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Behavior and Use of Lactic Streptococci and Their Bacteriophages1
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varying degrees of specificity) what is trying to be accomplished. Before Extension can state its educational objectives with a particular group, we must know the educational objectives of the group. Hence, a necessary step for Extension in its own program planning is to help groups of clientele spell out their objectives. The objectives need to be quite specific. 2. W h a t priority is attached to the attainment of each of the stated objectives? Likely, there will be many objectives for each group. These objectives are not equally important. Also, it is unlikely that all objectives can be achieved simultaneously. These situations make it highly desirable that priority be attached to attainment of objectives and that we allocate resources according to the priorities established. 3. W h a t resources are needed to accomplish the stated objectives? This is a process of (1) analyzing the resources needed; (2) analyzing resources already available; and (3) spelling out additional resources that must be created or acquired to help insure p r o g r e s s - dollars, labor, land, management, skills, markets, roads, communications, knowledge, and many more. 4. W h a t are the major obstacles or road blocks making difficult or preventing the attainment of stated objectives? What is their cause ? This is a key step to success. I t is almost axiomatic that the so-called felt causes are not really the bottlenecks. Also, exploring lags in use if technology is insufficient. Many of the obstacles lie in attitudes, mores, and tradition. This step enables the educator to use a rifle instead of a shotgun approach. 5. W h a t alternative actions might be taken to eliminate completely or reduce substantially the magnitude and intensity of effect of the identified obstacles and their causes? Usually, once the real problem has been identified, there are several possible alternative actions that might be taken toward solving these problems. I t then becomes a matter of working vigorously to identify, describe, and analyze various alternative solutions or actions which might be taken to eliminate completely these causes, or BEHAVIOR
at least reduce the intensity or magnitude of them, thereby permitting attainment of the objectives. 6. W h a t single action or combination of actions looks most promising for eliminating completely or reducing substantially the magnitude or intensity of the obstacles and their causes ? Searching for the answer to this question helps prevent going off in all directions in an attempt to overwhelm the problem, rather than sifting and sorting and then engaging in the most promising of the alternative actions. This is simply good management and it is usually wiser to think and then act than to act on a trial-and-error basis and then to think about what was done. 7. W h a t is the recommended time schedule for initiating and carrying out the most promising alternative actions? Quite often in an extension action program, certain things have to be done at certain times, sometimes simultaneously, and sometimes sequentially. I t is sound planning to think through the program before action is launched. 8. To whom has certain responsibility been assigned for initiating and making sure that the most promising actions are carried out? Once it has been decided precisely what kind of action should be taken to overcome these causes and problems, someone should specify precisely who is going to be responsible for doing what. Careful planning is needed at this point, so that there is no question about who does what and when. Once all of these questions have been asked and answered, of course, the next step is to initiate and carry out the action program. 9. W h a t evaluation should be conducted to determine what progress has been made toward attaining the specified objectives? The final step in this whole program-planning process is that of evaluating the progress that has been made toward attaining the objectives specified in the beginning. Basically, the success of the action taken is examined in the light of the progress made toward the objectives. Success can not be measured in terms of busyness.
AND USE OF LACTIC STREPTOCOCCI THEIR BACTERIOPHAGES 1
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E. B. COLLI~'S Department of Food Science and Technology, University of California, Davis Many types of bacteria are important in foods. Many are unwanted because they produce defects or diseases. But in the manufacture of cultured dairy products certain bacteria 1 Seminar presented at Department of Bacteriology, University of Wisconsin, Madison, November 7, 196.1.
are essential. I t is some of these that I want to talk about; specifically, the behavior of lactic streptococci and the bacteriophages active against them. The discussion will be divided into five p a r t s : The lactic streptococci, the action of bacteriophages on lactic streptococci, the composition of cultures in use, problems from mixing lactic streptococci, and a culture
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program devised for circumventing bacteriophage action in manufacturing cheese and buttermilk. People interested in the subject are aware that the manufacture of cheese requires lactic streptococci to produce lactic acid from lactose. They also know that in manufacturing products such as buttermilk and cultured butter the cultures must contain, in. addition to lactic streptococci, organisms that can produce diacetyl and certain other flavor compounds. Commonly, these flavor-compound-producing organisms are Leuconostoc citrovorum or Streptococcus diacetilactis. They must be present in cultures for manufacturing cultured buttermilk, but they are not necessary in cultures for manufacturing cheese. ThE LACTIC STBEPTOCOCCI This discussion of organisms will be limited to the lactic group of the genus Streptococus. The lactic group has two species, lactis and eremoris, commonly known as the lactic streptococci. These species are quite similar in most of the characteristics important in cheese cultures. More important to the cheese industry than the particular species are the differences among the strains of either species. I will define different strains as isolates from different sources--isolates sufficiently alike to be classified as belonging to the same genus and species. Single-strain culture will mean a strain or culture whose cells have a common origin, determined by source and single-colony isolation. Mixed-strain culture, on the other hand, will be a culture composed of two or more single strains. Systems of classification are man-made. Organisms that resemble each other in certain important characteristics are placed in the same genus and species. Beyond those characteristics there usually are differences--differences whose importance will depend on the viewpoint or the job to be done by the organism. Regarding use in making cultured dairy products, there are important differences between single-strain cultures. Three important differences are rate of acid production, production of antibiotics, and bacteriophage sensitivity. Most single-strain cultures of S. lactis and S. cremoris are too slow in acid production to be used satisfactorily in making cultured dairy products. Some strains produce antibiotics; others do not. W i t h regard to bacteriophage sensitivity, there is strain specificity, though not nearly as much as we would like. We have accunmlated 20 single-strain cultures that produce sufficient acid, that do not produce antibiotics, and that are unrelated in phage sensitivity. Finding more of such strains has proved to be very difficult. ACTIOK OF BACTERIOPHAGES ON LACTIC STREPTOCOCCI
Now that I have more or less introduced the
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organisms, let us take a look at the bacteriophages active against them. In size and shape these phages are similar to the well-known Escherichia coli phages--heads about 90 m;z in diameter or a little smaller and tails about 150 m/z long. They are harder to kill with chemicals and physical agents than their hosts. I n practice, this means that, once a culture is infected with an active bacteriophage, little can be done to prevent the bacteriophage from destroying the culture. One exception to this general rule is removal of calcium from the growth medium. I n 1950, we found that most bacteriophages active against lactic streptococci require calcium for multiplication and that the calcium of milk can be made unavailable by adding phosphate or citrate and heating (13). Reiter, in England, developed a phage-resistant medium (called P R M ) - - a milk from which calcium was removed by ion exchange (29). Unfortunately, something else apparently was removed, since many cultures did not grow well in PRM ( ! ) Recently, U S D A workers have experimented further with adding various phosphate salts and heating to precipitate the calcium in milk (16, 17). The method works, but is quite expensive i f applied to bulk-starter milk and, of course, is not applicable to vats of milk from which cultured products are to be made. Multiplication of lactic phages is by the usual four steps: adsorption, invasion, latent period, and lysis. We assume that, in invasion, deoxyribonucleic acid passes from the phage head through the tail into the bacterial c e l l - the same as Hershey and Chase established for E. coli phages (18). The latent period is a little longer for lactic phages---about 40 rain, compared to about 25 min for E. coli phages. The burst size is possibly a little l a r g e r - - a b o u t 90 particles, compared to about 60 or 70. With various lactic phages there are differences in latent period and burst size, resulting in differences in multiplication rate. The action of a bacteriophage on a singlestrain culture is drastic and obvious, with the bacteria multiplying by only doubling in n u m ber about every 40 to 45 min, whereas the bacteriophage multiplies about 100-fold every 45 to 50 min. That is true both in plant and in laboratory. To be more explicit--cultures of S. cremoris and S. lactis are normally propagated in milk. Normal growth of a culture can be determined by observing inoculated milk to see if it coagulates after a normal incubation time. Obviously, there are many factors that influence coagulation time: the capabilities of the culture, the per cent inoculum, heat treatment of the milk, incubation temperature, etc. Pasteurized milk inoculated with 5% of a good culture and incubated at 32 C will normally coagulate after about 4 hr and have a titratable acidity of about 0.70% after 4.5 hr. The same culture inoculated with an active phage will be lysed after 2 or 3 hr. Soon after this mass
