REVIEWS OF INFECTIOUS DISEASES. VOL. I, NO.2. MARCH-APRIL 1979 © 1979 by The University of Chicago. 0162-0886 79/0102-0009$00.95

Bacteriophages of Bacteroides From the Anaerobe Laboratory, Virginia Polytechnic Institute and State University, Blacksburg, Virginia

S. J. Booth, R. L. Van Tassell, J. L. Johnson, and T. D. Wilkins

Sixty-eight bacteriophages specific for nine species (DNA homology groups) of Bacteroides were isolated from sewage. Four distinct morphological types were isolated, three of which had not previously been described. Attempts to use these phages to transduce Bacteroides [ragilis were unsuccessful. A total of 91 phage-susceptible strains of Bacteroides were used with these phages in a study of the feasibility of developing a scheme for identification of Bacteroides species. Blind trials were performed with 10 B. tragilis-specific phages and a collection of over 200 bacterial strains, including 144 strains of R. [ragilis. Of the strains of B. tragi/is, 78% were identified correctly within 24 hr. A phage-carrier state, or pseudolysogeny, was observed with most of the phage-host systems, and this state was studied in detail with B. fragilis phage Bf-I. The presence of a thick capsule around some cells in a pure culture of a host strain appears to render these cells resistant to phage infection, thus perpetuating the carrier state. It is suggested that such capsules may playa role in the virulence of strains of Bacteroides.

The Bacteroides fragilis group of bacteria includes all the species of what were formerly called subspecies of B. [ragilis. Although these bacteria are very similar to one another both morphologically and phenotypically, DNA homology studies have demonstrated that they are genetically distinct groups. For this reason the former subspecies of B. fragilis have been reinstated to species rank [1]. These species include B. fragilis, Bacteroides thetaiotaomicron, Bacteroides ovatus, Bacteroides distasonis, and Bacteroides uulgatus. Two previously recognized species, Bacteroides eggerthii and Bacteroides uniiormis, along with homology group 3452-A and B. fragilis subspecies a, are also included in this group of species [2, 3]. Species of Bacteroides are the anaerobes most frequently isolated from clinical specimens [4], and B. fragilis is the most frequently isolated species [5]. Yet precise identification of these Bacteroides species can be costly and time-consuming because of the biochemical tests and the chromatographic analysis that are required. Final

verification may even depend on DNA homology determinations. One possible aid in dealing with this problem of identification may be the susceptibility of Bacteroides species to highly specific bacteriophages. A phage identification scheme could be quicker and less costly than the methods presently used and could be useful in determining the role that less well-characterized species of Bacteroides play in disease processes. The initial isolation of a bacteriophage for a Bacteroides species was briefly reported by Sabiston and Cohl in 1969 [6]. These investigators isolated a virulent phage from sewage that was most active against B. distasonis. A year later Prevot et al. [7] described the isolation of several bacteriophages from clinical sources that showed extreme specificity for Ristella pseudoinsolita (B. fragilis). This report also gives evidence for a lysogenic strain of R. pseudoinsolita. In 1972 Nacescu et al. reported on two bacteriophages from sewage that were specifically lytic for 23 of 68 strains of B. fragilis [8]. Strains belonging to other species, both aerobic and anaerobic, were not susceptible to either phage. Additional evidence for lysogenic strains of B. fragilis was also presented. Although examination of pure cultures for inducible phage yielded consistently negative results, eight phage isolates were obtained from mixed cell cultures, presumably as a result of an induced prophage. This observation was not further substantiated.

This research was supported by grant no. AI-12137-03 from the National Institute of Allergy and Infectious Diseases. We thank Drs. L. V. Holdeman and W. E. C. Moore for characterizing many of the bacteria used in this study. Please address requests for reprints to Dr. T. D. Wilkins, Anaerobe Laboratory, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061.

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Also in 1972 one of these two phages, 9>Al, was further characterized as to its morphological and biological properties by Brandis et al. [9]. Phage 9>Al belonged to Bradley's morphological group B [10] and exhibited no peculiar properties with respect to latency, heat and pH stability, or absorption kinetics. Brandis et al. [9] were the first to mention the possibility of using the extreme specificity of Bacteroides phages in a typing scheme for this genus. In 1974 Keller and Traub characterized a group of bacteriophages that were isolated from animal sera and active on B. fragilis [11]. All 25 phages were specific for B. [ragilis, a fact that implied their usefulness in identification of species of Bacteroides. These authors also provided the first details of a phage-host carrier state, or pseudolysogeny. In the present work we characterized several bacteriophages specific for B. [ragilis, examined the potential for an identification scheme that uses a number of newly isolated phages, and briefly examined the carrier state. Materials and Methods

