from their lysogenic host strains. ... different host ranges, whether strains with different origins ..... Computer-assisted numerical analysis of host range data.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1992, p. 1011-1018 0099-2240/92/031011-08$02.00/0 Copyright © 1992, American Society for Microbiology
Vol. 58, No. 3
Comparative Study of 35 Bacteriophages of Lactobacillus helveticus: Morphology and Host Range LAURENT SECHAUD, MICHELINE ROUSSEAU, BLANDINE FAYARD, MARIA LUISA CALLEGARI,t PASCAL QUENEE, AND JEAN-PIERRE ACCOLAS*
Station de Recherches Laitieres, Institut National de la Recherche Agronomique, 78350 Jouy-en-Josas, France Received 3 May 1991/Accepted 16 December 1991
This survey included 23 phages isolated from cheese whey and 12 temperate phages induced with mitomycin from their lysogenic host strains. All of the phages had an isometric head and a tail with a contractile sheath. In addition, short-tailed (160-nm-long) and long-tailed (260-nm-long) phages were distinguished. Short-tailed phages were by far the most widespread in French cheese factories (32 of the 35 phages studied). The study of phage relationships enabled two large groups of strains to be distinguished: those not or slightly sensitive to phages and those very sensitive to phages. There was an obvious relationship in the first group between phage sensitivity (or resistance) and the geographic origin of the strains. The second group contained primarily strains from large international collections and those isolated from commercial starters. The relationships among short-tailed phages, either temperate or isolated as lytic, suggest that lysogenic strains could be the major source of phages in French cheese factories. In spite of their potential negative impact on dairy technology, phages of thermophilic dairy lactic acid bacteria (Lactobacillus delbrueckii subsp. bulgancus, L. delbrueckii subsp. lactis, L. helveticus, and Streptococcus salivarius subsp. thermophilus) have received less attention than those of lactococci. The biology, taxonomy, ecology, and origin of isolated phages of L. delbrueckii subsp. lactis, L. delbrueckii subsp. bulgaricus, and S. salivarius subsp. thermophilus, however, have been the subject of a number of investigations for the past several years (7, 12, 16, 20). In contrast, L. helveticus phages remain relatively neglected. Phage outbreaks affecting L. helveticus starters were reported for the first time in Finland, in 1955 (9). Since then, four other reports (1, 14, 18, 19) have shown that all L. helveticus phages had a contractile sheath, i.e., belonged to Bradley's group A (2) or to the Myoviridae family (11) of the International Committee on Taxonomy of Viruses. In addition, five L. helveticus phages were included in a taxonomic analysis of various Lactobacillus phages (10) and were found to be closely related in terms of their DNA. Nevertheless, no extensive survey of L. helveticus phages has been completed to date. Phage 0241, the first known temperate phage of L. helveticus, was previously shown to be related to virulent phage 832-Bl isolated from cheese whey (15). This relationship suggested that virulent phages found in French dairy factories could originate from prophages harbored by lysogenic starter strains. The present report is a study of 35 L. helveticus phages, either induced from lysogenic strains or isolated from cheese whey. Our aims were the following: (i) to investigate the morphological characteristics of the 35 phages and to evaluate the frequency of each morphotype in French cheese factories; (ii) to ascertain the role of lysogenic strains as a source of phages in French factories; and (iii) to determine whether different phage morphotypes also have different host ranges, whether strains with different origins
have different phage sensitivity patterns, and whether resistant strains are widespread among L. helveticus strains. MATERIALS AND METHODS
Bacterial strains and bacteriophages. The strains of L. helveticus and the phages used in the present work are listed in Tables 1 to 4. Media. MRS (Difco) broth and agar were supplemented with 10 mM CaCl2* 6H2O (MRS-Ca2+) when specified. They were routinely used to grow or plate bacteria (measured as number of CFU per milliliter) or to propagate and count phages (measured as number of PFU per milliliter) as previously described (1, 3). Cheese whey samples. Cheese whey samples were collected in 1987 from different French dairy factories by the method of Reddy (13). Sterile bottles containing 1 g of CaCO3 and 2 ml of maleate buffer were prepared. Each bottle received 30 ml of a given whey sample. Each sample was inoculated with 0.6 ml of the lactobacillus starter culture used in the cheese factory at the time of sampling. Inoculation of whey samples with starter bacteria enabled the phages already present in the samples to further grow in the bacteria during transport. Whey samples were collected in 85 different factories located in Brittany (western France) and in the regions of Franche-Comte and Rh6ne-Alpes (eastern France). Fifteen mixtures, each containing wheys from five or six different factories of the same region, were examined. Each mixture was centrifuged (Sorvall centrifuge, GSA rotor, 8,000 x g for 30 min), and the supernatant (about 200 ml) was sterilized by vacuum filtration through Seitz EKS filters. Previously collected phages. Fifteen phages from various origins, previously isolated over a 20-year period between the early 1950s and the 1970s but which remained uncharacterized, were also included in the present survey. All data relating to these phages are shown in Tables 2 and 4. Isolation of phages from cheese whey in 1987. Twentyseven strains of L. helveticus from the collection of the Institut National de la Recherche Agronomique Research Center, Jouy-en-Josas, France (CNRZ), were chosen as the
* Corresponding author. t Present address: Istituto di Microbiologia, Universita Cattolica del Sacro Cuore, 29100 Piacenza, Italy.