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lysis, tho titratable acidity will stop increasing, and after 4.5 hr it will be only about 0.2 or 0.3%. Coagulation will not occur until it is caused by secondary growth, that is, by growth of phage-resistant mutants. This may require continued incubation for 15 to 25 hr. The influence of a bacteriophage on a nfixedstrain culture may or may not be drastic. I t is determined greatly by the per cent of sensitive bacteria. When less than half of the good acid producers are sensitive, the influence on acid production is hardly detectable (4). Let us, at this time, take a broad look at lactic streptococci and their bacteriophages to consider the possible results of a meeting between an mrknown strain of lactic streptococci and an unknown lactic bacteriophage in, for example, a cheese plant. (a) One possibility of their meeting would be for nothing to happen. That is, the culture nfight be resistant and not harmed in any way by the phage. (b) A second possibility would be for the culture to be sensitive and lysed, nmltiplying the bacteriophage. These are the extreme possibilities, usually expected, easily recognized, and easily understood, but there are others. (c) The third possibility was first described by Evans, in 1934 (15). He reported that certain bacteriophages were quite unusual in their action on certain strains of bacteria. They appeared able to lyse mixtures of sensitive and resistant strains of bacteria, although the same bacteriophages appeared to have no effect whatsoever on the resistant strains growing alone. This phenomenon, dubbed the nascent phenomenon, went without explanation until 1952, when we found that certain lactic phages produced this nascent effect. We studied the phenomenon and offered an explanation (3). The resistant strain is not completely resistant. Particles of the supposedly inactive phage are rapidly adsorbed, and cells of the supposedly resistant strain are quickly killed, whether or not the sensitive strain is present. Experimental values were 99% of the phage particles adsorbed in 15 min and over 90% of the bacteria killed in 20 rain. A f t e r the killing, however, the process of phage multiplication stops for some unknown reason, and no detectable progeny results. When this so-called resistant strain is mixed with a sensitive strain and the phage is added, the sensitive strain does not cause the phage to do anything that it ordinarily would not have d o n e - - i t serves only as a means of producing large numbers of phage particles which, in turn, kill essentially all of the cells of the so-called resistant strain. This explanation of the nascent phenomenon was confirmed in 1953 by the results of Whitehead, East, and McIntosh of New Zealand (33). This killing differs from lysis from without by the fact that a multiplicity of infection nmeh greater than one phage particle per bacterium is not required. (d) The fourth possibility is similar to the
second in effect but differs in mechanism. I t was reported in 1956 by Naylor and Czulak of Australia (25). Instead of the phage acting directly on the heterologous strain, the action is indirect. The phage particles are not adsorbed by the heterologous bacteria and do not kill detectible numbers of them during a 20-rain contact period. But, in the process of lysing the homologous strain, a heat-sensitive lysin is produced. This lysin, rather than the phage particles, acts upon and kills the heterologous bacteria. (e) We reported the fifth possibility in 1953 (5, 9). I t is host-controlled variations. Luria and Human, in 1952, defined host-controlled variations as "phenotypic, transient changes in bacteriophages that are produced by a single growth cycle on a new host" (24). We found certain bacteriophages to be restricted in activity against certain strains of S. cremoris. One growth cycle on the restrictive host gave progeny particles fully active on the cells of that host. The restriction can be explained by thinking in terms of individual phage particles and individual cells of the host. Adsorption of restricted particles was found in our study to produce: no detected consequence, or bacterial death without phage liberation, or bacterial death followed by bacteriophage reproduction. The occurrence of each of the last two possibilities was found to be influenced by the multiplicity of infection (9). This is different from findings with E. coll. I n E. coli the restriction can be explained as acceptance and multiplication of the phage only by rare, exceptional, active cells (2, 22). I n S. cremoris this explanation is unreasonable, because by multiple infection the incidence of fruitful cells was increased from about one p e r 105 cells to about ten or 15 per 100 cells. The mechanism by which multiple infection influences the results of adsorption is unknown. Fruitfulness after introduction of the material supplied by the bacteriophage particles might result either from a combination of a limiting essential (a minute quantity being furnished by each infecting particle) or from neutralization of some bacteriophage-growth-inhibiting mechanism. In any event, the progeny particles are not restricted by the previously restrictive host. They now, however, may be restricted by the original host, and their activity against related hosts might be changed. F o u r of these five possibilities form the salient features of that which I call the phage activity spectrum--ranging from no action through the nascent effect, host-controlled variations and, finally, full action. Obviously, the action of bacteriophages on lactic streptococci is not in all cases an all-or-nothing phenomenon. Further, there are some indications that host-range mutations occur, though there are no critical data regarding this probability. CO~POSITrO~ Of CULTURES I~ VSE With this introduction to lactic streptococci
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and the bacteriophages active against them, let us now consider the composition of the cultures available for use in manufacturing cultured dairy products. Those in the United States have been mixed-strain cultures, containing two or more strains of S. cremoris or lactis and usually species of L. citrovorum (or other citrate-fermenting species). These cultures were sold and used in manufacturing cultured dairy products long before bacteriophages were known to influence their growth, and long before the actions of bacteriophages were understood. The acid-producing bacteria in the available cultures have thus been mixtures of unknown strains of S. cremoris or lactis, i.e., unknown in terms of phage sensitivity. There has been little infornmtion to indicate whether the strains of bacteria in any given mixed-strain culture were different from each other in sensitivity to bacteriophages, and little information to indicate that the strains of bacteria in a culture being used were different from those in a supposedly different culture being held in reserve. With this situation, recommendations for controlling bacteriophage action have, by necessity, been prinmrily recommendations regarding: sanitation, using care to avoid contamination, and switching to a different unknown culture in the event of difficulty. Eventually, there was a general recommendation that cultures should be rotated, but there was no specific information to guide the dairy plant in selecting cultures to be used in rotation. Outside the United States there was one exception to this general situation. I n the early 1930's, H. R. Whitehead, at the Dairy Research Institute of New Zealand, was trying to alter the open texture of New Zealand Cheddar cheese. The obvious answer was to get away from the Leuconostoc-containing mixed-strain cultures, which had been developed primarily for manufacturing cultured butter. The procedure was to isolate strains of S. lactis and S. cremoris from available cultures and to use the isolated strains in manufacturing cheese. The problem of open texture was solved immediately, but plants using the single-strain cultures soon started having trouble with dead vats. Whitehead and Cox found, and reported in 1935, that the dead vats were caused by the action of bacteriophages (32). I n 1939, Nelson, 14arriman, and Hammer recognized and reported that the inhibitory principle in some butter cultures also was bacteriophages (26). Subsequent to finding bacteriophages, New Zealand workers accumulated several different single-strain cultures and introduced their use, two per day, in a four-day rotation (30). This culture program, used in conjunction with special sanitary precautions, was considered quite successful, and was soon used in practically all of the cheese plants in New Zealand. Several years ago this program was established in Australia (14), but its use did not spread to America.
