Penicillin-Induced Lysis of Streptococcus multans

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1 INFECTION AND IMMUNITY, Nov 1984, p /84/ $2/ Copyright 1984, American Society for Microbiology Vol 46, No 2 Penicillin-Induced Lysis of Streptococcus multans TIMOTHY A KRAL* AND MATTHEW D CALLAWAY Department of Botany and Microbiology, University of Arkansas, Fayetteville, Arkansas 7271 Received 19 December 1983/Accepted 1 August 1984 Treatment of Streptococcus mutans GS-5 cells with concentrations of penicillin G within a relatively narrow range resulted in substantial lysis This penicillin-induced lysis was dependent upon cell density and ph of the lysis medium Other oral streptococci (Streptococcus sobrinus, Streptococcus rattus, and Streptococcus cricetus) also demonstrated substantial levels of penicillin-induced lysis under appropriate conditions Lesser degrees of lysis were seen in a related organism, Streptococcus ferus Penicillin represents one of the most effective and widely prescribed antibiotics ever discovered; yet its mode of action has evaded attempts to define it One of the key questions that remains to be answered is how penicillin induces lysis in susceptible bacteria An older model, one accepted for many years, has suggested that penicillin-induced cell lysis is a direct result of unbalanced growth: cell mass increases while cell wall synthesis is inhibited by penicillin, and wall rupture and lysis follow (7) Recent evidence suggests other models One new model proposes that penicillin-induced lysis results as an indirect consequence of the reaction of penicillin with the penicillinbinding proteins of the cell This reaction inhibits peptidoglycan synthesis, resulting in inhibition of bacterial growth This primary interaction somehow triggers the release of teichoic acids, which activates one or more autolytic enymes These enymes then sever the covalent bonds of the cell wall, leading to the loss of osmotic integrity and, finally, cellular lysis (1, 11) Evidence for this new model has been supported by the discovery of mutant cells, derived from a penicillin-susceptible strain, with defects in the autolytic enyme systems Penicillin treatment leads to inhibition of growth without cellular lysis (9) This unusual response is referred to as antibiotic tolerance (9) Some naturally isolated, wild-type bacteria have also been shown to demonstrate this tolerance to penicillin Work done with a number of strains of oral streptococci appears to indicate that these microorganisms demonstrate growth inhibition without cellular lysis when treated with a wide range of penicillin G concentrations (1, 3-5) Additionally, Streptococcus rattus FA-1 (4) and Streptococcus mutans GS-5 (1) have demonstrated a penicillin G-induced, dose-dependent inhibition of peptidoglycan, RNA, DNA, and protein syntheses We studied the effects of a wide range of penicillin G concentrations on the growth of S mutans GS-5 The results indicate that these cells did indeed demonstrate tolerance to penicillin G at relatively high concentrations However, substantial levels of cellular lysis (up to 95%) have been observed within a lower concentration range This penicillin-induced lysis was dependent on both ph and cell density The present study defines conditions necessary for maximal lysis of S mutans GS-5 by penicillin G MATERIALS AND METHODS Organisms and growth conditions S mutans GS-5 and S rattus BHT were obtained from A S Bleiweis, Department * Corresponding author 442 of Microbiology and Cell Science, University of Florida, Gainesville Streptococcus cricetus HS6 and Streptococcus ferus 8S1 were obtained from A Coykendall, University of Connecticut School of Dental Medicine, Farmington S mutans Ingbritt was obtained from J van Houte, Forsyth Dental Center, Boston, Mass, and Streptococcus sobrinus 1 was obtained from W Little, National Caries Program, National Institute of Dental Research, Bethesda, Md Experiments were initiated by transferring 5 pl of a freshly-thawed culture with a pipette (Finnpipette; Arthur H Thomas Co, Philadelphia, Pa) into a cuvette (diameter, 18 mm) containing 5 ml of Todd Hewitt broth (Difco Laboratories, Detroit, Mich), supplemented with glucose (1%) Froen cultures were prepared by growing cells to 5 adjusted optical density (AOD) units (8) (an AOD unit is equivalent to ca 39 jig of cells [dry weight] per ml [6]), measured at 675 nm (Spectronic 2; Bausch & Lomb, Rochester, NY) in Todd Hewitt broth containing glycerol (3%), quick froen in dry ice-ethanol, and stored at -25 C All cultures were grown in Todd Hewitt broth supplemented with glucose (1%) unless stated otherwise From the firsttransfer culture, 1-2, 1-4, and 1-6 dilutions were made into the same medium The cultures were incubated at 37 C From an overnight culture measuring