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TABLE 1. L. helveticus strains used and their phage sensitivity patterns Strain
(CNRZ code) 32b 34 35b
57"
Temperate phage 032 034 035
58" 65C
065
66" 67" 223b,c
240 241b 243b 244b
0240 0241 0243 0244
248c 249b,c 303b
0303
328b 414 450 465b
0465
493b 762
0762
828b
829b 830b 831C 832b
833C
834b
835b,c 890 891d
892 1085 1086C 1087 1093 1094 1095b
01086e
1096c
1097b 109b,c,d 1102c 1103 1104 1106 1107 1108 1110 lllld
1112C 1113d
1117b
1118b 1146b 1147 1148 1181
01117
Synonyms, origin, and date of isolationa acs, 1960 acs, 1960 acs, 1960 NCDO 87, Metchnikoffs strain
NCDO 99, unknown origin, before 1954 acs, 1957 NCDO 30, French Gruyere de Comte cheese, before 1954 NCDO 384, acs, before 1954 ATCC 15009T, Orla-Jensen's Thernobacterium sp. strain 12, Swiss Emmenthal cheese, before 1919 acs, 1963 acs, 1963 acs, 1963 acs, 1963 acs, 1964 acs, 1964 acs, 1964 ATCC 15807, Emmenthal cs, Finland, early 1950s Cow milk koumiss, Moscow, USSR, 1971 ccs, France, 1963 acs, 1974 Emmenthal cs, Finland, received from E. Tybeck (Helsinki) in 1974 acs, 1971 IMPC 25C8, nws, 1968 IMPC Z18, nws, 1968 IMPC B23, nws, 1968 ccs, France, 1974 ccs, France, 1974 ccs, France, 1974 NZDRI 5001
NZDRI 5018 ccs, France, 1979 ccs, France, 1979 NCDO 261, from Switzerland acs, 1971 acs, 1971 acs, 1971 NCDO 262, from S. Orla-Jensen NCDO 766, Emmenthal cs, Finland, 1955 NCDO 1244, Emmenthal cs, Finland, 1958 NCDO 1844, Emmenthal cs, Finland, 1965 ATCC 10797, Swiss cs, from R. P. Tittsler NCDO 481, Denmark, before 1954 ISLC 11G, nws, received in 1988 ISLC 33G, Grana cheese whey, received in 1988 CSLM 46, Grana cheese milk, received in 1988 ISLC 36A, nws, received in 1988 ISLC 6G, nws, received in 1988 CSLM 179, nws, received in 1988 ccs, France, 1963 ccs, France, 1970 ccs, France, 1970 ccs, France, before 1974 ccs, France, before 1974 Beaufort artisan cheese starter, Rh6ne-Alpes, France, 1986 Artisan cheese starter, Rh6ne-Alpes, France, 1986 ccs, France, 1987 ccs, France, 1987 ccs, Italy, 1979 ATCC 12046, from E. E. Snell, before 1950
Phage sensitivity pattern 4)1 424204 328-Bl Insensitive 034 835-Bll 034 035 0240 0241 0243 0244 0465 0762 01117 4)8 4)9 223-B2 223-B3 328-Bl 835-B1l Insensitive 0762 (11 4)2 4)204 328-Bl 832-Bl Insensitive
Insensitive 032 034 035 065 0240 0241 0243 0244 0303 0465 0762 01117 b2 hb hw hwl 4D1 4)2 43 4)4 (D5 4)6 (D8 (19(1204 223-B2 223-B3 832-Bl 834-B3 835-B1l 1097-B12 1097-B14 (18 (19 328-Bl 832-Bl 1097-B12 Insensitive 0244 hw (1D 832-Bl 832-Bl 1097-B12 (D1 (12 (1204 223-B3 328-Bl Insensitive (D1 (D2 4)5 (1204 832-Bl 1097-B12 1097-B14 hv, NCDO 01244, 328-Bl 035 Same as that for CNRZ 223, but insensitive to 034 and 035 Insensitive Same as that for CNRZ 328 1097-B12 1097-B14 Insensitive Insensitive Insensitive Same as that for CNRZ 223, but insensitive to 034 and 035 Same as that for CNRZ 223, but insensitive to 034 and 035 Same as that for CNRZ 223, but insensitive to 034 Same as that for CNRZ 223, but insensitive to 034, 035, and 0762 Same as that for CNRZ 223, but insensitive to 034 Same as that for CNRZ 223, but insensitive to 034 and 035 Same as that for CNRZ 223, but insensitive to 034 and 035 Same as that for CNRZ 223 Insensitive 034 Insensitive Same as that for CNRZ 223, but insensitive to 034 Same as that for CNRZ 328 Same as that for CNRZ 328 Same as that for CNRZ 328 Same as that for CNRZ 223, but insensitive to 034 and 035 328-Bl Insensitive hw Insensitive Insensitive Insensitive Insensitive Same as that for CNRZ 450; in addition, sensitive to 328-Bl Insensitive Same as that for CNRZ 223, but insensitive to 034 and 035 Same as that for CNRZ 223, but insensitive to 034 and 035 Same as that for CNRZ 223, but insensitive to 034 and 035 15 832-Bl 1097-B12 Insensitive Insensitive Same as that for CNRZ 223, but insensitive to 034 and 035 Sensitive to 34 phages; insensitive to 035 Same as that for CNRZ 223, but insensitive to 034 and 035 Continued on following page