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I t is interesting to speculate as to why Americans did not go for the New Zealand program. Perhaps one reason is that the cheese industry of America was not under pressure to make a close-textured cheese. Another reason seems to be that Americans were not convinced that the Leuconostocs are unnecessary in Cheddar cheese. Even today some cheese makers maintain that the Leuconostocs are important. A very important reason seems to be that no commercial laboratory made Dr. Whitehead's cultures available on a routine basis. I n New Zealand, the Dairy Research Institute makes the cultures available to industry. This is done by the Dairy Research Section of the Commonwealth Scientific and Industrial Research Organization in Australia. Getting back to the unknown mixtures used in America--investigations have gradually indicated the reasons why bacteriophages give trouble. The reasons indicate the limitations and complications of using unknown mixtures. PROBLEMS FRO]~ I~IXIlgG LACTIC STRF,PTOCOCCI
Several problems may result from mixing lactic streptococci. (a) One problem results from piecemeal elimination of strains (6). Experiments have shown that when a bacteriophage gets into a mixed-strain culture by contamination, the strain of bacteria that is sensitive to the phage is lysed and for all practical purposes eliminated from the culture, usually without a noticeable influence on acid production. Subsequent contaminations with different bacteriophages over a period of time can in this way luther reduce the number of s t r a i n s - finally to one. Then a single strain of bacteriophage can cause trouble. The importance of this type of piecemeal elimination of strains becomes evident upon recognizing that dairy plants tend to keep and use a good culture just as long as it does a satisfactory job. The almost inevitable result is, sooner or later, slow acid production, with losses probable. (b) A second problem is strain domination (27). Some strains of lactic streptococci dominate others in mixtures, soon becoming responsible for most of the acid produced. Domination among strains actually seems to be the rule rather than the exception. I t may develop in only one or two propagations, or it may develop slowly, becoming pronounced only after a mixture has been propagated for several days or weeks, depending on the cultures mixed. When strain domination has occurred, a single bacteriophage active against the dominant strain can cause failure in the production of lactic acid. I n 1955, Lightbody and Meanwell of England studied domination and concluded that the reason for rapid domination is the production of nisin-like antibiotics by the strains that dominate (21). They suggested that slow domination might result from undetected small amounts of the antibiotics. Our results substantiate their
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conclusion regarding the production of antibiotics by cultures that dominate rapidly (that is, within two or three days), but we do not agree with their suggestion about slow domination (7, 12). Our results indicate that most cultures do not produce antibiotics, and that domination among them, as well as domination among cultures that produce the same antibiotic, should be attributed to differences in competitive growth ability. Competitive growth ability may be determined by such factors as differences in lag phase, growth rate, tolerance to fermentation end-products, nutritional requirements, etc. We have found it possible to arrange nonantibiotic-produeing cultures according to ability to dominate. The production of antibiotics by some cultures of S. cremo~s and S. lactis is interesting. Hirsch, in 1952, from work with a limited number of antibiotic-producing strains of S. lactis and S. cremoris~ concluded that S. lactis and S. cremoris produce distinct antibiotics, each directed primarily against the other (19). I n our more recent studies we used antibioticproducing strains of S. cremoris, S. laetis, and 8. diacetilactis, and several nonantibiotic-produeing strains of each species (12). Ten out of 33 strains produced one or the other of what appeared to be only two different antibiotics. Our results indicate that S. laetis and S. cremoris do not produce distinct ~ntibiotics directed primarily against each other. There was not a known species difference that determined either the type of antibiotic produced or antibiotic sensitivity. Each antibiotic-producing strain was resistant to its own antibiotic, but this resistance did not constitute protection against the other antibiotic. (c) A third problem is sensitivity changes (10). The bacteriophage sensitivities of lactic streptococci do sometimes change during continued daily propagations. This can happen after cultures are mixed. Changes in sensitivity involve alteration of bacteriophage adsorption characteristics, assumed to reflect changes in cell surface structure. When we found and studied such changes in sensitivity, we gave much consideration to the possibility that they could have resulted from cross contamination of cultures, followed by growth and domination. But our results were quite inconsistent with this possibility. A more tenable explanation was based on the results of Luria and Delhruek (23) and Noviek and Szilard (28). Luria and Delbruck showed that bacteriophage-resistant mutants arise in cultures independently of bacteriophage action. They appear to differ from sensitive cells by only a simple, gene-controlled difference in surface structure that prevents adsorption. Novick and Szilard found sporadic cycles in the numbers of such resistant mutants in a culture of E. coll. This was in the absence of bacteriophage action. Decreases in the proportion of resistant bacteria were ascribed to periodic displacement
of the entire bacterial population by a new sensitive population arising from adaptive, sensitive mutants. Thus, phage-resistant mutants and adaptive mutants normally occur as independent mutations in separate bacterial cells in the predominant (or sensitive) component of a bacterial population. This does not rule out the possibility that there may be an occasional adaptive mutant with altered surface structure. Such a mutant would be expected to develop into a resistant population that displaces the sensitive population. In 1947, Hunter, in New Zealand, suggested that the constant action in nature of a variety of bacteriophages on strains of lactic streptococci probably accounts for some of the differences in sensitivity encountered among isolates (20). Our results suggest that when strains of lactic streptococci are propagated for long periods in the absence of bacteriophage action, and in the absence of single colony isolation and selection, differences in sensitivity tend to disappear as a res'alt of mutations followed by natural selection. In addition to the fact that unknowns were mixed in the first place, changes in sensitivity may in part account for the fact that it has been difficult to find mixed-strain cultures entirely different from each other. Once we isolated 37 single strains from 14 commercially used cultures, with a procedure that excluded the possibility of isolating one strain twice from the same mixed-strain culture. We found 28 of the strains to represent only five different phage types (8). CULTURE PROGRA~-~ FOR CIRCUMVENTING BACTERIOPHAGE ACTION
Our studies of cultures and bacteriophages active against them gradually convinced us that strict sanitation is not sufficient to keep plants that make cheese and buttermilk out of trouble. We found, as others have found, that it is extremely difficult, if not impossible, to eliminate a bacteriophage from a cheese plant as long as a sensitive strain of bacteria is being used in the plant. We felt that dairy plants needed cultures with known different phage sensitivities, and that they needed a good plan for using the different cultures--these to be used in addition to good sanitation. Consequently, a few years ago we set about trying to develop what we considered to be a logical culture program for use in making cheese. When the program for cheese bad been developed, it was offered to dairy plants by a commercial laboratory that had helped in its development. We immediately started experimenting with a similar program for use in making cultured buttermilk. The New Zealand program, mentioned earlier, is based upon the thought that all bacteriophages are present or may be present in the cheese plant, that they cannot be eliminated, and that cheese must, therefore, be made in their presence. Excellent culture-propagation facilities
