THOMPSON, N. (1959). J. uppl. Buct. 22 (2). 287-293.

THE INFLUENCE OF ACIDITY ON THE MULTIPLICATION O F LACTIC STREPTOCOCCAL

BACTERIOPHAGE IN LIQUID MEDIA

BY NORAH THOMPSON

United Dairies Research Laboratories, Wood Lane, London, W.12

SUMMARY: Mass lysis of lactic streptococci infected with bacteriophage a t 30" was prevented at p H 5.10. At lower pH values no multiplication of phage followed infection, and prolonged incubation at 30" resulted in loss of phage particles from unlysed samples. Adsorption of phage particles on host cells was unaffected by acidity, but no phage penetration of host cells took place. Host cell properties were apparently unchanged by adsorption of phage particles in acid whey.

ALTHOUGH in 1926 d'Herelle had stated that an acid medium was unfavourable to the development of bacteriophage, little detailed work was reported for several years. Collins (1951) reported that a large phage infection caused lysis of Streptococcus lactis cells in milk coagulated by rennet and calcium chloride at the time of infection, but an equal phage infection in milk coagulated by lactic acid did not cause lysis within 7 hr. Overcast, Nelson & Parmelee (1951) found that with five strains of lactic streptococci and homologous phages a pH range of 55-7.5 allowed considerable proliferation of phage. Phage multiplication was not altogether prevented at pH values as low as 5.0. Cherry & Watson (1949) reported that lactic streptococcal phages showed only 5447% adsorption at pH 7, while mass lysis of host cells occurred only a t pH 7. Gold & Watson (1950) found that a t pH 5-1 lysis of Clostridium madisonii by phage was prevented and they concluded that phage penetration of the host cells was limited a t this pH. Meanwell & Thompson (1959) have shown that acid produced by the host culture affected the size of the foci produced by phage multiplication in a gel. The present paper describes an investigation of the mechanism by which acidity may limit the multiplication of phage in the presence of viable host cells.

MATERIALS AND METHODS The cultures and phages studied, the methods of maintenance and measurement of numbers were those given by Meanwell & Thompson (1959).

Media. The sterile milk used was autoclaved separated milk. To prepare sterile whey, raw separated milk was rennetted at 40°, the curd cut into + in. squares after 1 hr, and the whey strained through muslin before heating to 90". It was then cooled, filtered through a Buchner funnel and autoclaved a t 5 lb/in.2 for 15 min.

Tryptone-Yeastrel-lactose-beef extract broth (TYLB, Meanwell & Thompson, 1959) was the subculturing medium used for preparing the whey inocula.

288 Norah Thompson

Measurement of phage adsorption. Phage was allowed to adsorb on host cells in whey a t 30" for 10 min and the inoculated whey then diluted 10-1 into sterile whey held in iced water. After centrifuging a t 3,000 rev/min for 10 min the free phage in the supernatant of this dilution was determined and the percentage adsorption calculated.

into whey and incubated a t 30". The time of increase in the number of phage particles wa8 determined by plaque counts a t 5 min intervals.

One step growth experiment. An adsorption mixture was diluted

EXPERIMENTS AND RESULTS

Prevention of mass lysis at low pH values The experiments of Meanwell & Thompson (1959) demonstrated that a gel was necessary to prevent mass lysis in milk only a t low acidities. However, a t higher acidities destruction of the agar gel was quickly followed by acid coagulation of the milk. To distinguish the physical effects of coagulation on phage attack from the effect of acidity, milk was conveniently replaced by whey, the rates of cell and phage multiplication in whey and milk being similar.

Preliminary experiments indicated that lysis of host cells in whey was prevented a t pH 5-50-5.10. Thus it was seen that prevention of mass lysis was independent of coagulation but appeared closely linked to the acidity developed before the number of phage particles was equal to the number of cells. A more detailed study was then made to determine the pH a t which mass lysis of host cells was prevented.

Table 1. The effect of the pH of whey at the time ofphage addition on phage multiplication and the occurrence of mass lysis of host culture 818

since phage ,- A , addition 5.14* 4.95t 4.78t 4.70t 4.66t

0 8.18 8.18 8.15 8.15 8.28 0.5 9.70 9.42 8.11 7.99 7.67 0.75 10.06 9.38 8.98 7.46 7.58 1.5 9.93 9.36 9.16 7.90 7.51

1'3.0 9.81 <6.00 5.30 4.78 5.79

Time (hr) Log,, phage no./ml in wheys infected at pH

* Mess lysis occurred 45 min after infection. t No lysis.