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TABLE 1-Continued Strain
(CNRZ code) 1267 1268
Temperate phage
Synonyms, origin, and date of isolationa
Phage sensitivity pattern
CNRZ 892 lysogenized with temperate phage 0240, 034 0762 141 (12 (D5 18 19 0204 328-Bl 832-Bl 1097-B12 1097-B14 this work, 1988 CNRZ 892 lysogenized with temperate phage 0241, Same as that for CNRZ 1267, but insensitive to 328-Bl this work, 1988 Insensitive IMPC HLM2, nws, before 1973 IMPC S36.2, nws, from D. Matteuzzi, before 1972 Insensitive 035 0240 0241 0243 0244 0303 0465 0762 01117 11 12 13 14 (5 Cured derivative of CNRZ 241, this work, 1988 016 08 (19 (204 223-B2 223-B3 832-Bl 835-Bll 1097-B12 1097-B14 (not tested: 032, 065, hw, and NCDO 01244)
0240 0241
1315 1316 1500
a Abbreviations: ATCC, American Type Culture Collection; CNRZ, Institut National de la Recherche Agronomique Research Center, Jouy-en-Josas, France; CSLM, Centro per lo Studio Technologico, Bromatologico e Microbiologico del Latte, Milan, Italy; IMPC, Istituto di Microbiologia, Universita Cattolica del Sacro Cuore, Piacenza, Italy; ISLC, Istituto Sperimentale Lattiero-Caseario, Lodi, Italy; NCDO, National Collection of Dairy Organisms; NZDRI, New Zealand Dairy Research Institute, Palmerston, New Zealand; acs, artisan cheese starter from the Jura district, France; cs, cheese starter; ccs, commercial cheese starter; nws, natural whey starter culture (coltura naturale in siero) from Italian Grana cheese factories; T, type strain. b Strain used to isolate phages from whey samples in 1987. Strain which gave a bell-shaped growth curve when induced with MC. d Supernatant of this strain, treated with MC, had a bacteriocinlike activity. Temperate phage 01086 is morphologically similar to 034. It remains to be further studied since it has only been recently detected and isolated.
most representative of the diversity of the species and were used as test strains for the isolation of phages from whey samples. Each whey filtrate (0.5 to 2 ml) and 0.1 ml (108 CFU) of a fresh culture of the test strain were added to 10 ml of MRS-Ca2' broth and incubated at 37°C. For each test
strain, a control without whey was also incubated. The turbidity of each tube was regularly compared with that of the control by visual examination. Once growth of the
control became clearly visible, both cultures were simultaneously transferred to fresh MRS-Ca2" broth. If no lysis occurred in the tube, three sequential subcultures were systematically done to detect a possibly delayed lysis. Eight additional phages, 223-B2, 223-B3, 328-Bl, 832-Bl, 834-B3, 835-Bll, 1097-B12, and 1097-B14, were thus isolated in 1987 from French Emmenthal cheese whey samples (Table 2). Each of them bears the CNRZ code of the strain
TABLE 2. Short-tailed phages used (isolated from whey) and their host ranges Phage
strain Propagation (CNRZ code)
Date and area of isolation
b2
892
1963, France
hb hw hwl (14
892 892 892 892
1974, eastern France (Franche-Comte) 1974, eastern France (Franche-Comte) 1975, eastern France (Franche-Comte) 1975, western France (Brittany)
02
892
1976, eastern France (Lorraine)
13 (4 05
892 892 892
1976, eastern France (Burgundy) 1976, France 1978, eastern France (Lorraine)
06 08
892 892
1976, eastern France (Franche-Comte) 1973, France
19
892
1973, France
1204
892
1976, western France (Brittany)
223-B2 223-B3 328-Bl
892 892 328
1987, eastern France (Rh6ne-Alpes), this work 1987, eastern France (Rh6ne-Alpes), this work 1987, eastern France (Rh6ne-Alpes), this work
832-Bl
892
1987, eastern France (Rh6ne-Alpes), this work
834-B3 1097-B12
892 892 892
