates. The mixture was incubated at 37 C for 10 min. ba K value has not been calculated because the percentage of neutralization is outside the range of neutralization (90 to 99%) necessary for the valid determination of a K value. ' Incubation was for 15 min. 0 z w -E LA- a- 0 w o z J. CLIN. MICROBIOL TIME (MIN) OF INCUBATION WITH ANTISERUM FIG. 1. Neutralization of isolates UK1, UK2, LK1, and LK2 by antiserum prepared against UK1. Symbols: (0) UK1; (A) UK2; (0) LK1; (O) LK2. ofn. perflava (90 C in SSC), which corresponds to a guanine plus cytosine content of 49%. Evidence from one-step growth experiments, neutralization, host range, and morphological and nucleic acid studies has shown that our six isolates probably represent the same phage for N. perflava LS. We have designated the phage NP-1. This double-stranded DNA phage has a minimum latent period of 35 min and an average burst size varying from 34 to 63. By electron microscopy the phage has a head measuring 75 nm in diameter and a tail 300 nm in length. This phage is extremely specific for N. per/lava LS and will not form plaques on the other strains ofneisseria and the one species ofbranhamella tested. We gratefully acknowledge several discussions with Ovidio J. Mira concerning the immunological aspects of this study. We wish to express our appreciation to Susan Talley for her valuable assistance. This research was supported by a Public Health Service grant AI from the National Institute of Allergy and Infectious Diseases.

5 VOL. 4, 1976 N. PERFLAVA BACTERIOPHAGE 91 * rp 4L #L Downloaded from U J LITERATURE CITED 1. Adams, M. H Bacteriophages. Interscience Publishers, Inc., New York. 2. Burton, K A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem. J. 62: Kellogg, D. S., Jr., W. L. Peacock, Jr., W. E. Deacon, L. Brown, and C. I. Pirkle Neisseria gonorrhoeae. I. Virulence genetically linked to clonal variation. J. Bacteriol. 85: Mandell, J. D., and A. D. Hershey A fractionating column for analysis of nucleic acids. Anal. Biochem. 1: Marmur, J., and P. Doty Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature. J. Mol. Biol. FIG. 2. Electron micrograph of isolate LK1. (x62,000) t. 5: Phelps, L. N Isolation and characterization of bacteriophages for Neisseria. J. Gen. Virol. 1: Sparling, P. F Genetic transformation of Neisseria gonorrhoeae to streptomycin resistance. J. Bacteriol. 92: Stone, R. L., C. G. Culbertson, and H. M. Powell Studies of a bacteriophage active against a chromogenic Neisseria. J. Bacteriol. 71: Yamamoto, K. R., B. M. Alberts, R. Benzinger, L. Lawhorne, and G. Treiber Rapid bacteriophage sedimentation in the presence of polyethylene glycol and its application to large scale virus purification. Virology 40: Yehle, C. O., and R. H. Doi Stabilization of Bacillus subtilis phage with dimethylsulfoxide. Can. J. Microbiol. 11: on October 5, 2018 by guest