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have been developed for preventing contamination of cultures. Culture rotation and good sanitation procedures are depended upon to minimize the concentration of bacteriophages in the cheese vats. With use of the culture program in New Zealand, it is estimated that slow acid production is experienced on the average in making about 1 or 2% of the cheese (31). Were the product Cottage cheese instead of Cheddar cheese, losses would undoubtedly be greater, because bacteriophages are a greater potential hazard in making Cottage cheese than in making Cheddar cheese. Our culture program, on the other hand, is based upon the thought that the presence of bacteriophages can be avoided in making cheese - - a t least to a very great extent. This thought followed an experiment whose results indicated that the presence of bacteriophages must be avoided in making Cottage cheese (11). The experiment was performed a few years ago in a Cottage cheese plant that followed excellent sanitation procedures. We permitted sensitive strains of S. cremoris (each mixed with a selected resistant strain) to be used in rotation, after certain bacteriophages were known to have entered the plant. Numbers of bacteriophage particles in the cheese wheys were determined daily. With rotation, each sensitive strain was used only every fourth day; yet, the numbers of bacteriophage particles that developed in the vats on the days a sensitive strain was used were sufficient to have caused slow acid production if a resistant strain had not been present to maintain acid development. The results indicate the great probability of losses in making Cottage cheese if bacteriophages active against all strains in a culture are present. On the other hand, information accumulated during about 5 yr from plants using our culture program substantiates our thought that bacteriophages can be avoided in making cheese by correct manipulations of culture composition plus good plant sanitation. A primary advantage of culture rotation is that cheese making for the next three days is not dependent upon a culture that was slow in the vats today. Time is available for setting in motion a predetermined plan for making corrections before the culture used today will come up for use again. A few changes have been made in the cheeseculture program since the original publication (11). The program in its present form is, briefly, as follows: We were able to accumulate 20 capable single-strain cultures that do not produce antibiotics and that are unrelated in phage sensitivity. All possibilities for combining the cultures without domination during 2 wk were determined. The culture'program involves determining which phages are present in a dairy plant, combining appropriate strains for use in rotation, replacing those combinations routinely to avoid strain domination, examining wheys for bacteriophages routinely, and changing the composition of cultures when different phages
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are found in the plant whey. I t is strictly a program for circumventing phage action, utilizing the culture rotation feature of the New Zealand program as an additional safety measure. Normally, a cheese plant receives four combined cultures. Each combined culture contalus three or more strains of S. cremoris or S. lactis or both. Since the strains used in combining cultures for a particular plant are determined by the bacteriophages found in wheys from that plant, cultures must be composed specially for each plant. For making cultured buttermilk we first tried mixing available strains of S. diacetilactis with lactic streptococci. Too often the buttermilk was raw, or just not excellent in flavor. Subsequently, we tried various cultures of L. citrovorum. Results were excellent with certain strains of this species mixed with the lactic streptococci. When correctly manufactured and properly controlled, the resulting buttermilk was uniform in quality and had excellent flavor. The buttermilk culture program ultimately decided upon is very similar to the cheese culture program. I t differs primarily in that each butermilk culture contains L. citrovorum in addition to three or more selected strains of lactic streptococci; controls are the same. I t is possible that some strains of S. diacetilactis will serve as satisfactorily as the good strains of L. citrovorum, but we could not find them. Today these culture programs are used in manufacturing large amounts of Cottage cheese, Cheddar cheese, Brick cheese, Monterey cheese, and buttermilk. The initial product manufactured by the program was Cottage cheese. This product is still the largest-volume p r o d u c t - about 100,000,000 lb per year. Plants being served are located throughout the United States and Canada. With air mail, distance from the service laboratory seems no problem. Of great importance is the fact that slow acid production and milk losses resulting from action of bacteriophages are rare among the plants using the programs. Additionally, plants report that their products are more uniformly good in quality. REFEREN CES (1) BABISL, :F. J. New Developments in the Prop-
(2) (3)
(4)
(5)
agation of Lactic Cultures: Culture Media and Bacteriophage Inhibition. J. Dairy Sci., 41: 697. 1958. BE~ANI, G., Awl) WzmT.~., J. J. Host Controlled Variations in Bacterial Viruses. J. Bacteriol., 65: 113. ]953. COLLINS, E. B. Action of Bacteriophage on Mixed Strain Starter Cultures. I. Nature and Characteristics of the ~'Nascent Phenomenon." J. Dairy Sci., 35: 371. ]952. COLbISS, E. B. Action of Bacteriophage on MLxed Strain Starter Cultures. II. Relation to Acid Production of the Proportion of Resistant Bacteria. J. Dairy Sci., 35: 381. 1952. COI,LINS, E. B. Influence of Host on Adaptations of Bacteriophages Acitve Against
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(6)
(7)
(8)
(9)
(10) (11)
(12)
(13)
(14)
(15) (16)
(17)
(18)
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Lactic Streptococci. J. Dairy Sci., 36: 563. :1953. COLLINS, E. B. Action of Bacteriophage on Mixed Strain Cultures. I I I . Strain Domination Due to the Action of Bacteriophage and Variations iu the Acid Production of Secondary Growth Bacteria. Appl. Microbiol., 3: 137. 1955. COLLINS, E. B. Action of Bacteriophage on Mixed Strain Cultures. IV. Dondnation Among Strains of Lactic Streptococci. Appl. Microbiol., 3: 141. 1955. COLLI~S, E. B. Action of Bacteriophage on Mixed Strain Cultures. V. Similarities Among Strains of Lactic Streptococci in Commercially Used Cultures and Use of a Whey Activity Test for Culture Selection and Rotation. App[. Microbiol., 3: 145. 1955. COLLI.XS, E. B. Host-Controlled Variations in Bacteriophages Active Against Lactic Streptococci. Virology, 2: 261. 1956. COLLINS,E. B. Changes in the Bacteriophage Sensitivity of Lactic Streptococci. J. Dairy Sci., 41: 41. 1958. COLLIXS, E. B. Manufacturing Cottage Cheese with Pairs of Single Strain Cultures of Lactic Streptococci. J. Dairy Sci., 41: 492. 1958. COLLINS, E. B. Domination Among Strains of Lactic Streptococci with Attention to Antibiotic Production. Appl. Microbiol., 9: 200. 1961. COhI~NS, E. B. NgLSO.~-, F. E., A.XD P,~RMEC. F. The Relation of Calcium and Other Constituents of a Defined Medium to Proliferation of Lactic Streptococcus Bacteriophage. J. Bacteriol., 60: 533. 1950. C Z ~ K , JR., AND NAYLOR, J. Host-Phage Relationship of Cheese Starter Organisms. I I I . Significance in Selection and Maintenance of Starter Cultures in Commercial Use. J. Dairy Research, 23: 131. 1956. EVAXS, A. C. Streptococcus Bacteriophages: A Study of Four Serological Types. U. S. Public Health Rept., 49: 1386. 1934. H A t , ROVE, R. E. A Simple Method for Eliminating and Controlling Bacteriophage in Lactic Starters. J. Dairy Sci., 42: 906. 1959. ItA~XmOVE, R. E., McDoNo1:6H, F. E., AND TITTSLEI~,R. P. Phosphate Heat Treatment of Milk to Prevent Bacteriophage Proliferation in Lactic Cultures. J. Dairy Sci., 44: 1799. 1961. HERSHEY,A. D., ANDCIIASE,M. Independent
Functions of Viral Protein and Nucleic Acid in Growth of Bacteriophage. J. Gen. Physiol., 36: 39. 1952. (19) HIRSCH, A. The Evolution of the Lactic Streptococci. J. Dairy Research, 19:290.
1952. (20) HVNTF~, G. J. E. Phage-Resistance and Phage-Carrying Strains of Lactic Streptococci. J. Hygiene, 45: 307. 1947. (21) LIGH~ODY, L. G., AND M~ANW~bb, L. ft. The Growth of Lactic Acid Streptococci in MLxed Starter Cultures. J. Appl. Bacteriol., 18: 53. 1955. (22) Lu~I.t, S. E. Ho~-Induced Modifications o f Viruses. Cold Spring Harbor Symposia Quant. Biol., 18: 237. 1953. (23) LURIA, S. E., _tN1> DEI21~UCK, M. Mutations of Bacteria from Virus Sensitivity to Virus Resistance. Genetics, 28: 490. 1943. (24) L~'RL~, S. E., AND HUMA~r, J. L. A Nonhereditary, Host-Induced Variation of Bacterial Viruses. J. Baeteriol., 64: 557. 1952. (25) _N.~LO~, J., AND CzUL~K, J. Host-Phage Relationship of Cheese Starter Organisms. I L Effect of Phage Activity on Heterologous Strains of Lactic Streptococci. J. Dairy Research, 23: 126. 1956. (26) NELSON, F. E., H.~__g~I~tAX,L. A., AND HAM~iE~, B. W. Slow Acid Production by Butter Cultures. Iowa Agr. Expt. Sta., Research Bull., 256: 219. 1939. (27) NICHOLS, A. A., AN1) IXF~SON, P. J. Cheese Starter ' ' Recovery" A f t e r Attack by Bacterophage (Strain Dominance in Multiple Strain Starters). J. Dairy Research, 15: 99. 1947. (28) NOVlCK, A., AND SZlL~m), L. Experiments with the Chomostat on Spontaneous Mutations of Bacteria. Proc. Natl. Acad. Sci. U. S., 36: 708. 1950. (29) RI~ITER,B. hflfibition of Lactic Streptococcus Bacteriophage. Dairy Inds., 21: 877. 1956. (30) W~I~gH~I), H. R. Bacteriophage in Cheese Manufacture. Bacteriol. Revs., 17: 109. 1953. (31) WHI~EH~A]), H. R. Personal communication. 1959. (32) W H I ~ n E . ~ , H. R., ~\n) Cox, G. A. The Occurrence of Bacteriophage in Cultures of Lactic Streptococci. New Zealand J. Sci. Technol., 16: 319. 1935. (33) WHITFA=tEAD,H. R., E.~ST, A., AND MCINTOSH, L. The So-called " N a s c e n t " Bacteriophage Phenomenon. J. Dairy Research, 21: 60. 1953.