1987, eastern France (Rh6ne-Alpes), this work 1987, western France (Brittany), this work 1987, western France (Brittany), this work
1097-B14
892
1987, eastern France (Franche-Comte), this
835-Bll
work
Sensitive strains (CNRZ code)
223, 450, 831, 832, 833, 834, 835, 890, 891, 892, 1093, 1097, 1109, 1111, 1112, 1113, 1147, 1148, 1181 Same as that for b2 Same as that for b2; in addition, it lyses 243 and 1103 Same as that for b2 Same as that for b2; in addition, it lyses 32, 65, 243, 248, 303, 1267, and 1268 Same as that for b2; in addition, it lyses 32, 65, 248, 303, 1267, and 1268 Same as that for b2 Same as that for b2 Same as that for b2; in addition, it lyses 303, 1117, 1267, and 1268 Same as that for b2 Same as that for b2; in addition, it lyses 57, 240, 1267, and 1268 Same as that for b2; in addition, it lyses 57, 240, 1267, and 1268 Same as that for b2; in addition, it lyses 32, 65, 248, 303, 1267, and 1268 Same as that for b2; in addition, it lyses 57 Same as that for b2; in addition, it lyses 57 and 248
32, 57, 65, 240, 248, 328, 493, 1094, 1095, 1096, 1098,
1109, 1148, and 1267 Same as that for b2; in addition, it lyses 65, 240, 243, 244, 303, 1117, 1267, and 1268 Same as that for b2 Same as that for b2; in addition, it lyses 35 and 57 Same as that for b2; in addition, it lyses 240, 244, 303, 762, 1117, 1267, and 1268 Same as that for b2; in addition, it lyses 303, 762, 1267, and 1268
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TABLE 3. Short-tailed temperate phages used (isolated from lysogenic strains) and their host ranges Sensitive strains host strain Lysogenic Propagation (CNRZ code) (CNRZ code) (CNRZ code)
Phage Phage
032 035 065 0240 0241 0243 0244 0303 0465 0762
1112 57 1112 892 892 892 892
1093 892 892
32, this work 35, this work 65, this work 240, this work 241, this work 243, this work 244, this work 303, this work 465, this work 762, this work
01117
1093
1117, this work
a
Same as that for b2" 57, 223, 414, 833, 835, 892, 1093 Same as that for b2 Same as that for b2; in addition, it lyses 57 Same as that for b2; in addition, it lyses 57 Same as that for b2; in addition, it lyses 57 Same as that for b2; in addition, it lyses 57 and 243 Same as that for b2 Same as that for b2; in addition, it lyses 57 Same as that for b2, with the exception of 834; in addition, it lyses 57, 65, 1267, and 1268 Same as that for b2; in addition, it lyses 57
For strains sensitive to phage b2, see Table 2.
on which it was isolated, then the letter B (for bacteriophage), and finally the number of the whey mixture. For example, phage 223-B2 was isolated with strain CNRZ 223 from whey mixture no. 2. Isolation of temperate phages. Sixty-three strains of L. helveticus were treated with 0.5 pug of mitomycin (MC) ml-' as previously described (3). This MC concentration was chosen because it triggered optimal phage induction in strain CNRZ 241 at 37°C (15), although mass lysis of the culture was not apparent. After 5 h at 37°C with MC, cultures were sterilized by filtration through 0.45-,um-pore-size Millipore membranes, and 0.5 ml of each filtrate was added to a broth culture of each strain of our collection. These cultures and their controls without filtrate were incubated and subcultured in the same way as was done for phage isolation from whey. Each of 13 infectious temperate phages was thus isolated and designated by the CNRZ number of its lysogenic host strain preceded by 0 (see Tables 3 and 4). For example, temperate phage 0241 originated from strain CNRZ 241. Eleven of the corresponding lysogenic strains developed normally at 37°C when grown with 0.5 ,ug of MC ml-', whereas the two remaining lysogenic strains, CNRZ 65 and CNRZ 1086, gave bell-shaped growth curves. In addition, 10 other strains were also efficiently lysed in the presence of MC, but no infectious temperate phage was found in culture
supernatants.