less than 5 AOD units, fresh, prewarmed media were inoculated at a starting AOD of ca 25 Cultures were incubated at 37 C, and optical density measurements were recorded every 3 min for 8 h and again at 24 h Addition of antibiotics When the cultures reached appropriate cell densities (AOD), 5-ml portions were transferred to cuvettes containing sterile penicillin G (from a fresh 1-pg/ml stock solution) In one experiment, ampicillin was used instead of penicillin G Penicillin G (potassium salt) and ampicillin were purchased from Sigma Chemical Company, St Louis, Mo Antibiotics were dissolved in distilled water and sterilied by membrane filtration (polycarbonate, 4- pm pore sie; Nuclepore Corp, Pleasanton, Calif) After antibiotic treatment, all cultures were further incubated at 37 C, and optical density measurements were recorded every 3 min for at least 8 h A 24-h measurement was also recorded Variation in ph ph values were varied in three ways In one type of experiment, cells were simply inoculated into media with different ph values and allowed to grow to a defined density and then treated with the antibiotic ph was adjusted with either 1 M HCI or 1 M NaOH (In one of these ph experiments, a chemically defined medium [FMC] [6] was also used Cells were grown in this medium for at least 2 generations before antibiotic treatment Immediately Downloaded from on April 21, 218 by guest

2 VOL 46, 1984 after inoculation into FMC medium, 1 [LI of a fresh, sterile, sodium bicarbonate solution [84 mg/ml] was added per 1 ml) In a second approach, ph was adjusted by aseptic addition of 1 M HCl or 1 M NaOH directly to growing cultures at defined densities Antibiotic treatment followed immediately In the last type of ph experiment, growing cells were centrifuged (5, x g for 15 min) and suspended at defined densities in media with the ph values already adjusted Again, antibiotic treatment followed immediately A ph meter (Accumet, model 61; Fisher Scientific, Pittsburgh, Pa) was used to determine ph values Dilutions and spent media In some experiments, cultures at different cell densities were diluted with fresh prewarmed medium to defined densities; antibiotic treatment followed In other experiments, cultures at different cell densities were centrifuged (5, x g for 15 min), and the spent media were used to suspend pelleted lower-density cells Lysis determinations Percent lysis was estimated from loss of absorbance by using the equation: % lysis = (highest AOD - final AOD/highest AOD) x 1 The highest AOD value was determined during a 45-min period, starting two generations after penicillin addition, at which point optical density remained relatively constant before lysis The final AOD measurement was recorded after 24 h of incubation The percent lysis determinations reflect lysis of cell mass (Optical density is a measurement of mass [influenced by cell sie as well as number], and observations [data not shown] indicate that the average sie of S mutans GS-5 cells changes during incubation in the presence of lytic concentrations of penicillin G) The equation may underestimate the extent of lysis, since fully lysed suspensions may exhibit some absorbance so 25 Ul MINUTES FIG 1 Effects of different concentrations of penicillin G (no antibiotic [], 125,ug/ml [], 75,ug/ml [H], and 5 pg/ml [O]) on the growth of S mutans GS-5 in Todd Hewitt broth All cultures shared a common optical density measurement (±5 AOD units) for ca two generations after antibiotic treatment [*] The arrow indicates the time at which the antibiotic was added PENICILLIN-INDUCED LYSIS OF S MUTANS 443 RESULTS Treatment of exponential-phase, Todd Hewitt-grown cultures of S mutans GS-5 with penicillin G demonstrated three different effects on growth, depending on the concentration used At penicillin concentrations below 25 jig/ml, cultures showed only a slight inhibition of growth Concentrations between 25 and 25 jig/ml effected substantial decreases in culture turbidity (lysis) Cultures treated with penicillin G within this concentration range showed no apparent effects on growth for ca two generations Inhibition of growth began two generations after treatment, followed by initiation of lysis after an additional generation At higher concentrations, above 25 jig/ml, culture turbidity became stationary ca two generations after antibiotic treatment, followed eventually by lesser degrees of lysis (up to 25%) Figure 1 shows the effects of one concentration within each of the three ranges described Previous observations (1, 3-5) have indicated that the lytic phenomenon does not occur in cultures grown in a defined medium (FMC) even within the lysis concentration range described above Because of the differences in initial ph of Todd Hewitt broth (ph 765) and FMC medium (ph 65), an experiment was designed