J O U R N A L OF D A I R Y SCIENCE
varying degrees of specificity) what is trying to be accomplished. Before Extension can state its educational objectives with a particular group, we must know the educational objectives of the group. Hence, a necessary step for Extension in its own program planning is to help groups of clientele spell out their objectives. The objectives need to be quite specific. 2. W h a t priority is attached to the attainment of each of the stated objectives? Likely, there will be many objectives for each group. These objectives are not equally important. Also, it is unlikely that all objectives can be achieved simultaneously. These situations make it highly desirable that priority be attached to attainment of objectives and that we allocate resources according to the priorities established. 3. W h a t resources are needed to accomplish the stated objectives? This is a process of (1) analyzing the resources needed; (2) analyzing resources already available; and (3) spelling out additional resources that must be created or acquired to help insure p r o g r e s s - dollars, labor, land, management, skills, markets, roads, communications, knowledge, and many more. 4. W h a t are the major obstacles or road blocks making difficult or preventing the attainment of stated objectives? What is their cause ? This is a key step to success. I t is almost axiomatic that the so-called felt causes are not really the bottlenecks. Also, exploring lags in use if technology is insufficient. Many of the obstacles lie in attitudes, mores, and tradition. This step enables the educator to use a rifle instead of a shotgun approach. 5. W h a t alternative actions might be taken to eliminate completely or reduce substantially the magnitude and intensity of effect of the identified obstacles and their causes? Usually, once the real problem has been identified, there are several possible alternative actions that might be taken toward solving these problems. I t then becomes a matter of working vigorously to identify, describe, and analyze various alternative solutions or actions which might be taken to eliminate completely these causes, or BEHAVIOR
at least reduce the intensity or magnitude of them, thereby permitting attainment of the objectives. 6. W h a t single action or combination of actions looks most promising for eliminating completely or reducing substantially the magnitude or intensity of the obstacles and their causes ? Searching for the answer to this question helps prevent going off in all directions in an attempt to overwhelm the problem, rather than sifting and sorting and then engaging in the most promising of the alternative actions. This is simply good management and it is usually wiser to think and then act than to act on a trial-and-error basis and then to think about what was done. 7. W h a t is the recommended time schedule for initiating and carrying out the most promising alternative actions? Quite often in an extension action program, certain things have to be done at certain times, sometimes simultaneously, and sometimes sequentially. I t is sound planning to think through the program before action is launched. 8. To whom has certain responsibility been assigned for initiating and making sure that the most promising actions are carried out? Once it has been decided precisely what kind of action should be taken to overcome these causes and problems, someone should specify precisely who is going to be responsible for doing what. Careful planning is needed at this point, so that there is no question about who does what and when. Once all of these questions have been asked and answered, of course, the next step is to initiate and carry out the action program. 9. W h a t evaluation should be conducted to determine what progress has been made toward attaining the specified objectives? The final step in this whole program-planning process is that of evaluating the progress that has been made toward attaining the objectives specified in the beginning. Basically, the success of the action taken is examined in the light of the progress made toward the objectives. Success can not be measured in terms of busyness.
AND USE OF LACTIC STREPTOCOCCI THEIR BACTERIOPHAGES 1
AND
E. B. COLLI~'S Department of Food Science and Technology, University of California, Davis Many types of bacteria are important in foods. Many are unwanted because they produce defects or diseases. But in the manufacture of cultured dairy products certain bacteria 1 Seminar presented at Department of Bacteriology, University of Wisconsin, Madison, November 7, 196.1.
are essential. I t is some of these that I want to talk about; specifically, the behavior of lactic streptococci and the bacteriophages active against them. The discussion will be divided into five p a r t s : The lactic streptococci, the action of bacteriophages on lactic streptococci, the composition of cultures in use, problems from mixing lactic streptococci, and a culture
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program devised for circumventing bacteriophage action in manufacturing cheese and buttermilk. People interested in the subject are aware that the manufacture of cheese requires lactic streptococci to produce lactic acid from lactose. They also know that in manufacturing products such as buttermilk and cultured butter the cultures must contain, in. addition to lactic streptococci, organisms that can produce diacetyl and certain other flavor compounds. Commonly, these flavor-compound-producing organisms are Leuconostoc citrovorum or Streptococcus diacetilactis. They must be present in cultures for manufacturing cultured buttermilk, but they are not necessary in cultures for manufacturing cheese. ThE LACTIC STBEPTOCOCCI This discussion of organisms will be limited to the lactic group of the genus Streptococus. The lactic group has two species, lactis and eremoris, commonly known as the lactic streptococci. These species are quite similar in most of the characteristics important in cheese cultures. More important to the cheese industry than the particular species are the differences among the strains of either species. I will define different strains as isolates from different sources--isolates sufficiently alike to be classified as belonging to the same genus and species. Single-strain culture will mean a strain or culture whose cells have a common origin, determined by source and single-colony isolation. Mixed-strain culture, on the other hand, will be a culture composed of two or more single strains. Systems of classification are man-made. Organisms that resemble each other in certain important characteristics are placed in the same genus and species. Beyond those characteristics there usually are differences--differences whose importance will depend on the viewpoint or the job to be done by the organism. Regarding use in making cultured dairy products, there are important differences between single-strain cultures. Three important differences are rate of acid production, production of antibiotics, and bacteriophage sensitivity. Most single-strain cultures of S. lactis and S. cremoris are too slow in acid production to be used satisfactorily in making cultured dairy products. Some strains produce antibiotics; others do not. W i t h regard to bacteriophage sensitivity, there is strain specificity, though not nearly as much as we would like. We have accunmlated 20 single-strain cultures that produce sufficient acid, that do not produce antibiotics, and that are unrelated in phage sensitivity. Finding more of such strains has proved to be very difficult. ACTIOK OF BACTERIOPHAGES ON LACTIC STREPTOCOCCI
Now that I have more or less introduced the
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organisms, let us take a look at the bacteriophages active against them. In size and shape these phages are similar to the well-known Escherichia coli phages--heads about 90 m;z in diameter or a little smaller and tails about 150 m/z long. They are harder to kill with chemicals and physical agents than their hosts. I n practice, this means that, once a culture is infected with an active bacteriophage, little can be done to prevent the bacteriophage from destroying the culture. One exception to this general rule is removal of calcium from the growth medium. I n 1950, we found that most bacteriophages active against lactic streptococci require calcium for multiplication and that the calcium of milk can be made unavailable by adding phosphate or citrate and heating (13). Reiter, in England, developed a phage-resistant medium (called P R M ) - - a milk from which calcium was removed by ion exchange (29). Unfortunately, something else apparently was removed, since many cultures did not grow well in PRM ( ! ) Recently, U S D A workers have experimented further with adding various phosphate salts and heating to precipitate the calcium in milk (16, 17). The method works, but is quite expensive i f applied to bulk-starter milk and, of course, is not applicable to vats of milk from which cultured products are to be made. Multiplication of lactic phages is by the usual four steps: adsorption, invasion, latent period, and lysis. We assume that, in invasion, deoxyribonucleic acid passes from the phage head through the tail into the bacterial c e l l - the same as Hershey and Chase established for E. coli phages (18). The latent period is a little longer for lactic phages---about 40 rain, compared to about 25 min for E. coli phages. The burst size is possibly a little l a r g e r - - a b o u t 90 particles, compared to about 60 or 70. With various lactic phages there are differences in latent period and burst size, resulting in differences in multiplication rate. The action of a bacteriophage on a singlestrain culture is drastic and obvious, with the bacteria multiplying by only doubling in n u m ber about every 40 to 45 min, whereas the bacteriophage multiplies about 100-fold every 45 to 50 min. That is true both in plant and in laboratory. To be more explicit--cultures of S. cremoris and S. lactis are normally propagated in milk. Normal growth of a culture can be determined by observing inoculated milk to see if it coagulates after a normal incubation time. Obviously, there are many factors that influence coagulation time: the capabilities of the culture, the per cent inoculum, heat treatment of the milk, incubation temperature, etc. Pasteurized milk inoculated with 5% of a good culture and incubated at 32 C will normally coagulate after about 4 hr and have a titratable acidity of about 0.70% after 4.5 hr. The same culture inoculated with an active phage will be lysed after 2 or 3 hr. Soon after this mass