Bacteriocinlike activity. Some strains produced inhibitory compounds that interfered with the growth of some test strains. This bacteriocinlike effect and phage lysis were distinguished by observing the cultures (phages allow the strain to grow until lysis occurs, whereas inhibitory compounds do not), by dilution (inhibition rapidly disappeared with 10-fold dilutions of the inhibitor, whereas highly diluted phage suspensions could still lyse the culture), and by
plaquing (phages generally produced plaques on lawns of indicator strains, whereas inhibitors did not). Supernatants from strains CNRZ 891, CNRZ 1098, CNRZ 1109, CNRZ 1111, and CNRZ 1113 inhibited the growth of some other L. helveticus strains. These sensitive strains included CNRZ 1095 (NCDO 1244), previously used as a bacteriocin indicator when the bacteriocin-producing ability of strain NCDO 481 (CNRZ 1098) was studied (8). The four other strains demonstrating bacteriocinlike activity in our survey were isolated from commercial starters between 1963 and 1979 (Table 1). Propagation, purification, and concentration of phages. Once detected, phages were purified three times on MRSCa2' agar by the standard plaque method. Phage 834-B3, which did not form plaques under the conditions used, was purified by three successive multiplication steps in MRSCa2' broth by the limiting-dilution method. Thirty-four phages were concentrated in CsCl gradients as described previously (15), with the following modification: before the gradients were prepared, phage pellets were overlaid with 3 ml of CsCl solution (d = 1.3 instead of 1.7 as previously described) and then stored at 4°C for 48 h to allow the phages to resuspend slowly before the resulting suspension was loaded on top of the gradient. After centrifugation (100,000 x g, 1 h), a blue band of phage particles was obtained in the gradient at a density of 1.5, i.e., 5 M CsCl. Phages were sensitive to osmotic shock during dialysis of the band. Dialysis against buffer (10 mM Tris-HCl [pH 7.4], 10 mM MgSO4) without NaCl caused contraction of the sheaths and expulsion of phage DNA from the capsid of most of the phages and led to a concomitant increase of the viscosity of the suspension (data not shown). Virulent phage 832-Bl, previously described (15), was resistant to this treatment. Finally, three NaCl-supplemented buffers were used: (i) 0.5 M NaCl buffer to obtain almost all short-tailed phages
TABLE 4. Long-tailed phages used and their host ranges Phage
strain Propagation (CNRZ code)
. Source and date of isolationa
strains Sensitive (CNRZ code)
hv NCDO 01244 034 (temperate) 01086 (temperate)
493 493 35 35
Emmenthal cs, Finland, early 1950s Emmenthal cs, Finland, early 1950s Lysogenic strain CNRZ 34, this work Lysogenic strain CNRZ 1086, this work
328, 493, 1094, 1095, 1096, 1148 Same as that for hv 35, 57, 223, 892, 1086, 1148, 1267, 1268 Not yet determined
a
cs, cheese starter.
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undamaged; (ii) 3 M NaCI buffer for short-tailed phage 328-Bl; and (iii) 5 M NaCI buffer for the three long-tailed phages, hv, NCDO 01244, and 034. The previous protocol did not enable a high-titer preparation of phage 834-B3 to be obtained. The morphological study of this phage was therefore investigated as follows: 40 ml of crude lysate was filtered through a 0.45-,um-pore-size Millipore membrane and centrifuged (L8-55 Beckman ultracentrifuge, 70 Ti rotor, 40-ml Quick-Seal tubes, 100,000 x g for 30 min). Phage pellets were resuspended in 200 RI of dialysis buffer containing 0.5 M NaCl. Electron microscopy. Phage morphology was studied by electron microscopy of CsCl-purified phage preparations as previously described (15). Means of 10 particles per shorttailed phage and 20 particles per long-tailed phage were measured. Among the different negative stains tested, uranyl acetate was found to be the most suitable, although positive staining sometimes occurred. After phosphotungstic acid staining, phage particles generally appeared disrupted. Ammonium molybdate had a less drastic effect than phosphotungstic acid, but phage heads still appeared spherical and distended. Uranyl acetate-positive stained particles were not measured because they appeared to be reduced in size. Furthermore, the spreading of particles and the quality of the staining process were highly dependent on the brand of copper grid employed. Isolation of a prophage-cured derivative of strain CNRZ 241. Strain CNRZ 241 was treated with five doses of MC (0.05, 0.1, 0.2, 0.5, and 1 Sig ml-') under the conditions described for induction, and antiphage 0241 serum (15) was added to the cultures at a final dilution of 2 x 10-2. Antiphage serum was used to neutralize the phage particles which otherwise could lyse the sensitive cured cells that arose in MC-treated cultures, since phage 0241 can lyse a sensitive strain even in the absence of Ca2` ions (15). These cultures were incubated for 5 h at 37°C and then diluted in MRS broth (diluted 1/10 to avoid precipitation of antibodies) containing antiphage 0241 serum (final dilution, 10-3) and plated in a double layer of MRS soft agar containing antiphage serum at the same concentration. Plates were incubated in anaerobic jars (BBL Microbiology Systems) at 37°C with GasPak H2 + CO2 for 2 days. Twenty-five colonies per culture (i.e., a total of 125 clones) were examined. Only one negative clone of the 125 isolates was found to be sensitive to temperate phage 0241 and not inducible by MC after three successive reisolations on MRS agar. This clone was named CNRZ 1500. To ensure that it was not a contaminant, Southern blots of EcoRI digests of total DNA extracts from strains CNRZ 241, CNRZ 892, and CNRZ 1500 were hybridized as previously described (6) with a DNA-specific probe of L. helveticus, the 2-kb f-fragment of pCG 36 (6a). As anticipated, strains CNRZ 241 and CNRZ 1500 showed closely related profiles which differed from that of strain CNRZ 892 (data not shown). Furthermore, the phage sensitivity pattern of strain CNRZ 1500 was very specific (Table 1). These results strongly suggested that CNRZ 1500 was truly a prophage-cured derivative of strain CNRZ 241. Phage-bacteria relationships. The sensitivity of 63 strains of L. helveticus to 35 phages was determined as follows. Each strain was infected with each phage at a multiplicity of infection of between 0.01 and 1. Cultures were incubated at 37°C, and the turbidity of phage-infected cultures was regularly compared with that of a control without phage. Three subcultures were systematically prepared when lysis was