Bacterial strains and media. The strains of Bacteroides used for phage isolation were those from the culture collection at the Virginia Polytechnic Institute and State University Anaerobe Laboratory. They included isolates from clinical and normal human fecal flora and were from both domestic and foreign sources. These species of Bacteroides were identified by Drs. L. V. Holdeman and W. E. C. Moore. [12]; identifications were verified by DNA homology studies performed by one of us. The species and/or DNA homolo.gy groups and the number of strains (in parentheses) used for phage isolation included B. fragilis (79), B. vulgatus (10), B. ovatus (14), B. distasonis (12), B. thetaiotaomicron (12), B. unijormis (12), 3452-A DNA homology group (12), B. eggerthii (two), and subspecies a (two). The results of detailed studies of DNA homology of all groups have been published [2]. One hundred thirteen clinical isolates were received from and identified by Sharon Hansen at the Veterans Administration Hospital, Baltimore, Md. The identities of all these strains were verified by DNA homology experiments, and the strains were used

Booth et al.

in our identification trial. They were maintained in chopped meat broth and subcultured in brainheart infusion broth (Difco, Detroit, Mich.j supplemented with 5 /Lg of hemin/nil and 0.5% yeast extract (BHI-S broth) [12]. Isolation of bacteriophages. Samples of sewage water were collected from various sites (primary and secondary settling tanks, raw sewage, etc.) within the Blacksburg, Va., sewage treatment plant. The samples were pooled and passed through cheese cloth before being sequentially filtered through membrane filters (pore sizes, 0.65 and 0.45 /Lm, respectively; Millipore Corp., Bedford, Mass.). This filtrate was used to rehydrate brain-heart infusion broth (Difco, Detroit, Mich.), which was supplemented to make BHI-S broth. For bacteriophage enrichment, 10 ml of an overnight culture of a selected species and strain of Bacteroides was added to 100 ml of BHI-S in a 125-ml Erlenmeyer flask, which was then sealed with a rubber stopper. After incubation overnight at 37 C, the cells were removed by centrifugation (10,000 g for 20 min), and the supernatant was assayed for phages by a modification of the double-agar overlay method [13J. Some of the sewage samples were directly assayed for phages after membrane filtration. For some sewage samples antiserum prepared against one of the phages (Bf-I) was incorporated (final concentration, I: 1,000) in the media used for the enrichment and assay procedures. Each phage was purified by at least three successive single-plaque transfers and was designated by its numerical order of isolation (e.g., Bf-I, Bf-2, etc.). Propagation of phages. Stock cultures of phages (titers, from 108 to 5 X 1010 pfu/ml) were prepared either by growth of infected cells in broth or by harvesting from soft agar plates. For broth culture, 0.2 ml of an 18-hr BHI-S culture of cells was mixed with 0.2 ml of phage (1081010 pfu/ml), held anaerobically at 37 C for I hr to allow adsorption of phages, and transferred to BHI-S medium containing 10-3 M CaCl 2 plus 10-3 M MgCI 2. After incubation for 24-48 hr in 02-free CO 2 or N 2, cells were removed by centrifugation (10,000 g for 20 min), and the supernatant containing the phage was stored at 4 C over a small volume of chloroform. For those phages that produced low titers «10 8 pfu rml)

Bacteriophages of Bacteroides

when grown in broth, confluently lysed lawns of soft BHI-S agar (0.7% agar) were harvested as described by Adams [14]. Plates were incubated overnight at 37 C in an anaerobic glove box [I3, 15]. Phage assay. Phage dilutions were made in 0.01 M Hepes buffer (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; Sigma Chemical Co., St. Louis, Mo.) containing 10-3 M MgCl 2 plus 10-3 M CaCI 2, pH 7 (Hepes-Mg-Ca buffer). Plaque counts were obtained by the soft-agar overlay procedure [13, 14]. Plates containing BHI-S agar (1.5% agar) were overlayed with 3.5 ml of BHI-S soft agar (0.7% agar), which had been seeded with 0.2 ml of an 18-24-hr BHI-S culture of the assay organism. All manipulations were performed aerobically. The plates were incubated overnight at 37 C in an anaerobic glove box. Host range and phage sensitivity. Phage titers were adjusted to 100 times their routine test dilutions [16] as assayed on their propagating hosts, and each phage was spotted (0.05 ml) onto BHI-S agar plates and allowed to dry. Use of a Steers replicator [17] allowed 31 preparations of phage to be spotted simultaneously onto a large number of plates. Each plate was overlayed with the test organism as described above. Susceptibility to the phages was recorded after incubation at 37 C for 18 hr in an anaerobic glove box. Test organisms included all nine species of Bacteroides previously described, as well as the following anaerobic species or genera: Bacteroides melaninogenicus, Fusobacterium) Peptococcus, Peptostreptococcus, Clostridium) and Streptococcus. In a similar experiment, 185 strains of anaerobic bacteria were coded and then identified by determination of phage susceptibility. Of the 144 strains of B. fragilis used in this test, 113 were recent clinical isolates that had never been used in phage work. The remaining 72 non-B. fragilis strains consisted of five other species of Bacteroides and five non-Bacteroides species. The B. fragilis-specific phages Bf-13, Bf-15, Bf-20, Bf25, Bf-29, Bf-30, Bf-31, Bf-32, Bf-51, and Bf-52 were used as the test mixture in these identification trials. The phages were selected for this test on the basis of their wide host spectra. Electron microscopy. Soft-agar scrapings

