htternational Journal of Food Microbiology, 15 ( 1992 ) 109- I 19

109

~9 1992 Elsevier Science Publishers B.V. All rights reserved 0168-1605/92/ 3 . 0 0 FOOD 011464

Sites of action by propionate on Listeria monocytogenes Mahmoud M. Buazzi and Elmer H. Marth Department of Food St'iellce, Unicersity of Wisconsin-Madison, Madison, WI. USA (Received 7 May 1991: accepted 26 December 1991 )

Exposure of Listeria monoo'togenes to a solution of sodium propionate (Sr~ w / v ) for 60 min caused 87% of the population to be injured. Injury was evidenced by inability of the bacterium to tolerate 6% sodium chloride in tryptose agar as compared to ability to grow on tryptose agar with no added salt. Injured cells were allowed to repair in tryptose broth and the repair process was studied by addition to tryptose broth of sublethal amounts of metabolic or synthetic inhibitors. Repair of injured cells did not requile electron transport or synthesis of cell wall components, mRNA or protein. No changes which may have occarred in the cell membrane of injured cells, allowed leakage of proteins or nueleotides into the medium. Exogenous cation salts enhanced the rate of recovery of injured cells. The specific activity of lactic dehydrogenase was reduced in propionate-injured L. monocytogenes. Key words: Listeria monoo'togenes; Propionate; Injury

Introduction Sodium propionate or calcium propionate are used extensively to prevent growth of molds and bacteria in baked goods and dairy products. Propionic acid develops naturally in Swiss cheese at levels up to 1% (Langsrud and Reinbold, 1973). It is a weak inhibitor of many foodborne pathogens. Sodium propionate at levels of 0.1-5% delayed growth of Staphylococcus aureus by 5 days (Wolford and Anderson, 1945). More than 2000 ppm sodium propionate was required to inhibit growth of Listeria monocvtogenes in tryptose broth at pH 5.0 (EI-Shenawy and Marth, 1989). Propionic acid is thought to retard microbial growth by inhibiting synthesis of pantothenic acid (Wyss, 1948). The bacteriostatic action of sodium propionate on Escherichia colt is overcome by addition of small amounts of alanine to the medium (Wright and Skeggs, 1946), but inhibition of Bacillus subtilis by propionic acid could not be reversed by this treatment. Correspondence address: E.H. Marth, Department of Food Science, University of Wisconsin, 1605 Linden Drive, Madison, WI 53706, USA.

110

Exposure of L. monocytogenes to propionate in foods is likely to cause injury of cells. Although propionate is widely used in foods, little is known about how the chemical either injures or inactivates bacteria. Therefore, this study was designed to estimate the extent of such injury, to determine the conditions for resuscitation of propionate-injured cells and to characterize the mechanism of injury by sodium propionate.

Materials and Methods

Test orgaizism L. monocytogenes strain California (CA), serotype 4b, an isolate from Mexicanstyle cheese implicated in a 1985 listeriosis outbreak in California, was employed throughout the study. The stock culture, obtained from the Food Microbiology Laboratory Culture Collection, Department of Food Science, University of Wisconsin-Madison, was maintained through monthly transfers on slants of tryptose agar (Difco Laboratories, Detroit, MI), incubated aerobically for 24 h at 35°C, and stored at 4°C until transferred for use in experiments.

Preparation of inoculum lnocula from stock cultures were seeded to tryptose broth (Difco) and incubated aerobically at 35°C for 24 h. Culture activation was done by two subsequent transfers to sterile tubes of tryptose broth. After incubation, 40 ml of culture was centrifuged (Model RC-5, Sorvall, Chicago, IL) at 5000 × g at 5°C for 10 min. The supernatant fluid was decanted and the cell pellet was washed and suspended in sterile 0.1% peptone water. An initial cell population of ca. 1 × 107 cfu/ml, as determined with a spectrophotometer (Spectronic 2000, Bausch and Lomb, Rochester, NY), was obtained.