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delayed or did not occur, as was done for the isolation of phages from whey and from MC-treated cultures. A matrix was constructed in which the results were noted as 1 when a phage was active on a given strain or as 0 when the phage was inactive. Computer-assisted numerical analysis of host range data. Phages and strains were compared, two at a time, with a "distance" calculated as 1 - SI, where SI is the similarity index of Sokal and Michener (17): SI = I (1,1) + (0,0) /E (1,1) + (0,0) + (1,0) + (0,1) The distance between two given phages was thus calculated by using the 63 pairs of data obtained for those phages. Similarly, the distance between two given strains was calculated with the 35 pairs of data obtained for the two strains. Thus, two distance matrices were obtained and used to build two derived dendrograms through minimal linkage clustering, one for phages and the other for bacteria. Strain CNRZ 1500, a prophage-cured derivative of lysogenic strain CNRZ 241, is not included in the strain dendrogram because it was obtained late and its phage sensitivity pattern was determined after the present work was completed.
RESULTS AND DISCUSSION Morphology. All 35 phages had a contractile sheath and thus belonged to Bradley's group A (2) or to the Myovindae family of the International Committee on Taxonomy of Viruses (11). They were divided into two distinct morphological groups, short-tailed and long-tailed phages. The short-tailed phage group contained 32 morphologically identical phages (two are shown in Fig. 1). All had isometric heads (50 to 54 nm in diameter) and rigid tails (160 nm in length) with contractile sheaths (17 by 150 nm). The sheath contained about 40 rows of transverse striations, indicative of a helical organization of protein subunits. Some very short, thin, and labile fibers were sometimes visible at the tip of the tail in micrographs. The long-tailed group contained the two phages hv and NCDO 01244, isolated in Finland in the early 1950s, and temperate phage 034, borne by a strain isolated in France. Each of the three long-tailed phages had an isometric head (56 to 60 nm in diameter) and a slightly flexible 260-nm-long tail with a contractile sheath (17 by 250 nm). The sheath had a criss-cross appearance which was definitely different from the striated sheaths of the shorttailed phages (Fig. 1). Phage 034 possessed a conspicuous cluster of fibers at the distal end of the tail, whereas a tuft of thin and delicate fibers was sometimes present at the tail extremity of NCDO 01244 or hv particles. The contractile tails of the 35 L. helveticus phages distinguish them from known phages of other thermophilic dairy lactobacilli, L. delbrueckii subsp. lactis and L. delbrueckii subsp. bulgancus. The latter phages, with the sole exception of a tail-sheathed phage of L. delbrueckii subsp. bulgancus (14), belong to Bradley's group B or to the Siphoviridae family of the International Committee on Taxonomy of Viruses (16). On the other hand, phages with a contractile sheath and which are specific to other lactobacillus species have been described previously (4, 5, 21). Phage host ranges. Computerized host range data led to the delimitation of three phage groups (PGs) (Fig. 2), PG1, PG2, and PG3. PG1 included 30 short-tailed phages active on many strains (between 19 and 27). It consisted of temperate phages carried by lysogenic strains and of phages isolated as lytic from whey between 1963 and 1987 in France. These 30 phages formed a homogeneous group and displayed very
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FIG. 1. Morphology of phages of L. helveticus. (a) Short-tailed phage 835-B1l. (b) Short-tailed phage 4)4. The particle on the left shows a contracted sheath. (c) Long-tailed phage hv. (d) Long-tailed phage NCDO 01244 with a contracted sheath showing some fibers at the tip of the tail core. (e) Long-tailed phage 034 with a cluster of fibers at the distal tail tip. Bar, 50 nm. (The same magnification factor was used for all of the panels.)
close host ranges. However, temperate short-tailed phages 0762 and 0244 lysed some lysogenic strains which carried similar short-tailed temperate phages. This means that different immunity groups exist among the short-tailed phages, in spite of the apparent homogeneity observed at first glance. PG2 consisted of the two long-tailed phages hv and NCDO 01244 isolated in Finland and short-tailed phage 328-Bl isolated in France. These three phages were the only ones capable of multiplying on the five strains CNRZ 328, CNRZ 493, CNRZ 1094, CNRZ 1095, and CNRZ 1096 from Finland. They also attacked strain CNRZ 1148 (see below), and
phage 328-Bl was active on eight additional strains (see Table 2). The third group, PG3, included only two temperate phages, long-tailed 034 and short-tailed 035. Both phages attacked strains which were also sensitive to PG1 phages, and their host ranges were related to those of PG1 phages. On the other hand, PG2 and PG3 phages appeared closely clustered (Fig. 2) only because phages of both groups were inactive on many strains [i.e., high numbers of (0,0) pairs which accordingly reduce the distances calculated as 1 SI]. If only the (1,1) pairs were used, phages 034 and 035
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FIG. 2. Dendrogram of the 35 phages derived from minimal linkage clustering. Three phage groups, PG1, PG2, and PG3, were delimited. The more the host ranges of phages are related, the shorter the distances between these phages are and the more the phages are clustered on the left of the dendrogram.