to determine whether ph was a factor in the lytic phenomenon (Fig 2, Table 1) At ph values below 7, culture lysis is low or absent in both media This would seem to account for the lack of lysis previously seen in FMC-grown cultures Percent lysis reaches a maximum at ph 75 in FMC medium and at ph 8 in Todd Hewitt broth Much reduced levels occurred above ph 8 in both media (Strain GS-5 grows well between ph 5 and 85, and it grows very slowly, with a reduced cell yield, at ph 45 or 9) In both Fig 1 and 2, penicillin treatment was initiated at culture turbidities of between 1 and 15 AOD units To determine whether the lytic phenomenon was dependent on cell density, samples from growing cultures at different cell densities were treated with penicillin (Fig 3, Table 2) Lysis (88 to 94%) was observed in cultures between 5 and 175 AOD units Only 45% lysis was evident at 2 AOD units, and no lysis was seen above 4 AOD units Because of reduced accuracy of density measurements below 5 AOD units, cultures with cell densities below 5 AOD units were not tested in these experiments Results from the previous experiments indicated that maximal lysis could be effected in Todd Hewitt-grown cells at cell densities between 1 and 125 AOD units with a penicillin concentration of 75,ug/ml The ph in those cultures within that density range was ca 75 (down from 765, owing to fermentative metabolism) The amount of penicillin per cell (or per cell mass [AOD unit]) does not appear to be critical to affect cell lysis (Table 31) Cells at different culture densities were treated with either 75,ug of penicillin per ml, the concentration effecting maximal lysis at a culture density of 1 to 125 AOD units, or 75,ug of penicillin per AOD unit, a concentration derived from treatment of 1 AOD cultures with 75,ug of penicillin per ml Also, because ph apparently affects lysis, one group of cultures was treated with penicillin without adjusting the culture ph, while a second group was treated after the culture ph was adjusted to 765, the ph of fresh Todd Hewitt broth The results indicate that cell lysis occurred only at relatively low culture densities (1 to 2 AOD units), in agreement with previous results No substantial lysis was seen at higher densities with either set of concentrations In an additional experiment, cells at Downloaded from on April 21, 218 by guest

3 444 KRAL AND CALLAWAY TABLE 1 ph scale No of % SD tests Cl, - CD ~i w 4 lu ph FIG 2 Effects of ph on penicillin-induced lysis of S mutans GS-5 growing in Todd Hewitt broth [] and a defined medium [H] Cells were treated with a concentration of 75,ug of penicillin per ml at a culture density of 1 to 125 AOD units The ph values of the media were adjusted before inoculation (See Table 1) higher densities were treated with a range of penicillin concentrations between 25 and 12,ug/ml without any lysis (data not shown) To determine whether cell density or some factor related to phase of growth was actually responsible for the lysis observed only at low densities, cells derived from differentdensity cultures, representing different phases of growth, were diluted to 1 AOD units in fresh medium and treated with penicillin at an optimal lytic concentration (Fig 4) Cells from phases of growth in which active cell division occurs showed substantial levels of lysis when diluted and treated with penicillin Cells from stationary-phase cultures showed reduced levels of lysis Since lysis occurred only in actively dividing cells, an experiment was designed to determine whether higher-density cells (5 AOD units), maintained in an actively dividing phase by dilution, would lyse in the presence of penicillin Cells were grown to 5 AOD units, treated with penicillin (75 jxg/ml), and diluted with the same medium plus penicillin (75,ug/ml) to ca 5 AOD units every 15 min for 9 min After 9 min, the optical density no longer increased (The optical density reached a maximum of 745 AOD units between dilutions) Cells treated in this manner demonstrated 93% lysis Figure 5 shows the results of this experiment compared with those seen in Table 3 with a constant penicillin concentration and unadjusted p11 Cells diluted from older cultures will experience an increased ph in fresh Todd Hewitt broth To determine whether some factor other than relatively low ph was responsible for the reduced levels of lysis seen in older cultures, cells from a 1-AOD culture were centrifuged and suspended in different levels of spent media (determined by different cell densities before clearing) with adjusted (765) and unadjusted ph values (Fig 6) ph was adjusted immediately before penicillin treatment The amount of cell lysis rapidly decreased with increasing ages of spent media when the ph values were unadjusted However, in spent media adjusted to ph 765, lysis was