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from confluent areas of lysis were placed into Brinkman micro-test tubes (Brinkman Instruments, Westburg, N.Y.) with 0.5 ml of HepesMg-Ca buffer and were homogenized by mixing on a vortex mixer. After incubation at room temperature (about 24 C) for 3 hr or at 4 C overnight, the tubes were centrifuged at 12,000 g for 2 min in a Brinkman model no. 3200 microcentrifuge. A drop of the supernatant was placed onto Parlodion" (Mallinckrodt, New York, N.Y.) and carbon-coated grids. After 10 min the drops were blotted off with filter paper, and the grids were washed twice with 0.01 M phosphate-buffered saline (PBS), pH 7.0, and once with doubly distilled water. The grids were finally stained with 1% uranyl acetate for 3 min and viewed with a JEM 100 B electron microscope (] apan Electronic Optics, Japan) operated at 80 kV. Cells were prepared for sectioning by fixing with 3% glutaraldehyde for I hr, postfixing with 1 109 pfu I ml) were prepared according to the plate method described by Adams [14]. Propagating hosts included B. fragilis strain 12256 (ATCC 29768; American Type Culture Collection, Rockville, Md.), which was naturally resistant to high levels of clindamycin (512 /Lg/ml), and B. fragilis strain 2044R3 (ATCC 29762), which was a spontaneous rifampicin-resistant mutant (MIC, >25 /Lg/ml) of B. fragilis strain 2044 (ATCC 29771), which was isolated in our laboratory [18a]. Phages propagated on these two host strains included Bf-l, Bf-Il, Bf-13, Bf-15, Bf-29, Bf-31, Bf40, and Bf-56 for strain 12256; and Bf-l, Bf-Il, Bf-13, Bf-34, Bf-35, Bf-40, and Bf-51 for strain 2044R3. These lysates were used to examine the potential for phage-mediated transfer of antibiotic resistance. Recipient strains included B. fragilis strains 4147, 3390, 6057B, and 4082 and a wildtype strain of B. [ragilis, 2044. All recipient strains were phage hosts with extensive phage susceptibility patterns. Similarly, high-titer lysates used to examine the potential for transfer of auxotrophic markers in B. fragilis were propagated on B. fragilis strains 4147, 4082, and 3390 as well as strains

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12256 and 2044. Recipients used in these experiments were several auxotrophic mutants of strains 12256 and 2044R3 recently isolated by one of us [18aJ. Their strain designations were 12256I A12 (ATCC 29769, arg-), l2256/PI8 (ATCC 29770, ade: and gua-), 2044R3/M32 (ATCC 29765, mer), 2044R3/PI2 (ATCC 29769, ade: and gua-), 2044R3/T55 (ATCC 29767, trp-), 2044R3/A2 (ATCC 29763, arg-), and 2044R3/ H6 (ATCC 29764, his-). Phages used as vectors for DNA transfer were those listed previously. Results

Isolation of phages. A total of 68 phages were isolated from sewage water over a three-year period. Although the phages were numbered Bf-lBf-82, indicating 82 isolates, 14 were subsequently identified as bacteriocins or were lost during subculturing or storage. Most of the phages were isolated by the simple enrichment procedure; however, three phages were isolated when antiserum to phage Bf-l was incorporated into each step of the enrichment procedure. Although morphologically indistinguishable from phage Bf-I, Table 1. Number and morphological types bacteriophages isolated on species of Bacteroides. No. of strains of Bacteroides Species or ON A homology group

Used for isolation

of

Bacteriophages

MorPhagesusNo. phological type" ceptible * isolated

fragilis

78

70

44 1 1

ovatus

14

5

8

B

1 0 1

All

vulgatus distasonis thetaio tao micron uniformis Homology group

10 12 12 12 12

0 2 7 4 3

2 2 154

0 0 91

5

5 2

3452-A eggerthii Subspecies a Total

*Strains susceptible to at least one phage. tBradley's classification (m) [10]. tPhage Bf-41. §Phage Bf-42. [Phage Bf-71.