shifted and then became a subgroup of PG1, but the corresponding dendrogram was otherwise less meaningful (data not shown). Most of the phages propagated on strain CNRZ 892, with the exception of the five temperate phages (032, 035, 065, 0303, and 01117) which propagated better on another strain (Table 3). The host ranges of the latter phages, however, were similar to those of phages propagated on CNRZ 892 and thus were not dramatically modified by a different propagating host. Strain sensitivity patterns. Numerical analyses of the 63 strain sensitivity patterns led to two bacterial groups (BGs), BG1 and BG2 (Fig. 3). The BG1 strains were either sensitive to only a small number of phages (the most sensitive strain of this group, CNRZ 1267, was attacked by only 12 phages) or resistant to all of them. They can be divided into three subgroups, a, b, and c. BGla contains lysogenic strains isolated from Franche-Comte artisan cheese starters in the 1970s and two
FIG. 3. Dendrogram of the 63 strains of L. helveticus derived from minimal linkage clustering. Two broad bacterial groups were created, BG1 and BG2. The closer the phage sensitivity patterns of strains are, the smaller the distances between these strains are and the more the strains are gathered on the left of the dendrogram.
laboratory-made lysogenic derivatives from strain CNRZ 892, strains CNRZ 1267 and CNRZ 1268, which carry temperate phages 0240 and 0241, respectively. GBlb contained strain CNRZ 1098, sensitive only to phage 328-Bl, and the five strains from Finland which were sensitive to long-tailed phages hv and NCDO 01244 and to short-tailed phage 328-Bl. The third subgroup, BG1c, contained 26 strains, either sensitive to only one or two phages (Table 1, 4 strains) or resistant to the 35 phages (Table 1, 22 strains, i.e., about 35% of the strains). This third subgroup included the remaining lysogenic strains from Franche-Comte artisan cheese starters (CNRZ 34, CNRZ 35, CNRZ 241, CNRZ 465, and CNRZ 1086) and 11 strains that were apparently nonlysogenic, from Italian Grana cheese factories using natural cheese whey starters (colture naturali in siero). Ten
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of the latter strains were resistant to all phages, whereas the 11th, CNRZ 1103, was sensitive only to phage hw. BG2 contained 20 highly sensitive and apparently nonlysogenic strains. Two subgroups were delimited. The first, BG2a, contained only strain CNRZ 57, isolated at the beginning of the century in Metchnikoff's laboratory. Strain CNRZ 57 was found to be sensitive to 15 phages, including 9 temperate phages. The second subgroup, BG2b, contained highly sensitive strains attacked by 30 to 34 phages. The BG2b strains were either obtained from large international collections (for example, CNRZ 223 [the type strain for L. helveticus], CNRZ 892 [one of the best indicator strains used to propagate almost all of the short-tailed phages], CNRZ 1093, CNRZ 1097, and CNRZ 1181) or isolated from commercial starters. One of the latter strains, CNRZ 1148, definitely behaved as the most phage-sensitive strain in our collection (34 phages) and could be tested as a possible substitute for CNRZ 892 to propagate most of the phages. It is noteworthy that strain CNRZ 892 belonged to BG2b whereas its lysogenic derivatives, CNRZ 1267 and CNRZ 1268, belonged to BGla. Moreover, virulent phage 832-Bl formed large clear plaques on lawns of CNRZ 892 (15) but only tiny plaques on lawns of CNRZ 1267 or CNRZ 1268. On the other hand, CNRZ 1500, a prophage-cured derivative of CNRZ 241 (not shown in the dendrogram), was found to be sensitive to many phages (Table 1) and should consequently be clustered in the BG2b subgroup, whereas the original strain, CNRZ 241, was resistant to all phages and appeared in BG1c. Thus, lysogeny definitely appeared to enhance the phage resistance of strains. A molecular comparison of the 35 phages, at the protein and DNA levels, is currently under way in our laboratory and will help to further establish the classification of L. helveticus phages. This will be presented and discussed in a forthcoming article. ACKNOWLEDGMENTS Sandra Carini, Marie-Christine Chopin, Roberta Lodi, Marc Bigret, and Vittorio Bottazzi are gratefully acknowledged for their kind gifts of some strains and phages. We thank Florence Prost, Jean-Francois Chamba, and their colleagues at the Institut Technique du Gruyere, as well as Gerard Oeuvrard and his colleagues of the Cooperative Laitiere de Beaufort, for providing us with the cheese whey samples analyzed in 1987. We also thank Tim Cogan, who kindly helped us in 1988 during the summer investigation of phage-host relationships; Claire Chabanet, who efficiently computerized our phage host data; and, finally, Hans-Wolfgang Ackermann, Charles Daly, and Gaetan Limsowtin, who kindly performed a critical reading of our manuscript. REFERENCES 1. Accolas, J.-P., and H. Spillmann. 1979. Morphology of bacteriophages of Lactobacillus bulgaricus, L. lactis and L. helveticus. J. Appl. Bacteriol. 47:309-319. 2. Bradley, D. E. 1967. Ultrastructure of bacteriophages and bacteriocins. Bacteriol. Rev. 33:230-314.