evident at all levels tested Figure 7 and Table 4 show the results of an experiment designed to examine both ph and cell density Exponentialphase cells were transferred to media with different ph values at a density of 15 AOD units and allowed to grow in those media At various cell densities, aliquots were removed and treated with penicillin The results show a strong correlation between cell density and ph with respect to amount of lysis As cell density increases from 5 to 2 TABLE 2 AOD scale AOD No of % SD tests , C1,, -6' w 4 w 2'- ' INFECT IMMUN AOD FIG 3 Effects of cell density on penicillin-induced lysis of S mutans GS-5 growing in Todd Hewitt broth The concentration of antibiotic was 75,ug/ml at each cell density Note that the AOD scale is logarithmic (see Table 2) Downloaded from on April 21, 218 by guest

4 VOL 46, 1984 TABLE 3 Lysis of S mutans GS-5 cells treated with a constant penicillin concentration and cells treated with a constant amount of penicillin per cell mass at different culture densities and ph" Treatment AOD ph % Lysis Constant penicillin G concn (75,ug/ml) , b b b 8 765b 1,6 765b Constant amt of penicillin G per cell mass (75,ug/AOD) , b b b 1,6 765b a This experiment was performed twice with similar results b ph was adjusted by direct aseptic addition of 1 M NaOH to the cultures AOD units, an increase in ph from 65 to 8 was necessary in order to achieve maximal cellular lysis No substantial lysis was seen above 25 AOD units at any ph (It is important to note that at the time of antibiotic treatment in this experiment, ph values have been decreased, owing to fermentation What is significant is the variation in ph values at any one cell density and the effect of the variation on lysis at that cell density) tl 6 C) w AOD BEFORE DILUTION FIG 4 Penicillin-induced lysis of Todd Hewitt-grown S mutans GS-5 cells diluted to 1 AOD units from a variety of higher cell densities Antibiotic concentration was 75,ug/ml This experiment was performed once PENICILLIN-INDUCED LYSIS OF S MUTANS 445 TABLE 4 Lysis rates of S mutans GS-5 cells treated with 75,ug of penicillin G per ml at different phs and culture densitiesa Lysis rates (h-') at AOD of: ph ± 8 23 ± 4 No lysis No lysis 7 45 ± 1 37 ± 1 29 ± 6 18 ± ± 8 36 ± 3 33 ± 3 21 ± ± 4 21 ± 7 24 ± 1 27 ± 5 a Lysis rates were determined by using the equation: K = In 2/LH, where K is the lysis rate and LH is the time (hours) required to decrease the AOD by half a 4 >111 I- wl 4- In a similar experiment (data not shown), cells from cultures at various cell densities were centrifuged and suspended at the initial densities in fresh Todd Hewitt broth with different ph values These cultures were immediately treated with penicillin The results were similar to those seen in Fig 7 (ph was adjusted in three different ways in these experiments [see above] It appears that penicillin-induced lysis is dependent only on the ph of the culture at the time of antibiotic addition and not on the method used to adjust ph) To determine whether strains of oral streptococci other than S mutans GS-5 demonstrate penicillin-induced lysis, 1-AOD Todd-Hewitt-grown cultures of another S mutans I * lat i-' v 16 jj U *1 a 2 o o a o o o *,% o * MINUTES FIG 5 Effects of adding penicillin G to S mutans GS-5 cells at different cell densities (no antibiotic [], 1 AOD units [K], 2 AOD units [O], 4 AOD units [D], 8 AOD units [], 16 AOD units [*]) In a separate experiment (performed twice with similar results), cells were treated with penicillin at 5 AOD units (*) and diluted back to that optical density every 15 min for 9 min The concentration of penicillin was 75 Lg/ml o a * Downloaded from on April 21, 218 by guest

5 446 KRAL AND CALLAWAY strain, Ingbritt, as well as strains of S sobrinus, S rattus, S cricetus, and S ferus were treated with penicillin in a range of concentrations between 125 and 1 pg/ml (Table 6) All strains except for S ferus 8S1 demonstrated lysis within a concentration range comparable to that optimum for strain GS-5 (The optimal concentration for strain 1 was actually slightly lower than that for GS-5, whereas the range for BHT extended somewhat higher than that for GS-5) The effects of a related antibiotic, ampicillin, were also tested on S mutans GS-5 Lysis was observed in the concentration range between 1 and 25 pug/ml with maximal lysis (95%) within the 1-to-125-pg/ml range DISCUSSION Previous reports (1, 3-5) have indicated that strains of S mutans are tolerant to the lytic effects of penicillin G at any concentration tested The data presented here indicate that S mutans cells treated with a concentration of penicillin G within a relatively narrow range, and only under certain conditions, do indeed demonstrate cellular lysis Coincidentally, concentrations below the lysis range appear to prevent