0

0 68

B

C+ A§

B B B B

Bacteriophages of Bacteroides

these three phages (Bf-5I, Bf-52, and Bf-53) were not serologically related to Bf-I. The number of phages isolated on each species of Bacteroides is shown in table 1. Strains of B. fragilis were used more often in the isolation procedures; therefore, the number of phages isolated on this species does not necessarily reflect the actual ratios of these phages in their natural environment. When raw, membrane-filtered sewage water was directly assayed on three strains of B. fragilis that were chosen for their wide spectrum of phage susceptibility, the titers ranged from 60 to 150 pfu / ml. No phages could be isolated on eight of the B. fragilis strains. Host ranges and phage specificity. Each phage was tested for its host range by spotting of a phage lysate onto soft-agar overlays, each preseeded with one of ]27 strains of Bacteroides representing nine different species (as determined by DNA homologies). Each phage was specific only for strains within a given species. For example, phage Bf-I, which was isolated on a strain of B. [ragilis, was specific for other strains of B. fragilis. Strains belonging to any of the other Bacteroides species, such as B. ovatus or B.

Figure 1. Electron micrograph of bacteriophage Bf-1 propagated on Bacteroides fragilis strain 3390 (x 100,000).

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uulgatus, were not susceptible to any of the B. Iragilis phages. Each phage that we have isolated was species-specific. Other genera were resistant to all of the phages. Within the species B. jragilis, no two phages had identical lytic patterns. However, there were groups of susceptibility patterns that were very similar, a fact indicating that these groups of phages may be closely related. Similarly, no two strains of B. fragilis were susceptible to the same phages although, again, some strains seemed to be more closely related than others, as indicated by phage susceptibility patterns. No specific patterns of phage susceptibility seemed to be associated with the source (i.e., clinical vs. fecal) of the B. fragilis strains or with the type of infection (e.g., bacteremia, liver abscess, or myocarditis). One of our primary interests was the possible use of these species-specific phages as an adjunct for the identification of species of Bacteroides, particularly B. fragilis. Ninety percent of B. fragilis strains from our stock collection (table 1) were susceptible to at least one phage. Although these data were collected from the host spectrum of all 46 of the B. fragilis-specific phages, only

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nine phages were necessary to attain the 90% susceptibility level. Subsequently, in experiments in which 10 selected phages and 185 strains of anaerobic bacteria were used, HI (77%) of 144 B. fragilis strains were correctly identified. Of the 113 recent clinical isolates of B. fragilisJ 7870 were correctly identified. Only two strains (both B. vulgatus) were incorrectly identified as B. fragilis because of weak positive responses, perhaps due to contaminating bacteriocins in the phage lysates. The inability to identify the remaining 22% of clinical isolates of B. fragilis was attributable to the nonsusceptibility of these strains to any of the 10 test phages. Electron microscopy. Of the 68 phages isolated, 65 were of the same general morphological type when viewed with an electron microscope. These 65 phages were morphologically similar to the previously described phages of Bacteroides [7, 9, H, 19, 20] and were similar to Bradley's group B bacteriophages [10]. A representative of this morphological type, phage Bf-l, is shown in figure 1. Phages of this general morphology were nearly indistinguishable from each other regardless of the species of Bacteroides used for isolation (table 1). Most phage heads ranged from 50 to 70 nm in diameter, and most tail lengths ranged from 120 to 200 nm. The remaining three phages (Bf-41, Bf-42, and Bf-71) were each of a distinctly different morphology that had not been described previously for species of Bacteroides.

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Phages Bf-41 and Bf-42 were isolated from the same enrichment culture of B. fragilis strain 0439 and are shown individually in figure 2. The fourth morphologically distinct phage, Bf-71 (figure 3), was isolated on B. ovatus strain 0038. The morphological characteristics of these bacteriophages are summarized in table 2. Serum neutralization. Sixty-five of the 68 phages, including those isolated on different species, could not be distinguished morphologically from one another. Using antiserum to phage Bf-l, we looked for any antigenic differences among these phages. The results (table 3) demonstrated that not all phages of the same morphotype (Bradley's group B) were serolo.gically related to phage Bf-l. The group B phages isolated on species other than B. fragilis were not serologically related to phage Bf-l although they were morphologically similar. On the other hand, most (41 of ~4) of the phages of this morphology that were isolated on B. fragilis strains were serologically related to phage Bf-l, yet all had different host ranges. Only the three B. fragilis-specific phages (Bf-51, Bf52, and Bf-53), which were isolated in the presence of antiserum to phage Bf-l, showed no relatedness to phage Bf-l. Growth characteristics. All of our phages appeared to be virulent, and we were unable to detect lysogeny in 83 strains of B. fragilis (authors' unpublished observations). However, a phage-carrier or pseudolysogenic state similar to

Figure 2. Left, bacteriophages Bf-41 (short tail) and Bf-42 (long tail) propagated on Bacteroidesfragilis strain 0489 (X 100,000). Center, bacteriophage B£-41 propagated on B. fragilis strain 0489 (x 100,000). Right, bacteriophage B£-42(with contracted sheath) propagated 'on B. fragz"lis strain 0489 (x 100,000).