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lysis, tho titratable acidity will stop increasing, and after 4.5 hr it will be only about 0.2 or 0.3%. Coagulation will not occur until it is caused by secondary growth, that is, by growth of phage-resistant mutants. This may require continued incubation for 15 to 25 hr. The influence of a bacteriophage on a nfixedstrain culture may or may not be drastic. I t is determined greatly by the per cent of sensitive bacteria. When less than half of the good acid producers are sensitive, the influence on acid production is hardly detectable (4). Let us, at this time, take a broad look at lactic streptococci and their bacteriophages to consider the possible results of a meeting between an mrknown strain of lactic streptococci and an unknown lactic bacteriophage in, for example, a cheese plant. (a) One possibility of their meeting would be for nothing to happen. That is, the culture nfight be resistant and not harmed in any way by the phage. (b) A second possibility would be for the culture to be sensitive and lysed, nmltiplying the bacteriophage. These are the extreme possibilities, usually expected, easily recognized, and easily understood, but there are others. (c) The third possibility was first described by Evans, in 1934 (15). He reported that certain bacteriophages were quite unusual in their action on certain strains of bacteria. They appeared able to lyse mixtures of sensitive and resistant strains of bacteria, although the same bacteriophages appeared to have no effect whatsoever on the resistant strains growing alone. This phenomenon, dubbed the nascent phenomenon, went without explanation until 1952, when we found that certain lactic phages produced this nascent effect. We studied the phenomenon and offered an explanation (3). The resistant strain is not completely resistant. Particles of the supposedly inactive phage are rapidly adsorbed, and cells of the supposedly resistant strain are quickly killed, whether or not the sensitive strain is present. Experimental values were 99% of the phage particles adsorbed in 15 min and over 90% of the bacteria killed in 20 rain. A f t e r the killing, however, the process of phage multiplication stops for some unknown reason, and no detectable progeny results. When this so-called resistant strain is mixed with a sensitive strain and the phage is added, the sensitive strain does not cause the phage to do anything that it ordinarily would not have d o n e - - i t serves only as a means of producing large numbers of phage particles which, in turn, kill essentially all of the cells of the so-called resistant strain. This explanation of the nascent phenomenon was confirmed in 1953 by the results of Whitehead, East, and McIntosh of New Zealand (33). This killing differs from lysis from without by the fact that a multiplicity of infection nmeh greater than one phage particle per bacterium is not required. (d) The fourth possibility is similar to the
second in effect but differs in mechanism. I t was reported in 1956 by Naylor and Czulak of Australia (25). Instead of the phage acting directly on the heterologous strain, the action is indirect. The phage particles are not adsorbed by the heterologous bacteria and do not kill detectible numbers of them during a 20-rain contact period. But, in the process of lysing the homologous strain, a heat-sensitive lysin is produced. This lysin, rather than the phage particles, acts upon and kills the heterologous bacteria. (e) We reported the fifth possibility in 1953 (5, 9). I t is host-controlled variations. Luria and Human, in 1952, defined host-controlled variations as "phenotypic, transient changes in bacteriophages that are produced by a single growth cycle on a new host" (24). We found certain bacteriophages to be restricted in activity against certain strains of S. cremoris. One growth cycle on the restrictive host gave progeny particles fully active on the cells of that host. The restriction can be explained by thinking in terms of individual phage particles and individual cells of the host. Adsorption of restricted particles was found in our study to produce: no detected consequence, or bacterial death without phage liberation, or bacterial death followed by bacteriophage reproduction. The occurrence of each of the last two possibilities was found to be influenced by the multiplicity of infection (9). This is different from findings with E. coll. I n E. coli the restriction can be explained as acceptance and multiplication of the phage only by rare, exceptional, active cells (2, 22). I n S. cremoris this explanation is unreasonable, because by multiple infection the incidence of fruitful cells was increased from about one p e r 105 cells to about ten or 15 per 100 cells. The mechanism by which multiple infection influences the results of adsorption is unknown. Fruitfulness after introduction of the material supplied by the bacteriophage particles might result either from a combination of a limiting essential (a minute quantity being furnished by each infecting particle) or from neutralization of some bacteriophage-growth-inhibiting mechanism. In any event, the progeny particles are not restricted by the previously restrictive host. They now, however, may be restricted by the original host, and their activity against related hosts might be changed. F o u r of these five possibilities form the salient features of that which I call the phage activity spectrum--ranging from no action through the nascent effect, host-controlled variations and, finally, full action. Obviously, the action of bacteriophages on lactic streptococci is not in all cases an all-or-nothing phenomenon. Further, there are some indications that host-range mutations occur, though there are no critical data regarding this probability. CO~POSITrO~ Of CULTURES I~ VSE With this introduction to lactic streptococci
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INDUSTRY
and the bacteriophages active against them, let us now consider the composition of the cultures available for use in manufacturing cultured dairy products. Those in the United States have been mixed-strain cultures, containing two or more strains of S. cremoris or lactis and usually species of L. citrovorum (or other citrate-fermenting species). These cultures were sold and used in manufacturing cultured dairy products long before bacteriophages were known to influence their growth, and long before the actions of bacteriophages were understood. The acid-producing bacteria in the available cultures have thus been mixtures of unknown strains of S. cremoris or lactis, i.e., unknown in terms of phage sensitivity. There has been little infornmtion to indicate whether the strains of bacteria in any given mixed-strain culture were different from each other in sensitivity to bacteriophages, and little information to indicate that the strains of bacteria in a culture being used were different from those in a supposedly different culture being held in reserve. With this situation, recommendations for controlling bacteriophage action have, by necessity, been prinmrily recommendations regarding: sanitation, using care to avoid contamination, and switching to a different unknown culture in the event of difficulty. Eventually, there was a general recommendation that cultures should be rotated, but there was no specific information to guide the dairy plant in selecting cultures to be used in rotation. Outside the United States there was one exception to this general situation. I n the early 1930's, H. R. Whitehead, at the Dairy Research Institute of New Zealand, was trying to alter the open texture of New Zealand Cheddar cheese. The obvious answer was to get away from the Leuconostoc-containing mixed-strain cultures, which had been developed primarily for manufacturing cultured butter. The procedure was to isolate strains of S. lactis and S. cremoris from available cultures and to use the isolated strains in manufacturing cheese. The problem of open texture was solved immediately, but plants using the single-strain cultures soon started having trouble with dead vats. Whitehead and Cox found, and reported in 1935, that the dead vats were caused by the action of bacteriophages (32). I n 1939, Nelson, 14arriman, and Hammer recognized and reported that the inhibitory principle in some butter cultures also was bacteriophages (26). Subsequent to finding bacteriophages, New Zealand workers accumulated several different single-strain cultures and introduced their use, two per day, in a four-day rotation (30). This culture program, used in conjunction with special sanitary precautions, was considered quite successful, and was soon used in practically all of the cheese plants in New Zealand. Several years ago this program was established in Australia (14), but its use did not spread to America.