APPL. ENVIRON. MICROBIOL. 3. Cluzel, P.-J., M. Veaux, M. Rousseau, and J.-P. Accolas. 1987. Evidence for temperate bacteriophages in two strains of Lactobacillus bulgaricus. J. Dairy Res. 54:397-405. 4. De Kierk, H. C., J. N. Coetzee, and J. T. Fourie. 1965. The fine structure of Lactobacillus bacteriophages. J. Gen. Microbiol. 38:35-38. 5. De Kierk, H. C., and N. Hugo. 1970. Phage-like structures from Lactobacillus acidophilus. J. Gen. Virol. 8:231-234. 6. de los Reyes-Gavilan, C. G., G. K. Y. Limsowtin, L. Sechaud, M. Veaux, and J.-P. Accolas. 1990. Evidence for a plasmid-linked restriction-modification system in Lactobacillus helveticus. Appl. Environ. Microbiol. 56:3412-3419. 6a.De los Reyes-Gavilan, C. G., G. K. Y. Limsowtin, P. Tailliez, L. Sechaud, and J.-P. Accolas. Submitted for publication. 7. Jarvis, A. W. 1989. Bacteriophages of lactic acid bacteria. J. Dairy Sci. 72:3406-3428. 8. Joerger, M. C., and T. R. Klaenhammer. 1986. Characterization and purification of helveticin J and evidence for a chromosomally determined bacteriocin produced by Lactobacillus helveticus 481. J. Bacteriol. 167:439-446. 9. Kiuru, V. J. T., and E. Tybeck. 1955. Characteristics of bacteriophages active against lactic acid bacteria in Swiss cheese. Suom. Kemistil. B 28:57-62. 10. Lahbib-Mansais, Y., M. Mata, and P. Ritzenthaler. 1988. Molecular taxonomy of Lactobacillus phages. Biochimie 70:429435. 11. Matthews, R. E. F. 1982. Classification and nomenclature of viruses. Fourth report of the International Committee on Taxonomy of Viruses. Intervirology 17:1-200. 12. Mercenier, A., and Y. Lemoine. 1989. Genetics of Streptococcus thernophilus: a review. J. Dairy Sci. 72:344-454. 13. Reddy, M. S. 1974. Development of cultural techniques for the study of Streptococcus thermophilus and Lactobacillus bacteriophages. Ph.D. thesis. Iowa State University, Ames. 14. Reinbold, G. W., M. S. Reddy, and E. G. Hammond. 1982. Ultrastructure of bacteriophages active against Streptococcus
thermophilus, Lactobacillus bulgaricus, Lactobacillus lactis and Lactobacillus helveticus. J. Food Prot. 45:119-126. 15. Sechaud, L., M. L. Callegari, M. Rousseau, M.-C. Muller, and J.-P. Accolas. 1989. Relationship between temperate bacteriophage 0241 and virulent bacteriophage 832-Bl of Lactobacillus helveticus. Neth. Milk Dairy J. 43:261-277. 16. Sechaud, L., P.-J. Cluzel, M. Rousseau, A. Baumgartner, and J.-P. Accolas. 1988. Bacteriophages of lactobacilli. Biochimie 70:401-410. 17. Sokal, R. R., and C. C. Michener. 1958. A statistical method for evaluating systematic relationships. Univ. Kans. Sci. Bull. 38:1409-1438. 18. Sozzi, T. 1977. L'infezione da batteriofago. Latte 2:31-36. 19. Sozzi, T., and R. Maret. 1975. Isolement et quelques caracteristiques des bact6riophages de Streptococcus thennophilus et de Lactobacillus helveticus de ferments d'Emmental. Lait 55: 269-288. 20. Sozzi, T., K. Watanabe, K. Stetter, and M. Smiley. 1981. Bacteriophages of the genus Lactobacillus. Intervirology 16: 129-135. 21. Trevors, K. E., R. A. Holley, and A. G. Kempton. 1983. Isolation and characterization of a Lactobacillus plantarum bacteriophage isolated from meat starter culture. J. Appl. Bacteriol. 54:281-288.