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Bacteriophages of Bacteroides

Figure~. Bacteriophage Bf-71 with noncontracted sheath (toP) and contracted sheath (bottom), propagated on Bacteroides fragilis strain 0038 (x 100,000).

that described by Keller and Traub [11] for phages of B. [ragilis was also observed for our phages. Although visible cell lysis was apparent on agar plates, as evidenced by plaque formation (clear to extremely turbid, depending on phage and I or host), none of our phages caused visible cell lysis in BHI-S broth cultures when their propagating hosts were infected. The relationship between phage Bf-I and B.

[ragilis strain 3390 was studied in more detail. Phage titers of Bf-l-infected cells ranged from -1 to 1011 pfu Iml after 24 hr. Spread plates of these infected cells resulted in viable cell counts of from I to 6 X 109 cfu/rnl. When single colonies were picked and grown in BHI-S broth, -80% continued to produce phages and had phage titers and cell counts similar to those of the originally infected cells. The phage-producing clones were not susceptible to phage Bf-l, as determined by plate assay. The non-phage-producing clones were susceptible to phage Bf-I when assayed by this method. All single-colony isolates from uninfected control cells were susceptible to phage Bf-I. The phage-producing' clones continued to produce phages on serial transfer, but could be cured by growth in the presence of antiserum to phage Bf-I. All clones isolated from cured cells were susceptible to phage Bf-I, as determined by plate assay, and each would readily revert to the pseudolysogenic state when reinfected. Wet mounts of infected and control cells were observed by the wet India ink method [21]. After 24 hr control cells were composed of a mixture of nonencapsulated and encapsulated cells (ratio, ~20: 1). Infected cells were composed of nearly all encapsulated cells and had a phageto-cell ratio of ~ I0: 1. Electron micro.graphs of sectioned cells (24 hr after infection) showed that very few cells in the population were infected and actively producing phages (figure 4). The cells could be cured of phages by growth in the presence of Bf-I-specific antiserum. Attempts at transduction. We attempted to transduce strains of B. fragilis but were unable to detect any transduced clones.

Table 2. Summary of morphological characteristics of bacterophages isolated on Bacteroides. Tail Phage

Bf-I *

Bf-41 Bf-42 Bf-71

Head diameter (nm)

70 106 90 100

Length (nm)

Width (nm)

190 50 167 150

16 16 24 22

Flexible Yes No No No

rresence of tail sheath No Yes (?)t Yes Yes

Presence of tail pins or fibers Fiber (?) Fiber Pins Pins (?)

·Of the 68 phages isolated. 65 were of this.general morphology. tThe question mark indicates that the characteristic appeared to be present in some micrographs but could not be determined with certainty.

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Table 3. Inactivation of phages by antiserum to phage Bf-l whose host was Bacteroides fragilis strain 3390.

Host

B. fragilis Bacteroides ovatus Bacteroides distasonis Bacteroides thetaiotaomicron Bacteroides uniformis Homology group 3452·A Total

No. of phages tested *

No. of phages inactivated

44 8 1 5 5

2

41 0 0 0 0 0

65

41

*Only those phages in Bradley's group B (m) [10] were tested.

Discussion

In general, bacteriophages specific for strains of B. tragilis were readily isolated. Several strains were particularly susceptible to phages, and with these strains we could routinely isolate phages from sewage water. However, there were eight

strains of B. [ragilis for which no phages could be isolated. There appeared to be no relationship between the source of the bacterial strains (i.e., clinical or fecal) and their pattern of susceptibility to the phages. Although a detailed search was not undertaken, it was still interesting that no phages were isolated from sewage water on 10 strains of B. uulgatus, particularly since B. vulgatus is the most common species of Bacteroides in the human colon [22, 23]. As reported by others [8], we also failed to isolate any phages directly from human feces (authors' unpublished observations), although our attempts to make such isolations were very limited. vVe isolated three morphological types not previously described for species of Bacteroides. Two of these phages (Bf-42 and Bf-71) belonged to Bradley's group A and one (Bf-41) to group C [l0]. The remaining 65 phages were essentially identical to each other morphologically and identical to previously described phages of Bacter-

Figure 4. Pseudolysogenic state of Bacteroides fragr:lis strain 3390 24 hr after infection with bacteriophage Bf-I. The bacteria were grown in brain-heart infusion broth supplemented with hemin and yeast extract (see Materials and Methods).