Preparation of control and injury media A sodium chloride (Sigma Chemical Company, St. Louis, MO), 8.0% (w/v), solution was prepared using deionized water as a diluent; the pH was adjusted to 7.0 using 0.1 N HCi or 0.1 N NaOH. The salt solution was filter-sterilized by passage through a sterile membrane with a pore size of 0.20 /xm (Nalgene disposable filter units, Type S CN, Nalgene Co., Rochester, NY). Sodium propionate (Sigma) solutions (w/v) of 2.0, 4.0, 6.0 and 8.0% were prepared using deionized water as the diluent; the pH was adjusted to 7.0 using 0.1 N HCI or 0.1 N NaOH. The solutions were filter-sterilized (Nalgene), kept in the dark at ca. 7°C and used within 2 weeks.

Cell injury procedure One milliliter of cell suspension was added to 9.0 ml of an 8.0 (w/v) sodium propionate solution. Controls were made by adding cells to peptone water or 8.0% sodium chloride solution. Tubes were incubated aerobically for 1 h at 35°C. At various times, 1-ml samples were serially diluted in 9.0 ml of peptone water and

111

plated in duplicate on tryptose agar, and tryptose agar with 6% (w/w) total NaCI. Tryptose agar and tryptose agar with NaCl were adjusted to p | I 7.1 with a predetermined amount of sterile 1 N NaOH immediately before pouring. Inoculated plates were incubated aerobically for a8 h at 35°C. Colonies were counted with the aid of a Quebec Colony Counter (American Optical Corporation, Buffalo, NY). Inability to form colonies on tryptose agar with 6% (w/w) total NaC! was considered evidence of injury (Ahamad and Marth, 1990). The total cell population was measured using tryptose agar, whereas the number of noninjured (or recovered) cells was measured using tryptose agar with 6% ( w / w ) total NaCl. The difference in the initial counts on tryptose agar and tryptose agar with NaC1 was deemed to be equal to the number of injured cells in the suspension. The extent of injury was determined as the ratio of counts on t~pvose agar with NaCI to those on tryptose agar. The extent of death was determined as the ratio of tryptose agar counts after various treatment times to initial tryptose agar counts.

Cell recouery procedure Samples (1.0 ml) of injured cells exposed to the 8.0% sodium propionate solution for 1 h at 35°C were transferred to 125-ml Erlenmeyer flasks containing 98 ml of tryptose broth recovery medium, plus 2 ml of deionized water in which 1 mg (1200 units) of penicillin (Sigma) was dissolved before filter sterilization. Flasks were shaken at 45 rpm for 5 rain at 35°C in a water bath (Precision Scientific Company, Chicago, IL). Samples for all trials were incubated aerobically at 35°C. Portions of the samples were taken periodically, serially diluted in 9.0 ml of 0.1% peptone water, and 0.1-ml quantities surface-plated onto both tryptose agar and tryptose agar with 6% ( w / w ) total NaCI which were incubated aerobically at 35°C for 48 h. Colony counts were determined as described earlier and percent recovery calculated.

Calculation of recovery by cells Recovery (%) was obtained through application of the following formula to values of recovery trials. Recovery(%) =

(S,

-

So)

(To-So)

× 100

where S O= colony count on plates of tryptose agar with 6% ( w / w ) total NaCI after injured cells were inoculated into the recovery medium, T0 = colony count on plates of tryptose agar after injured cells were inoculated into the recovery medium, S t = colony count on plates of tryptose agar with 6% (w/w) total NaCl after injured cells were incubated for 1 h in the recovery medium.

Effect of metabolic inhibitors on recovery by cells Low concentrations of different growth inhibitors (actinomycin D, rifampicin, chloramphenicol, and 2,4-dinitrophenol, each at 10 and 2 0 / z g / m l ) were separately added to the tryptose broth recovery medium to obtain information on synthetic

112

processes involved in cell iJ~juiy (Blankenship, 1980; Zayaitz and Ledford, 1985). The inhibitors were obtained from Sigma. Solutions of inhibitors were filter-sterilized and aseptically added to sterile tryptosc broth. Samples (1.0 ml) of injured cells, exposed to 8.0% sodium propionate for 1 h at 35°C, were transferred to tryptose broth containing various inhibitors and incubated aerobically at 35°C for 4 h. At zero time and after each additional hour, l-ml samples were withdrawn, serially diluted in 9.0 ml of sterile 0.1% peptone water, and 0.l-ml quantities were surface-plated on plates of tryptose agar and tryptose agar with 6% ( w / w ) ~otal NaCI. Colonies were counted after incubation of plates at 35°C for 48 h.