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I t is interesting to speculate as to why Americans did not go for the New Zealand program. Perhaps one reason is that the cheese industry of America was not under pressure to make a close-textured cheese. Another reason seems to be that Americans were not convinced that the Leuconostocs are unnecessary in Cheddar cheese. Even today some cheese makers maintain that the Leuconostocs are important. A very important reason seems to be that no commercial laboratory made Dr. Whitehead's cultures available on a routine basis. I n New Zealand, the Dairy Research Institute makes the cultures available to industry. This is done by the Dairy Research Section of the Commonwealth Scientific and Industrial Research Organization in Australia. Getting back to the unknown mixtures used in America--investigations have gradually indicated the reasons why bacteriophages give trouble. The reasons indicate the limitations and complications of using unknown mixtures. PROBLEMS FRO]~ I~IXIlgG LACTIC STRF,PTOCOCCI
Several problems may result from mixing lactic streptococci. (a) One problem results from piecemeal elimination of strains (6). Experiments have shown that when a bacteriophage gets into a mixed-strain culture by contamination, the strain of bacteria that is sensitive to the phage is lysed and for all practical purposes eliminated from the culture, usually without a noticeable influence on acid production. Subsequent contaminations with different bacteriophages over a period of time can in this way luther reduce the number of s t r a i n s - finally to one. Then a single strain of bacteriophage can cause trouble. The importance of this type of piecemeal elimination of strains becomes evident upon recognizing that dairy plants tend to keep and use a good culture just as long as it does a satisfactory job. The almost inevitable result is, sooner or later, slow acid production, with losses probable. (b) A second problem is strain domination (27). Some strains of lactic streptococci dominate others in mixtures, soon becoming responsible for most of the acid produced. Domination among strains actually seems to be the rule rather than the exception. I t may develop in only one or two propagations, or it may develop slowly, becoming pronounced only after a mixture has been propagated for several days or weeks, depending on the cultures mixed. When strain domination has occurred, a single bacteriophage active against the dominant strain can cause failure in the production of lactic acid. I n 1955, Lightbody and Meanwell of England studied domination and concluded that the reason for rapid domination is the production of nisin-like antibiotics by the strains that dominate (21). They suggested that slow domination might result from undetected small amounts of the antibiotics. Our results substantiate their
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conclusion regarding the production of antibiotics by cultures that dominate rapidly (that is, within two or three days), but we do not agree with their suggestion about slow domination (7, 12). Our results indicate that most cultures do not produce antibiotics, and that domination among them, as well as domination among cultures that produce the same antibiotic, should be attributed to differences in competitive growth ability. Competitive growth ability may be determined by such factors as differences in lag phase, growth rate, tolerance to fermentation end-products, nutritional requirements, etc. We have found it possible to arrange nonantibiotic-produeing cultures according to ability to dominate. The production of antibiotics by some cultures of S. cremo~s and S. lactis is interesting. Hirsch, in 1952, from work with a limited number of antibiotic-producing strains of S. lactis and S. cremoris~ concluded that S. lactis and S. cremoris produce distinct antibiotics, each directed primarily against the other (19). I n our more recent studies we used antibioticproducing strains of S. cremoris, S. laetis, and 8. diacetilactis, and several nonantibiotic-produeing strains of each species (12). Ten out of 33 strains produced one or the other of what appeared to be only two different antibiotics. Our results indicate that S. laetis and S. cremoris do not produce distinct ~ntibiotics directed primarily against each other. There was not a known species difference that determined either the type of antibiotic produced or antibiotic sensitivity. Each antibiotic-producing strain was resistant to its own antibiotic, but this resistance did not constitute protection against the other antibiotic. (c) A third problem is sensitivity changes (10). The bacteriophage sensitivities of lactic streptococci do sometimes change during continued daily propagations. This can happen after cultures are mixed. Changes in sensitivity involve alteration of bacteriophage adsorption characteristics, assumed to reflect changes in cell surface structure. When we found and studied such changes in sensitivity, we gave much consideration to the possibility that they could have resulted from cross contamination of cultures, followed by growth and domination. But our results were quite inconsistent with this possibility. A more tenable explanation was based on the results of Luria and Delhruek (23) and Noviek and Szilard (28). Luria and Delbruck showed that bacteriophage-resistant mutants arise in cultures independently of bacteriophage action. They appear to differ from sensitive cells by only a simple, gene-controlled difference in surface structure that prevents adsorption. Novick and Szilard found sporadic cycles in the numbers of such resistant mutants in a culture of E. coll. This was in the absence of bacteriophage action. Decreases in the proportion of resistant bacteria were ascribed to periodic displacement
of the entire bacterial population by a new sensitive population arising from adaptive, sensitive mutants. Thus, phage-resistant mutants and adaptive mutants normally occur as independent mutations in separate bacterial cells in the predominant (or sensitive) component of a bacterial population. This does not rule out the possibility that there may be an occasional adaptive mutant with altered surface structure. Such a mutant would be expected to develop into a resistant population that displaces the sensitive population. In 1947, Hunter, in New Zealand, suggested that the constant action in nature of a variety of bacteriophages on strains of lactic streptococci probably accounts for some of the differences in sensitivity encountered among isolates (20). Our results suggest that when strains of lactic streptococci are propagated for long periods in the absence of bacteriophage action, and in the absence of single colony isolation and selection, differences in sensitivity tend to disappear as a res'alt of mutations followed by natural selection. In addition to the fact that unknowns were mixed in the first place, changes in sensitivity may in part account for the fact that it has been difficult to find mixed-strain cultures entirely different from each other. Once we isolated 37 single strains from 14 commercially used cultures, with a procedure that excluded the possibility of isolating one strain twice from the same mixed-strain culture. We found 28 of the strains to represent only five different phage types (8). CULTURE PROGRA~-~ FOR CIRCUMVENTING BACTERIOPHAGE ACTION
Our studies of cultures and bacteriophages active against them gradually convinced us that strict sanitation is not sufficient to keep plants that make cheese and buttermilk out of trouble. We found, as others have found, that it is extremely difficult, if not impossible, to eliminate a bacteriophage from a cheese plant as long as a sensitive strain of bacteria is being used in the plant. We felt that dairy plants needed cultures with known different phage sensitivities, and that they needed a good plan for using the different cultures--these to be used in addition to good sanitation. Consequently, a few years ago we set about trying to develop what we considered to be a logical culture program for use in making cheese. When the program for cheese bad been developed, it was offered to dairy plants by a commercial laboratory that had helped in its development. We immediately started experimenting with a similar program for use in making cultured buttermilk. The New Zealand program, mentioned earlier, is based upon the thought that all bacteriophages are present or may be present in the cheese plant, that they cannot be eliminated, and that cheese must, therefore, be made in their presence. Excellent culture-propagation facilities