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Bacteriophages oi Bacteroides

oides [7, 9, 11, 20]. However, the phages were completely specific for the species on which they were isolated, and the serologic studies showed no cross-reactivity. We are now in the process of determining the DNA homologies of phages specific for different species to determine whether they are related. The use of a specific bacteriophage as a means of mediating exchange of genetic material between strains of Bacteroides species via transduction could prove valuable in the study of transfer of determinants of antibiotic resistance and virulence as well as in genetic mapping of chromosomal markers. Interspecies transfer of antibiotic resistance via conjugation between Escherichia coli and B. tragi lis has been described [24, 25], and the first evidence for conjugation between B. tragilis strains was recently established (F. L. Macrina, M. Sebald, and F. P. Tally, personal communication). Also, transfection, whereby DNA from B. thetaiotaomicron phage {31 was introduced into growing cells, with subsequent recovery of virulent phage particles, has been demonstrated [20]. However, no evidence of transduction has been reported to date. Our failure to transduce strains of B. [ragilis may have resulted from our failure to isolate a temperate phage. Similar problems with attempts to transduce Bacteroides species have also been encountered by others (D. R. Woods, personal communication). There have been reports of lysogeny in B. [ragilis [7, 8], but neither we nor D. R. Woods can confirm these reports. All of the phages isolated by us to date were virulent. We did find a common pseudolysogeny in all of the species we tested. This property appears to be due to the formation of a large capsule by some cells in a culture, and the interference by this capsule with attachment of phage to the cell wall. Babb and Cummins [26] demonstrated the large capsule on cells of several species of Bacteroides, and Burt et al. [27] showed that cultures segregate into phage-resistant encapsulated clones and phagesusceptible clones that have only a very small capsule. Kasper [28] has described a small carbohydrate layer that is present on the outside of B. iragilis cells only and not on other species of Bacteroides. This layer also has been termed a capsule, but evidently it is not the same as the

large capsules that we have observed. Onderdonk et al. [29] have suggested that the capsule they are working with may be a virulence factor for B. tragi lis; this idea also could apply to the large capsules that result in resistance to phage. The phage could be used as a mechanism for eliminating nonencapsulated cells from cultures, and we are initiating experiments to determine whether such pure populations of encapsulated cells are more virulent than the mixed population normally found in cultures. By use of 10 phages specific for B. [ragilis, 78% of 113 recent clinical isolates of B. tragi/is were identified within 24 hr. Isolation of more phages and their use either alone or in combination with biochemical tests (e.g., catalase production and resistance to bile and cephalothin [30]) could provide a means for rapid, positive identification of B. [ragilis and other species of Bacteroides. Perha ps even more important would be the use of the phage as a means for "typing" strains within a species of Bacteroides. In our project whereby we isolated auxotrophs of B. jragilis, the phages proved useful in confirming identity between parental and mutant strains. Because of the variety of phage-susceptibility patterns among strains (none of our 91 phage hosts had identical patterns), a "typing" scheme may prove an important tool for studies of the ecology of Bacteroides. We have already found it useful for determining whether antibiotic-resistant B. [ragilis strains that emerge during therapy are different strains or represent the development of resistance by the original susceptible strain. References 1. Cato, E. P., Johnson, J. L. Reinstatement of species rank for Bacteroides tragi/is, B. ovatus, B. distasonis, B. thetaiotaomicron, and B. vulgatus: designation of neotype strains for Bacteroides [ragilis (Veillon and Zuber) Castellani and Chalmers and Bacteroides thetaiotaomicron (Distaso) Castellani and Chalmers. Int. J. Syst. Bacteriol. 26:230-237,1976. 2. Johnson, J. L. Taxonomy of the Bacteroides. 1. Deoxyribonucleic acid homologies among Bacteroides [ragilis and other saccharolytic Bacteroides species. Int. J. Syst. Bacteriol. 28:245-256, 1978. 3. Johnson, J. L., Ault, D. A. Taxonomy of the Bacteroides. II. Correlation of phenotypic characteristics with deoxyribonucleic acid homology groupings for Bacteroides [ragilis and other saccharolytic Bacteroides species. Int. J. Syst. Bacteriol. 28:257-268, 1978.