Determination of nucleotide and protein leakage Suspensions of cells injured with 2.0, 4.0, or 8.0% sodium propionate for 1 h at 35°C, were centrifuged (Sorvall) at 5°C for 15 rain at 2000 x g. Supernatant liquids were passed through 0.20-/~m filters (Nalgene) and examined by scanning from 800 nm to 2(10 nm using a double beam spectrophotometer (Model DU-65, Beckman, Rochester, NY). An equal amount of control cells was suspended in sterile 0.85% saline solution. A subsequent saline solution wash of control and injured cells was filtered and scanned for leaked materials.

E]]'ect of cations on the repair of injured cells Solutions of sodium propionate, 2.0, 4.0, and 8.0%, were used to injure cell suspensions of L. monocytogenes. In addition, solutions A (0.025 M each of MgCI 2, CaCI 2, and KCI) and B (0.0125 M each of the aforementioned salts) were prepared to contain 0.005 M and 0.0025 M concentrations of salts, respectively. One ml of either solution A or B and 1.0 ml of L. monocytogenes cell suspension (1.0 X 10 4 cfu/ml of sterile 0.85% saline solution) were added to 8.0 mi of each of the aforementioned solutions of sodium propionate. Cells in the salts-propionate solution were incubated at 35°C for 1 h, centrifuged (Sorvail) at 5000 x g at 5°C for 10 min, resuspended in 10 ml of sterile 0.85% saline solutica for 30 rain at 35°C and plated in duplicate on tryptose agar with 6% ( w / w ) total NaCI. A second trial using the same treatment as just described was done, but 1 ml each of solutions A or B (instead of the propionate solution) were added to resuspended cells. A propionate-free control was included in all trials.

Preparation of cell-free extract Cells of L. nzonocytogenes from stock tryptose broth were transferred as previously described to activate the culture. Sterile tryptose broth (200 ml) was inoculated with 2 ml of the active culture and incubated at 35°C for 24 h. Cells were harvested by transferring the incubated medium to 100-ml centrifuge tubes and centrifuging (Sorvall) at 1500 x g for 20 rain at 5°C. Cells were washed, resuspended in 1 ml of potassium phosphate buffer (pH 7.0) (PPB), transferred to 9.0 ml of solutions of sodium propionate (2.0, 4.0, 6.0, or 8.0% final concentr:,:ion) (pH 7.0) prepared as described earlier, and incubated at 35°C for 1 h. Control cells were suspended in 9.0 ml of PPB. The cells were removed by eentrifugation (Sorvall) at 1500 x g for 10 min at 5°C, washed, and resuspended in 1 ml of PPB.

113

A cell-free extract was obtained by treatment of the cell suspension first with pancreatic lipase and then with lysozyme as described by Ghosh and Murray (19671. The pellet was suspended in 0.01 M Tris buffer (pH 8.1) containing 0.02 M MgC! 2 and 0.01 M glucose. Peilet suspensions were incubated at 7°C with frequent shaking for 30 min to allow complete lysis of cells and uniform dispcrsion of lysate. The cell-free extract (supernatant fluid) was obtained b~ centrifuging (Sorvall) the suspension of enzyme-treated cells at 23000 × g for 10 fnin at 7°C.