cell division at a time corresponding to the onset of the chromosome replication time, and higher concentrations appear to inhibit cell division at a time corresponding to protein synthesis inhibition by chloramphenicol (2) The observed lysis may represent cells trapped in the division cycle during a period of autolytic activity (2) The concentration of penicillin was only one of three factors critical to effect penicillin-induced cellular lysis A relatively low cell density and a ph correlated with a specific cell density were also important Any growing cells, whether diluted or concentrated (data not shown) to a proper 1 8 w 4 w AOD OF SPENT MEDIUM BEFORE CENTRIFUGATION FIG 6 Effects of spent media on penicillin-induced lysis of S mutans GS-5 cells Cells were grown to 1 AOD units in Todd Hewitt broth and centrifuged Cell pellets were suspended at 1 AOD units in spent Todd Hewitt media collected from cultures allowed to grow to different densities In one group of experiments, the ph values of the spent media were adjusted to 765 (A), whereas spent media in the second group were unaltered () Antibiotic concentration was 75,ug/ml This experiment was performed once TABLE 5 ph and AOD 91 SD at AOD of: ph U) 6 w 4 2 ' AOD FIG 7 Effects of cell density and ph (65 [], 7 [], 75 [U], 8 [O]) on penicillin-induced lysis of S mutans GS-5 cells growing in Todd Hewitt broth The concentration of antibiotic was 75,ug/ml in all cultures The ph values of the media were adjusted before inoculation This experiment was performed twice (See Table 5) density, could demonstrate lysis Both the amount of penicillin per cell and factors (other than ph) present in spent medium do not appear to be critical to this lytic phenomenon There does not appear to be any correlation between percent lysis and culture growth rate in cells doubling between 42 and 65 min The data presented in this paper represent results from 276 penicillin-treated cultures Of those, 257 had doubling times between 52 and 65 min with percent lysis ranging from to 95 The remaining 19 cultures grew less rapidly (doubling times, 68 to 195 min) and demonstrated much lower degrees of lysis ( to 26%) A correlation between slower growth rates and percent lysis remains to be elucidated The rate of lysis appeared to be correlated with percent lysis; the higher the percent lysis, the faster was the rate of lysis (linear regression correlation coefficient = 71) TABLE 6 INFECT IMMUN Penicillin-induced lysis in different strains of oral streptococci Organism and strain PenicillinG 91 concn (pg/ml) Lysis S mutans Ingbritt S sobrinuis S rattus BHT S (ricetus HS Sferus 8S <5() Downloaded from on April 21, 218 by guest

6 VOL 46, 1984 ACKNOWLEDGMENT This research was supported in part by Public Health Service grant BRSG S7 RR711 awarded by the Biomedical Research Support Grant Program, Division of Research Resources, National Institutes of Health LITERATURE CITED 1 Higgins, M L, T D McDowell, U B Sleytr, M Mychajlonka, and G D Shockman 198 Effects of penicillin on macromolecular synthesis and surface growth of a tolerant streptococcus as studied by computer reconstruction methods J Bacteriol 144: Kral, T A, and L Daneo-Moore 1982 Effects of cell wall inhibitors on cell division in Streptococcus mutans Infect Immun 41: Mattingly, S J, L Daneo-Moore, and G D Shockman 1977 Factors regulating cell wall thickening and intracellular iodophilic polysaccharide storage in Streptococcus mutans Infect Immun 16: Mychajlonka, M, T D McDowell, and G D Shockman 198 Inhibition of peptidoglycan, ribonucleic acid, and protein synthesis in tolerant strains of Streptococcus mutans Antimicrob Agents Chemother 17: PENICILLIN-INDUCED LYSIS OF S MUTANS Shockman, G D, L Daneo-Moore, J B Cornett, and M Mychajlonka 1979 Does penicillin kill bacteria? Rev Infect Dis 1: Terleckyj, B, N P Willett, and G D Shockman 1975 Growth of several cariogenic strains of oral streptococci in a chemically defined medium Infect Immun 11: Tipper, D J, and J L Strominger 1965 Mechanism of action of penicillins: a proposal based on their structural similarity to acyl-d-alanyl-d-alanine Proc Natl Acad Sci USA 54: Toennies, G, and D L Gallant 1949 The relationship between photometric turbidity and bacterial concentration Growth 13:7-2 9 Tomas, A, A Albino, and E Zanati 197 Multiple antibiotic resistance in a bacterium with suppressed autolytic system Nature (London) 227: Tomas, A, and J V Holtje Murein hydrolases and the lytic and killing action of penicillin, p In D Schlessinger (ed), Microbiology-1977 American Society for Microbiology, Washington, DC 11 Tomas, A, and S Waks 1975 Mechanism of action of penicillin: triggering of the pneumococcal autolytic enyme by inhibitors of cell wall synthesis Proc Natl Acad Sci USA 72: Downloaded from on April 21, 218 by guest