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4. Holland, J. W., Hill, E. 0., Altemeir, W. A. Numbers and types of anaerobic bacteria isolated from clinical specimens since 1960. J. Clin. Microbiol. 5:20-25, 1977. 5. Polk, B. F., Kasper, D. L. Bacteroides fragilis subspecies in clinical isolates. Ann. Intern. Med. 86:569-571, 1977. 6. Sabiston, C. B., Jr., Cohl, M. E. Bacteriophage virulent for species of the genus Bacteroides. J. Dent Res. 48: 599,1969. 7. Prevot, A. R., Vieu, J. F., Thouvenot, H., Brault, G. Etude de Ristella pseudoinsolita et de ses bacteriophages. Essai de lysotypie. Bulletin de l'Academie Nationale de Medecine 154:681-690, 1970. 8. Nacescu, N., Brandis, H., Werner, H. Isolierung von zwei Bacteroides fragilis-phagen aus Abwasser und Nachweis lysogener B. fragilis-Stamme. Zentralbl. Bakteriol. (Orig. A.) 219:522-529. 1972. 9. Brandis, H., Voight, W.-H., Viebahn, A. Morphologische und biologische Eigenschaften des Bacteroides fragilis-Bakteriophagen 4>AI. Zentralbl. Bakteriol. (Orig. A) 222:57-63, 1972. 10. Bradley, D. E. Ultrastructure of bacteriophages and bacteriocins. Bacteriol. Rev. 31:230-314,1967. II. Keller, R., Traub, N. The characterization of Bacteroides fragilis bacteriophage recovered from animal sera: observations on the nature of bacteroides phage carrier cultures. J. Gen. Virol. 24:179-189, 1974. 12. Holdeman, L. V., Moore, W. E. C. Anaerobe laboratory manual. 4th ed. Virginia Polytechnic Institute and State University, Blacksburg, Va., 1977, P: 3144, 117-152. 13. Booth, S. J., Johnson, J. L., Wilkins, T. D. Bacteriocin production by strains of Bacteroides isolated from human feces and the role of these strains in the bacterial ecology of the colon. Antimicrob. Agents Chemother. 11:718-724, 1977. 14. Adams, M. H. Bacteriophages. Interscience Publishers, New York, 1959, p. 450-457, 461-466. 15. Aranki, A., Freter, R. Use of anaerobic glove boxes for the cultivation of strictly anaerobic bacteria. Am. J. Clin. Nutr. 25:1329-1334,1972. 16. Blair, J. E., Williams, R. E. O. Phage typing of staphylococci. Bull. W.H.O. 24:771-784,1961. 17. Steers, E., Foltz, E. L., Graves, B. S. An inocula-replicating apparatus for routine testing of bacterial susceptibility to antibiotics. Antibiot. Chemother. 9: 307-311,1959.

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18. Yamamoto, K. R., Albert, B. M., Benzinger, R., Lawhorne, L., Treiber, G. Rapid bacteriophage sedimentation in the presence of polyethylene glycol and its application to large.scale virus purification. Virology 40:734-744, 1970. 18a. Van Tassell, R. L., Wilkins, T. D. Auxotrophs of Bacteroides fragilis. Can. J. Microbiol., 1979 (in press). 19. Silver, R. P., Chase, D. G., Tally, F. P., Gorbach, S. L. Bacteriophage-associated spherical bodies in Bacteroides fragilis.J. Virol.I5:894-897, 1975. 20. Burt, S. J., Woods, D. R. Transfection of the anaerobe Bacteroides thetaiotaomicron with phage DNA. J. Gen. Microbiol. 103:181-187, 1977. 21. Duguid, J. P. The demonstration of bacterial capsules and slime. J. Pathol. Bacteriol. 63:673-685, 1951. 22. Holdeman, L. V., Good, I. J., Moore, W. E. C. Human fecal flora: variation in bacterial composition within individuals and a possible effect of emotional stress. Appl. Environ. Microbiol. 31:359-375,1976. 23. Moore, W. E. C., Holdeman, L. V. Human fecal flora: the normal flora of 20 Japanese-Hawaiians. Appl. Microbiol. 27:961-979,1974. 24. Mancini, C., Behme, R. J. Transfer of multiple antibiotic resistance from Bacteroides fragilis to Escherichia coli. J. Infect. Dis. 136:597-600, 1977. 25. Burt, S. J., Woods; D. R. R factor transfer to obligate anaerobes from Escherichia coli. J. Gen. Microbiol. 93:405-409,1976. 26. Babb, J. L., Cummins, C. S. Encapsulation of Bacteroides species. Infec. Immun. 19:1088-1091,1978. 27. Burt, S., Meldrum, S., Woods, D. R., Jones, D. T. Colonial variation, capsule formation, and bacteriophage resistance in Bacteroides thetaiotaomicron. Appl. Environ. Microbiol. 35:439-443,1978. 28. Kasper, D. L. The polysaccharide capsule of Bactetoides fragilis subspecies fragilis: immunochemical and morphologic definition. J. Infect. Dis. 133:7987,1976. 29. Onderdonk, A. B., Kasper, D. L., Cisneros, R. L., Bart· lett, J. G. The capsular polysaccharide of Bacteroides fragilis as a virulence factor: comparison of the pathogenic potential of encapsulated and unencapsulated strains. J. Infect. Dis. 136:82-89,1977. 30. Draper, D. L., Barry, A. L. Rapid identification of Bacteroides jragilis with bile and antibiotic disks. J. Clin. Microbiol. 5:439-443, 1977.