Assay for lactic dehydrogenase actirity The protein content of the cell-free extract was determined according to the method of Lowry et al. (19511 with bovine serum albumin (1.0 mg/ml) (Sigma) as a standard. Lactic dehydrogenase (LDH) activity was determined by measuring the decrease in optical absorbance by reduced nicotine adenine dinucleotidc (NADH) (Sigma) at 340 nm using a double beam spectrophotometer (Model DU-65, Beckman). The reaclion mixture contained 2.0 ml of cell-free extract, 0.5 ml of 0.05 M pyruvate (Sigma), 0.1 ml of 0.001 M NADH, and 0.4 mi of PPB. Reaction reagents were refrigerated immediately after preparation. All reactions were done at 25°C in a total volume of 3.0 ml. One unit of LDH is arbitrarily defined as the amount of enzyme which will produce a AOD of 0.001 per min at 340 nm in 3.0 ml of assay mixture incubated at 25°C. The specific activities of LDH are reported as units per ml of protein in the cell-free extract.

Results

lzktimation of cell btjury There were no appreciable differences in colony counts of L. monocytogenes on tryptose agar and tryptose agar with 6% ( w / w ) total NaCI after cells were incubated in 8.0% NaCI (Fig. 1) or 0.1% peptone water. Differences in survival of L. monocytogenes were found when propionate-injured cells were plated on tryptose agar and tryptose agar with NaCI (Fig. 2). Colony counts on tryptose agar and tryptose agar with NaC! showed values of 87% for injury and 12% for death after exposure of cells to the 8.0% propionate solution for 1 h. Lower propionate concentrations (data not shown) gave less reproducible results. However, the rate of injury and death increased with an increasing propionate concentration from 2.0 to 8.0%, as measured by colony counts on tryptose agar with and without NaCI.

Estimation of recovery by injured cells Injured cells recovered their salt tolerance after incubation in tryptose broth (Fig. 3). Complete repair, as indicated by equal colony counts on plates of tryptose agar and tryptose agar with NaCi, was observed after approx. 3 h of incubation. Recovery was accompanied by a noticeable lag phase. The slight growth observed after complete recovery indicates inability of penicillin, 5 / z g / m l , to halt growth of uninjured cells and probably injured cells.

114 6.0x10 6

5.5x10 6

• b

5.0x106

6 I

4.5x10 ,

4.0x106 0

30

60

Minutes Fig. 1. Numbers of L. monocytogenes recovered during treatment with 8.0% (w/v) sodium chloride at 35°C. Cells were plated on TA ( • ) and TAS (o) (means of two experiments, each carried out in duplicate; standard deviations indicated by bars).

Recot'ery of cells in the presence of hzhibitors N o d e a t h o f cells, a s i n d i c a t e d by a n a p p r e c i a b l e d e c r e a s e in t r y p t o s e a g a r p l a t e c o u n t , was n o t e d in a n y trial u s i n g t h e s u b l e t h a l c o n c e n t r a t i o n s o f t h e a n t i b i o t i c s o r m e t a b o l i c i n h i b i t o r s l i s t e d in T a b l e I. R e c o v e r y o f cells i n j u r e d a f t e r t r e a t m e n t w i t h 8 . 0 % p r o p i o n a t e for 60 rain at 35°C w a s i n v a r i a b l y d e l a y e d by t h e s e i n h i b i t o r s

1.0x10 6.

t

:3 1.0x10 s in tj

1.0x104 30 60 Minutes Fig. 2. Numbers of L. monocytogenes recovered during treatment with 8.0% (w/v) sodium propionate at 35°C. Cells were plated on TA (11) and TAS (e) (means of two experiments, each carried out in duplicate; standard deviations indicated by bars). 0

115

1×105 t

lx104

J

u_ lx10 3

Ixi0: 0

2 Hours

3

4

Fig. 3. Numbers of L. monocytogenes on TA (11) and TAS (e) plates after injury with 8.0% ( w / v ) sodium propionate for ! h at 35°C and incubation in TB with penicillin (10/.tg/ml) at 35°C (means of two experiments, each carried out in duplicate; standard deviations indicated by bars).

(Table I). Propionate-injured cells recovered substantially in the presence of all inhibitors tested.

Absence of leaked intracellular substances There was no evidence of membrane damage since released proteins or nucleotides were absent from supernatant fluids of propionate-injured cells. Supernatant liquids Gf control suspensions also showed no absorbance in the tested range.