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have been developed for preventing contamination of cultures. Culture rotation and good sanitation procedures are depended upon to minimize the concentration of bacteriophages in the cheese vats. With use of the culture program in New Zealand, it is estimated that slow acid production is experienced on the average in making about 1 or 2% of the cheese (31). Were the product Cottage cheese instead of Cheddar cheese, losses would undoubtedly be greater, because bacteriophages are a greater potential hazard in making Cottage cheese than in making Cheddar cheese. Our culture program, on the other hand, is based upon the thought that the presence of bacteriophages can be avoided in making cheese - - a t least to a very great extent. This thought followed an experiment whose results indicated that the presence of bacteriophages must be avoided in making Cottage cheese (11). The experiment was performed a few years ago in a Cottage cheese plant that followed excellent sanitation procedures. We permitted sensitive strains of S. cremoris (each mixed with a selected resistant strain) to be used in rotation, after certain bacteriophages were known to have entered the plant. Numbers of bacteriophage particles in the cheese wheys were determined daily. With rotation, each sensitive strain was used only every fourth day; yet, the numbers of bacteriophage particles that developed in the vats on the days a sensitive strain was used were sufficient to have caused slow acid production if a resistant strain had not been present to maintain acid development. The results indicate the great probability of losses in making Cottage cheese if bacteriophages active against all strains in a culture are present. On the other hand, information accumulated during about 5 yr from plants using our culture program substantiates our thought that bacteriophages can be avoided in making cheese by correct manipulations of culture composition plus good plant sanitation. A primary advantage of culture rotation is that cheese making for the next three days is not dependent upon a culture that was slow in the vats today. Time is available for setting in motion a predetermined plan for making corrections before the culture used today will come up for use again. A few changes have been made in the cheeseculture program since the original publication (11). The program in its present form is, briefly, as follows: We were able to accumulate 20 capable single-strain cultures that do not produce antibiotics and that are unrelated in phage sensitivity. All possibilities for combining the cultures without domination during 2 wk were determined. The culture'program involves determining which phages are present in a dairy plant, combining appropriate strains for use in rotation, replacing those combinations routinely to avoid strain domination, examining wheys for bacteriophages routinely, and changing the composition of cultures when different phages
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are found in the plant whey. I t is strictly a program for circumventing phage action, utilizing the culture rotation feature of the New Zealand program as an additional safety measure. Normally, a cheese plant receives four combined cultures. Each combined culture contalus three or more strains of S. cremoris or S. lactis or both. Since the strains used in combining cultures for a particular plant are determined by the bacteriophages found in wheys from that plant, cultures must be composed specially for each plant. For making cultured buttermilk we first tried mixing available strains of S. diacetilactis with lactic streptococci. Too often the buttermilk was raw, or just not excellent in flavor. Subsequently, we tried various cultures of L. citrovorum. Results were excellent with certain strains of this species mixed with the lactic streptococci. When correctly manufactured and properly controlled, the resulting buttermilk was uniform in quality and had excellent flavor. The buttermilk culture program ultimately decided upon is very similar to the cheese culture program. I t differs primarily in that each butermilk culture contains L. citrovorum in addition to three or more selected strains of lactic streptococci; controls are the same. I t is possible that some strains of S. diacetilactis will serve as satisfactorily as the good strains of L. citrovorum, but we could not find them. Today these culture programs are used in manufacturing large amounts of Cottage cheese, Cheddar cheese, Brick cheese, Monterey cheese, and buttermilk. The initial product manufactured by the program was Cottage cheese. This product is still the largest-volume p r o d u c t - about 100,000,000 lb per year. Plants being served are located throughout the United States and Canada. With air mail, distance from the service laboratory seems no problem. Of great importance is the fact that slow acid production and milk losses resulting from action of bacteriophages are rare among the plants using the programs. Additionally, plants report that their products are more uniformly good in quality. REFEREN CES (1) BABISL, :F. J. New Developments in the Prop-
(2) (3)
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agation of Lactic Cultures: Culture Media and Bacteriophage Inhibition. J. Dairy Sci., 41: 697. 1958. BE~ANI, G., Awl) WzmT.~., J. J. Host Controlled Variations in Bacterial Viruses. J. Bacteriol., 65: 113. ]953. COLLINS, E. B. Action of Bacteriophage on Mixed Strain Starter Cultures. I. Nature and Characteristics of the ~'Nascent Phenomenon." J. Dairy Sci., 35: 371. ]952. COLbISS, E. B. Action of Bacteriophage on MLxed Strain Starter Cultures. II. Relation to Acid Production of the Proportion of Resistant Bacteria. J. Dairy Sci., 35: 381. 1952. COI,LINS, E. B. Influence of Host on Adaptations of Bacteriophages Acitve Against
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Lactic Streptococci. J. Dairy Sci., 36: 563. :1953. COLLINS, E. B. Action of Bacteriophage on Mixed Strain Cultures. I I I . Strain Domination Due to the Action of Bacteriophage and Variations iu the Acid Production of Secondary Growth Bacteria. Appl. Microbiol., 3: 137. 1955. COLLINS, E. B. Action of Bacteriophage on Mixed Strain Cultures. IV. Dondnation Among Strains of Lactic Streptococci. Appl. Microbiol., 3: 141. 1955. COLLI~S, E. B. Action of Bacteriophage on Mixed Strain Cultures. V. Similarities Among Strains of Lactic Streptococci in Commercially Used Cultures and Use of a Whey Activity Test for Culture Selection and Rotation. App[. Microbiol., 3: 145. 1955. COLLI.XS, E. B. Host-Controlled Variations in Bacteriophages Active Against Lactic Streptococci. Virology, 2: 261. 1956. COLLINS,E. B. Changes in the Bacteriophage Sensitivity of Lactic Streptococci. J. Dairy Sci., 41: 41. 1958. COLLIXS, E. B. Manufacturing Cottage Cheese with Pairs of Single Strain Cultures of Lactic Streptococci. J. Dairy Sci., 41: 492. 1958. COLLINS, E. B. Domination Among Strains of Lactic Streptococci with Attention to Antibiotic Production. Appl. Microbiol., 9: 200. 1961. COhI~NS, E. B. NgLSO.~-, F. E., A.XD P,~RMEC. F. The Relation of Calcium and Other Constituents of a Defined Medium to Proliferation of Lactic Streptococcus Bacteriophage. J. Bacteriol., 60: 533. 1950. C Z ~ K , JR., AND NAYLOR, J. Host-Phage Relationship of Cheese Starter Organisms. I I I . Significance in Selection and Maintenance of Starter Cultures in Commercial Use. J. Dairy Research, 23: 131. 1956. EVAXS, A. C. Streptococcus Bacteriophages: A Study of Four Serological Types. U. S. Public Health Rept., 49: 1386. 1934. H A t , ROVE, R. E. A Simple Method for Eliminating and Controlling Bacteriophage in Lactic Starters. J. Dairy Sci., 42: 906. 1959. ItA~XmOVE, R. E., McDoNo1:6H, F. E., AND TITTSLEI~,R. P. Phosphate Heat Treatment of Milk to Prevent Bacteriophage Proliferation in Lactic Cultures. J. Dairy Sci., 44: 1799. 1961. HERSHEY,A. D., ANDCIIASE,M. Independent
Functions of Viral Protein and Nucleic Acid in Growth of Bacteriophage. J. Gen. Physiol., 36: 39. 1952. (19) HIRSCH, A. The Evolution of the Lactic Streptococci. J. Dairy Research, 19:290.
1952. (20) HVNTF~, G. J. E. Phage-Resistance and Phage-Carrying Strains of Lactic Streptococci. J. Hygiene, 45: 307. 1947. (21) LIGH~ODY, L. G., AND M~ANW~bb, L. ft. The Growth of Lactic Acid Streptococci in MLxed Starter Cultures. J. Appl. Bacteriol., 18: 53. 1955. (22) Lu~I.t, S. E. Ho~-Induced Modifications o f Viruses. Cold Spring Harbor Symposia Quant. Biol., 18: 237. 1953. (23) LURIA, S. E., _tN1> DEI21~UCK, M. Mutations of Bacteria from Virus Sensitivity to Virus Resistance. Genetics, 28: 490. 1943. (24) L~'RL~, S. E., AND HUMA~r, J. L. A Nonhereditary, Host-Induced Variation of Bacterial Viruses. J. Baeteriol., 64: 557. 1952. (25) _N.~LO~, J., AND CzUL~K, J. Host-Phage Relationship of Cheese Starter Organisms. I L Effect of Phage Activity on Heterologous Strains of Lactic Streptococci. J. Dairy Research, 23: 126. 1956. (26) NELSON, F. E., H.~__g~I~tAX,L. A., AND HAM~iE~, B. W. Slow Acid Production by Butter Cultures. Iowa Agr. Expt. Sta., Research Bull., 256: 219. 1939. (27) NICHOLS, A. A., AN1) IXF~SON, P. J. Cheese Starter ' ' Recovery" A f t e r Attack by Bacterophage (Strain Dominance in Multiple Strain Starters). J. Dairy Research, 15: 99. 1947. (28) NOVlCK, A., AND SZlL~m), L. Experiments with the Chomostat on Spontaneous Mutations of Bacteria. Proc. Natl. Acad. Sci. U. S., 36: 708. 1950. (29) RI~ITER,B. hflfibition of Lactic Streptococcus Bacteriophage. Dairy Inds., 21: 877. 1956. (30) W~I~gH~I), H. R. Bacteriophage in Cheese Manufacture. Bacteriol. Revs., 17: 109. 1953. (31) WHI~EH~A]), H. R. Personal communication. 1959. (32) W H I ~ n E . ~ , H. R., ~\n) Cox, G. A. The Occurrence of Bacteriophage in Cultures of Lactic Streptococci. New Zealand J. Sci. Technol., 16: 319. 1935. (33) WHITFA=tEAD,H. R., E.~ST, A., AND MCINTOSH, L. The So-called " N a s c e n t " Bacteriophage Phenomenon. J. Dairy Research, 21: 60. 1953.