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Discussion DR. DAVID SNYDMAN. Did you look at changes in plaquing efficiency when you did the mutagenesis procedure? DR. TRACY WILKINS. No. DR. SNYDMAN. Have you looked at differences in phage patterns based on geographic locale or on clinical isolates from different sources (liver abscess, intraabdominal abscess, or blood isolates)? Are there regional patterns? DR. WILKINS. No. We haven't decided what patterns we should look for. Each one of the different strains had different patterns. DR. DENNIS KASPER. In relation to the capsule, we clearly are talking about different material. First, it disturbs me that the cells that are India ink-positive are the ones that seem to float in the centrifuge. That observation would indicate a high lipid content, rather than a high carbohydrate content. Polysaccharide is much denser than lipid, and encapsulated cells do not have any known tendency to float after centrifugation. Another reason for positive India ink preparations is the charge on the cell surface. India ink particles are repelled by similarly charged material on the surface of the bacterium. Most capsules are negatively charged so that we see positive India ink preparations with many encapsulated bacteria. The results of India ink preparations can be variable depending on the concentration of India ink, depending on whether the preparations are wet or dry, and depending on the fixation. The best evidence for there being a capsule is the isolation and purification of the material from organisms that give positive India ink preparations. Using India ink methods, Lindberg et al. have found that >80% of Bacteroides [ragilis strains have capsules, whereas only a minority of strains of other Bacteroides species are encapsulated. DR. WILKINS. Cummins has done some work on the capsular material. The encapsulated cells were lighter in the centrifuge, so that he found a fluffy layer of encapsulated cells on top of the centrifuged pellet. The "capsule" seems to be quite hydrated. After light sonic treatment, a viscous material is released that has the same chemicalcharacteristics as the cell wall. He found the

same sugars in the cell wall as in this material. The cells are quite viscous, and we can dissociate the cells from the capsular material, leaving a high-molecular-weight viscous material. I don't know whether it is a complex polysaccharide, or whether it is a single antigen. Changing the pH of the buffers had no effect on our results with I ndia ink. We do not believe that the effect is due to the charge in these preparations. When we looked at negatively stained encapsulated cells with the electron microscope, there was a lightstaining network that extended out. We couldn't see much, but we could see a delineated area like cobwebbing, which we think may be the capsule. DR. KASPER. Have you taken other bacterial species that are thought to be unencapsulated and grown them in the same medium to see if they give positive India ink preparations? Perhaps your observations are due to the medium. DR. WILKINS. For every strain of Bacteroides that we've looked at by these methods, there are some encapsulated cells. We have grown these organisms in several types of medium and get the same phenomenon. I realize that most bacteria have some structures, glycocalyx or whatever you want to call it, which extend out from the cell wall. These may not be a specific carbohydrate or protein, but they act like a capsule and should be discussed in this context. DR. ROGER VAN TASSELL We have looked at several species that do not form capsules when grown under conditions that show capsules in these Bacteroides organisms. Therefore, this phenomenon does not seem to be an artifact of growing these cells in an unusual medium. DR. KASPER. The buoyant density of outer membranes of unencapsulated gram-negative bacteria does not differ significantly from that of the outer membrane of encapsulated bacteria. Capsules are part of the outer membrane; therefore, there is no reason for encapsulated cells to float in a centrifuge. A capsule shouldn't change the density of the cell. I believe you may be looking at an experimental artifact. Simple hydration does not explain your results. DR. ANN BJORNSON. Can you do the converse

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and take few encapsulated organisms, cure the phage by antisera, and then see if you get a greater proportion of unencapsulated cells? DR. WILKINS. Yes. We have picked colonies from phage-carrier cultures; those that do not continue to carry the phage represent a population ofwhich~20% is encapsulated. You see some cells with very small capsules and many in between. At some point the phage can attach to the cell wall, and an infection then results. DR. BJORNSON. Can you cure them by means other than specific antisera, such as acridine orange? DR. WILKINS. We can cure them by random chance plating. Organisms picked from individual colonies have no phage in about 10% of the cells. DR. TOR HOFSTAD. Can you indicate anything about the nature of the phage receptors on the cell wall? DR. WILKINS. This has not been studied. DR. ANDREW ONDERDONK. Have you tried to add the isolated capsular material to a strain to see if it prevents phage adherence for phage infection?

Discussion

DR. WILKINS. No, the material is a viscous glob. Sometimes you don't see the capsules because of good technique. When cells are washed thoroughly, encapsulated cells that are on top of the cell pellet may be poured off. DR. SHERWOOD GORBACH. Have you been able to assign any function to the phage? We have some evidence that phage function may be related to bacteriocin production. We could never prove it because we couldn't plaque it, and therefore the results were always questionable. Is there any evidence for coding by the various phages? DR. WILKINS. We can get bacteriocins produced without phage being present, or at least without our being able to detect them. Quite a few different strains produced bacteriocins. We did work to see if bacteriocins were plasmid-coded. We haven't been able to show a definite relationship. We have several strains that produce bacteriocins and have plasmids. We would like to see which plasmids, if any, code for bacteriocin production.