TABLE I Recovery of propionate-injured L. monocytogenes inhibitors

at 35°C in tryptosc broth containing different

Inhibitor

/.tg/ml

Recovery in 3 h

Rifampicin

10 20

98 97

0 1.4

Actinomycin D

10 20

100 100

2.8 5.6

Chloramphenicol

10 20

87 81

8.4 2.8

2,4-Dinitrophenol

10 20

71 60

12,6 7.0

" Standard deviation of % recovci~ in duplicate trials.

SD "

116 T A B L E 11 N u m b e r of L. monocytogenes recovered on t~'ptose agar with 6c,~ ( w / w ) total NaCI from cell s u s p e n s i o n s treated with propionate and cation salts Sodium propionate ((k)

Treatment

2.0

4.0

8.0

Salts a d d e d with propionate Solution A " Solution B h

7.6 × 1114 4.5 × 10 4

1.8 × I() 4 3.7× 10 4

1.7 x I(13 2 . 0 × 10 3

Salts a d d e d with saline wash Solution A " Solution B h

5.7 × 1(14 3 . 0 × 104

1.0 × 10 4 1.1)× 10 4

5:3 x 10 3 2.1 × I() ~

N o salts added

5.3 x 104

2.0 × i(I 4

9.0 × 102

" A solution of MgCI 2, CaCI 2 and KCI in a final concentration of 0.(105 M. b A solution of M g C I , , CaCI2 and KC! in a final concentration of 0.0025 M.

Effect of salts on recot'ety of htjured cells MgCi 2, CaC! z, and KC! enhanced recovery of propionate-injured L. monocytogenes (Table II). Compared to the control, an average increase of 0.63 and 0.27 log in colony counts was observed when salts were added with the saline wash and the

propionate solution, respectively. Activity of lactic dehydrogenase (LDH) in cells Propionate-treated and control cells contained approx. 245 and 250 /.tg of protein per ml of cell-free lysate, respectively. Specific activities of LDH of

36

34 32" > 3O

~ 28 ~26 ° ~

,

24

22 20

0

2

4 6 S o d i u m propionate (%)

8

Fig. 4. Specific activity of lactic d e h y d r o g e n a s e in cell-free extract of L. monocytogenes various concentrations of s o d i u m propionate for l h at 35°C.

treated with

i17

propionate-injured cells decrcascd appreciably with increasing propionate concentrations (Fig. 4).

Discussion

Injury of L. monocytogenes caused by exposure to sodium propionate did not involve cellular functions related to electron transport, cell wall, cell membrane, m R N A or protein. The most likely cause of cellular damage appears to be inhibition of proper synthesis or functioning of lactic dehydrogenase. To ensure that only the primary site of action of sodium propionate was affected, time allowed for injury was limited to the first hour of incubation. Secondary sites of damage may arise in the first generation which evolves after approx. 40 min of incubation at 35°C in tryptose broth (Ahamad and Marth, 1989). Dissociation of propionic acid is inversely related to the pH of the medium (Russell, 1983). To determine the specific action of sodium propionate, effects of low pH should be excluded. Hence a high concentration (8%) of sodium propionate was used at a neutral pH. At pH 7.0, the 8.0% sodium propionate yielded approx. 0.36 M propionate ion, 0,36 M sodium ion, and 0.004 M propionic acid in the injury mixture. Treatment of L. monocytogenes with 8.0% NaCI alone (Fig. 1) resulted in no apparent injury when results were compared to those of control trials. Nevertheless, the sodium ion may have a greater impact on the ability of cells to maintain a proton gradient when cells are treated with sodium propionate rather than sodium chloride. Penicillin was added to the recovery medium to halt growth of L. monocytogenes; otherwise this growth may be considered as recovery of injured cells. The relatively slight growth observed upon complete recovery indicates inability of penicillin to effectively halt growth of uninjured cells and probably injured cells. However, the rate of recovery was much faster and greater than the rate of growth. An increase in colony counts in the presence of penicillin aiso was observed during recove,~ of acid-injured (Zayaitz and Ledford, 1985) and heat-injured (Hurst et al., 1973) cells of S. aureus. Penicillin was lethal to recovering cells of heat-injured Streptococcus faecalis (Clark et al., 1968). Antibiotics or metabolic inhibitors to which Gi~am-positive bacteria are sensitive were used to characterize cellular targets harmed by sodium propionate. There is no agreement in the literature on percentage of inhibition at which an antibiotic target is concluded as essential for recovery of injured cells. In our view, the recovery may not exceed 10% for an antibiotic to be concluded as inhibitory. Greater than 10% recovery indicates appreciable ineffectiveness of the inhibitor. The substantial recovery of injured cells in the presence of all test inhibitors indicates that propionate-injured cells of L. monocytogenes did not require R N A or protein synthesis for recovery. Rifampicin was used to inhibit initiation of R N A synthesis. It noncovalently binds to the /3-subunit of the DNA-dependent R N A polymerase (Waring and McQuillen, 1982). If propionate exerted a general in-