A bulk of whey inoculated with 1 % of a TYLB culture of strain 818 was divided into 5 portions, all of which were incubated a t 30". At intervals, a phage infection, similar in numbers to the host cell numbers, was introduced into individual samples and the pH was measured. In each portion, phage and cell numbers were determined from time to time following infection, and the pH values, the phage counts and the occurrence of lysis are recorded in Table 1. Mass lysis of the host cells followed phage infection only when the pH a t the time of infection was 5.14, though infection a t pH 4.95 was followed by multiplication of phage within 30 min. However, the increase was smaller than in the portion which showed mass lysis. When the pH a t phage infection was 4-78 the increase in phage was delayed until 45 min from

290 Norah Thompson

Experiments done in acid whey with both cultures 818 and 1059 showed that although phage concentration had no effect on the degree of adsorption a t the highest bacterial density, there was a sharp decrease in adsorption of phage particles when the number of host cells was reduced (Table 3). Similar results were obtained in neutral whey (pH 6.20). The attachment between phage and host cells in both wheys was irreversible. Thus it appeared that only those phage particles which had been irreversibly adsorbed on to host cells were destroyed in acid whey during incubation a t 30".

Viability and phage susceptibility of unlysed host cells in acid whey

Since irreversible attachment of phage particles to host cells normally causes cell death (Garen & Puck, 1951), studies were made on the viability Qf cells of culture 818 on which phage particles had irreversibly adsorbed in acid whey.

Table 4. The survival of cells of culture 818 in acid whey after incubation with phage for 17 hr at 30"

Time of Log,, (no./ml) of incubation 7->

(hr) Cells Phage 0 8.05 8.11

17 7.86 6.15

Counts of host cells and phage were done immediately, and again after 17 hr a t 30°, but because phage particles were known to interfere with colony development in agar poured plates, the dilutions were passed through 'Oxoid' membrane filters, which retained the cells but not the phage particles. The filters were then incubated on TYLB agar for 48 hr a t 30" and the colonies counted. The initial host cell numbers were 112 .: 106/ml and the phage adsorbed amounted to 139 x 106/ml, adsorption being maximal; yet as shown in Table 4, many (64gb) of the host cells were recovered after incubation for 17 hr a t 30". If adsorption of phage had caused death none of the cells should have been viable, since the host cell and phage concentrations were such that all the cells should have been infected. Following subculture from the acid whey to neutral whey the surviving unadsorbed phage attacked and lysed the viable cells, demonstrating that viability did not depend on acquired resistance to phage. Similar results were obtained with culture 105.9. Thus, it appeared that in acid whey the host cells were apparently unaffected by the irreversible attachment of phage particles.

The effect of acid whey on phage multiplication Since in acid whey adsorption of phage particles occurred but phage multiplication did not, it appeared that some step subsequent to adsorption was prevented. One step growth experiments were done to study this problem.

Acidity and phage multiplication 291

Phage was adsorbed on to host cells in acid whey and samples transferred to neutral whey (a dilution of l O P ) . At intervals one of these inoculated neutral wheys was further diluted into acid whey. One sample was further diluted directly into neutral whey following adsorption, i.e. there was in this case no more contact with acid whey. The burst time during incubation a t 30" was determined for each sample.

Fig.

C ._ Y - 3

0 C ._

; L - c .- E - i i=

1. The effect on phage release in acid whey of increasing contact time in neutral whey following adsorption in acid whey of (a), culture 818; (b), culture 1059. Hatched meas, contact with acid whey; blank areas, contact with neutral whey. Horizontal broken lines indicate the beginning of phage release, with completion at the upper limit of each column.

80

70

60

50

40

30

20

10

0

(bl

Fig. 3. The effect on phage release of increasing contact time in acid whey following penetration in neutral whey after adsorption in acid whey of (a), culture 818; (b), culture 1059. Hatched areas, contact with acid whey; blank areas, contact with neutral whey. Horizontal broken lines indicate the beginning of phage release, with completion at the upper limit of each column.

297- Norah Thompson

As shown in Fig. l a , contact with neutral whey before final transfer to acid whey allowed host cells of culture 818 infected in acid whey eventually to release new phage particles. For all periods of contact with neutral whey of 35 min or less, phage release was equally delayed, The burst times in acid and neutral whey coincided only when there was 40 min contact with neutral whey. The results obtained with culture 1059 (Fig. 1 b) confirmed that renewed contact with acid whey delayed the burst time, but only when transfer of infected cells was completed about 10 min before phage release was normally due to occur. Thus it appeared that a contact period in neutral whey of only 1 or 2 min was sufficient to allow phage particles adsorbed on host cells in acid whey to penetrate them.