118

hibitory effect on diffcrent enzymes, including RNA polymerases, actinomycin D was used to selectively encourage inhibition of RNA synthesis without altering RNA polymerases. Actinomycin D acts by binding to double stranded DNA thus preventing its use as a template for RNA synthesis (Waring and McQuillen, 1982). Actinomycin D effectively inhibited recovery of S. attretts after injury by lactic acid (Zayaitz and Lcdford, 1985). We used chloramphenicol to inhibit peptide bond formation between newly synthesized peptide~ inside 50S ribosomal subunits (Russcll, 1983). Protein synthesis may be a minor requirement for complete recovery of L. monoqvtogenes injured with sodium propionate. 2,4-Dinitrophenol was added to inhibit electron transport, and oxidative phosphorylation. Both may play a minor role in salt tolerance of propionate-injured L. monocytogenes. 2,4-Dinitrophenol strongly inhibited recovery of acid-injured E. coli (Przybyiski and Witter, 1979) and S. aureus (Zayaitz and Ledford, 1985). Injured cells frequently lose some of their cellular material through leakage into the surrounding medium. This leakage is primarily indicative of membrane damage, as that reported for mildly heated microbes (Hurst, 1977). No leakage of 260-280 nm absorbing materials has been reported for acid-injured S. aureus (Zayaitz and Ledford, 1985), S. bareilly (Blankenship, 1980), and E. coli (Pxzybylski and Witter, 1979). Cations are essential for proper functioning of enzymes and phospholipids of microorganisms (Hurst and Hughes, 1975). Recovery of heat-injured S. aureus depends on restoration of magnesium leaked during the course of heat treatment. Even though no leakage of cellular components was found in propionate-injured L. monocytogenes, propionate may affect functioning or availability of cations in the cell. The marked decrease in injured cells when cations were added to the suspending medium emphasizes the critical role these cations play in recovery of propionate-injured L. monocytogenes. There was a noticeable reduction in the specific LDH activity of propionate-injured L. monocytogenes. Because many enzymes function only in the presence of other subcellular components, addition of external LDH to a mixture of pyruvate, NADH, and propionate was not appropriate to test for changes in enzyme activity. Among these other subcellular components required for LDH activity is pantothenic acid, also known as coenzyme A (Dawes and Large, 1982). Indeed, propionic acid is thought to inhibit synthesis of pantothenic acid by bacteria (Wyss, 1948). The bacteriostatic action of sodium propionate in E. coli is overcome by addition of small amounts of alanine (Wright and Skeggs, 1946). Successful inhibition by propionic acid of microorganisms in low-alanine foods, like bread, may explain differences in this inhibition between foods of plant or animal origin. It would be of interest to compare rates of inhibition of L. monocytogenes by propionate in a minimal medium with or without excess pantothenic acid or alanine.

119

Acknowledgement Research supported, in part, by the College of Agricultural and Life Sciences, University of Wisconsin-Madison, Madison, WI, U.S.A.

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