To investigate the delayed burst in acid whey, host cells with phage adsorbed in acid whey were diluted into neutral whey for 2 min to allow phage penetration and then further diluted into acid whey. At intervals a final dilution was made into neutral whey (a total dilution of One control sample remained in neutral whey following adsorption and a second control sample remained in acid whey following the penetration period. The burst time was determined for all samples, and in none of the samples transferred back to neutral whey was the burst time prolonged to that of infected cells which had been kept entirely in acid whey following penetration (Fig. 3, a and b). When contact with acid whey continued beyond the time of phage release in neutral whey the infected cells burst on transfer to neutral whey. These results confirmed that uninterrupted contact with acid whey after penetration merely delayed the release of phage particles.

Thus although phage adsorption on to host cells was not followed by phage multi- plication in acid whey, transfer of such adsorbed phage to neutral whey permitted normal phage reproduction. Acid whey appeared primarily to prevent penetration of phage into host dells and subsequent contact with neutral whey was necessary for this to take place. Transfer of ‘penetrated’ cells back to acid whey revealed a secondary effect in the delayed release of phage particles. Intracellular phage multiplication and burst size were not affected.

DISCUSSION

The prevention of mass lysis in acid whey a t pH values below 5.10 has been shown to depend on the inability of phage to multiply freely in such conditions. Puck & Tolmach (1954) found that attachment of phage T, to host cells of Escherichia coli B fell to l0-20% a t pH 5.0; but using a similar technique to determine adsorption of lactic phages the present experiments demonstrated that phage adeorption in whey was independent of pH between 4.40 and 6.20. In acid whey the adsorbed phage particles were apparently unable to penetrate the host cells, but the mechanism of prevention is not yet known.

Continued incubation of phage and host cells adsorbed in acid whey led to a loss of phage particles. As the number of phage particles so destroyed was similar to the number adsorbed it is thought that the phage particles were either bound irreversibly to the host cells or were disrupted there. Puck (1953) reported rapid destruction of phage particles by the release of their contents into the medium following adsorption

Acidity and phage multiplication 293

on heat killed E. coli B cells. However, in the present work the reduction in the number of phage particles was gradual during the first 18 hr of incubation a t 30°, with a further decrease when incubation was continued for 17 hr, so the disappearance of phage probably did not result from that sort of disruption a t the cell wall.

Puck (1953) also stated that phage adsorption on to viable cells was followed by invasion of the cells by the particle contents, causing rapid cell death. In the present work there was apparently no penetration of the host cells by the phage following adsorption in acid whey, and cells with phage adsorbed on them remained viable even after 17 hr a t 30". They also remained susceptible to phage attack in conditions suitable for phage reproduction, so the interaction between phage and host cells in acid whey, in which penetration of the host cell was apparently prevented and the phage eventually died, had little detectable effect on the properties of the host cells.

In their investigations into the reactions at the host cell wall which permit successful invasion of E. coli B cells by phage T,, Kozloff & Lute (1957) found that zinc was essential. They suggested that before adsorbed phage can release its contents into the host cell an interaction must take place between the phage particle and a zinc protein in the cell wall. It is possible that the reactions between lactic strepto- coccal phages and their host cells which lead to penetration similarly involve essential elements or compounds and that a t low pH values the availability of these is affected.

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influencing quantitative measurement of the virus. J . Bact. 58, 601. COLLINS, E. B. (1951). The relation of different numbers of bacteriophage and bacteria to

population changes and acid production. J . Dairy Sci. 34, 894. GAREN, A. & PUCK, T. T. (1951). The mechanism of virus attachment to host cells. 11. The

first two steps of the invasion of host cells by bacterial viruses. J . ezp. Med. 94, 177. GOLD, W. & WATSON, D. W. (1950). Studies on the bacteriophage infection cycle. 11. Phage

infection and lysis of Clostridiurn rnadisonii, a function of pH. J . Bact. 59, 17. D'HERELLE, F. (1926). The Bacteriophage and its Behattiour. English translation. London:

BeilliPre, Tindall & Cox. KOZLOFF, L. M. & LUTE, M. (1957). Viral invasion. 11. The role of zinc in bacteriophage invasion.

J. biol. Chern. 228, 529. MEANWELL, L. J. & THOMPSON, N. (1959). The influence of rennet on bacteriophage infection

in the cheese vat. J . appl. Bact. 22, 381. OVERCAST, W. W., NELSON, F. E. & PARMELEE, C . E. (1951). The influence of pH on prolifera-

tion of lactic acid streptococcus bacteriophage. J. Bact. 61, 87. PUCK, T. T. (1953). The first steps of virus invasion. Cold Spr. Harb. Symp. quant. Biol. no. 18.

Viruses, p. 149. PUCK, T. T. & TOLMACH, L. J. (1954). The mechanism of virus attachment to host cells. N.

Physicochemical studies on virus and cell surface groups. Arch. Biochem. Biophys. 51, 229.

(Received 23 April, 1959)