Inactivation of Listeria Monocytogenes by Chlorine

Transcription

1 520 Journal of Food Protection, Vol. 51, No. 7, Pages (July 1988) Copyright International Association of Milk, Food and Environmental Sanitarians Inactivation of Listeria Monocytogenes by Chlorine SOUZAN E. EL-KEST and ELMER H. MARTH* Department of Food Science and the Food Research Institute, University of Wisconsin-Madison, Madison, Wisconsin (Received for publication December 18, 1987) ABSTRACT Cells of Listeria monocytogenes strain Scott-A were harvested from cultures, washed, and then treated with a solution of sodium hypochlorite at 25 C and ph 7. The cells were more resistant to chlorine when they were (a) harvested from a 24- rather than 48-h-old culture, (b) grown in tryptose broth rather than on a slant of tryptose agar, and (c) washed and suspended using a 20 rather than mm phosphate buffer solution. Cells of L. monocytogenes were exposed for 30 s-4 h to sodium hypochlorite solutions that contained ppm available chlorine. Generally, the number of survivors decreased rapidly during the first 30 s followed by a slower decrease during the rest of the exposure time. The initial count of L. monocytogenes in the suspension (Ixl xl0 8 /ml) decreased 0.49 to 6.4 orders of magnitude during the first 30 s of exposure to the chlorine solutions. The effect of the presence of organic substances on the strength of hypochlorite solutions was studied. Presence of 0.05 or 0.1% peptone caused a large and rapid loss of available chlorine. Glucose or lactose (up to 1%) had almost no effect on the concentration of available chlorine. Listeria monocytogenes is a pathogen that can infect pregnant women and their fetuses. Listeriosis, the disease caused by L. monocytogenes, manifests itself as a septicemia which can lead to death or mental retardation of newborns. Meningitis is the major manifestation of listeriosis in adults (9). During the last few years, there were several food-associated outbreaks of listeriosis as well as product recalls because L. monocytogenes was present in food. Chlorine is widely used in the food and dairy industry as a disinfectant (18). The effectiveness of chlorine against many pathogens is well known (4,15). However, information about inactivation of L. monocytogenes by chlorine is limited (3,13). The aims of this study were to investigate the behavior of L. monocytogenes when cells were exposed to different concentrations of chlorine and to determine consumption of chlorine during treatment of L. monocytogenes and in the presence of peptone or sugars. In this study, we also investigated the resistance to chlorine of L. monocytogenes cells produced on different media, of different ages, and suspended in a buffer having several concentrations of solute. MATERIALS AND METHODS Preparation of glassware Chambers' method (5) was followed to prepare zero chlorine-demand glassware. Dark-colored, 250-ml Erlenmeyer flasks each with a ground glass opening were used to treat L. monocytogenes with chlorine. Class A pipettes and volumetric flasks were used for final adjustment of the chlorine concentration. Preparation of hypochlorite solutions A sodium hypochlorite solution (Baker) which contained ca. 5% available chlorine was used as the source of chlorine. A stock solution (ca. 100 ppm available chlorine) was prepared using a phosphate buffer solution (20 mm and ph 7). This stock solution was held refrigerated and was made fresh every 2 weeks. The buffer also was used to prepare all other hypochlorite solutions. Deionized glass-distilled water was used to prepare the phosphate buffer. This water was considered to have zero chlorine-demand (16). Three solutions that contained approximate concentrations of available chlorine were prepared: one at about the desired concentration, the second at a higher and the third at a lower concentration than the first one. The desired concentration was then obtained by adjusting the strength of the first solution using one of the other two solutions, right before treatment of L. monocytogenes. The rest of this adjusted solution was kept to measure the concentration of available chlorine after treatment. Concentration of available chlorine was determined by one of two methods: the AOAC arsenious oxide titration method (10) or the chlorine electrode (Orion Model , Cambridge, MA) method. The AOAC method was used for the initial work. It also served as the "standard method", and was used with a wide range of chlorine concentrations. The chlorine electrode method was compared with the AOAC method. Results obtained on the same samples by the two methods were similar and the chlorine electrode method was more rapid, convenient, and economical to use than the AOAC method, particularly for solutions with low concentrations of chlorine. Neutralizer Sodium thiosulfate, 2.22 mm, plus % Bacto-peptone (ph 7.0) were used to stop the action of chlorine against L. monocytogenes after the desired time of exposure (16). Toxicity and effectiveness of this neutralizer were determined (5). It was non-toxic to the bacterium and effectively neutralized the activity of chlorine.

2 CHLORINE INACTIVATES LISTERIA 521 Preparation of the suspension ofh. monocytogenes L. monocytogenes strain Scott-A from a slant culture was transferred to a tube of Tryptose broth (TB) which was incubated 24 h at 35 C. This was followed by two additional similar transfers in TB on each of two consecutive days. Contents of the last culture tube (third transfer) were used to inoculate a series of tubes of TB. These tubes were incubated for 24 or 48 h (to make a 24- or 48-hold culture). This culture (or cell suspension) was centrifuged at 1100 x g for 7 min in a bench-top centrifuge (IEC clinical centrifuge, Damon/IEC Division, Needham Hts., MA). The supernatant liquid was discarded. The cell pellet was washed three times with phosphate buffer solution (PBS), 20 mm, (or otherwise as indicated) and cells were collected by centrifugation using the gravitational force just described. The cell pellet was suspended in PBS to yield ca cells/ml. This suspension was prepared right before treatment of L. monocytogenes. Treatment ofh. monocytogenes with chlorine The initial number of CFUs of Listeria/mX of control PBS was measured by surface-plating on Tryptose agar (TA). Plates were incubated at 35 C for 48 h before counting. The control flask was then kept at the same temperature, and for a time equal to the maximum exposure time of L. monocytogenes to chlorine in the treatment. Then the CFUs of L. monocytogenes /ml of control were determined again. One ml of the suspension of L. monocytogenes was mixed with the previously prepared hypochlorite solution (99 ml of the indicated strength). One ml of this mixture was taken after a desired time of exposure (between 30 s and 4 h) and immediately thoroughly mixed with the neutralizer (9 ml). Plating from the appropriate dilutions of the neutralized mixture was done to determine the CFUs of L. monocytogenes/ml that survived after a specific time of exposure. Plates of TA were used and incubated at 35 C for 48 h. Each experiment was repeated at least three times. Factors affecting inactivation ofh. monocytogenes The effects of the following factors on inactivation of L. monocytogenes by chlorine were studied. Age of culture. A 24- or48-h-old culture of L. monocytogenes was used to determine the effectiveness of 1 ppm available chlorine against cells of different ages. The previously described method of treatment with chlorine was used. Culturing and washing procedures. The following four suspensions of L. monocytogenes were made: (a) growth of L. monocytogenes from a Tryptose agar slant was washed with phosphate buffer dilution water (PBDW) following the AOAC method (10); then cells were harvested by centrifugation, washed thrice, and suspended using PBDW as described previously, (b) the suspension was prepared as in (a) except 20 mm phosphate buffer solution (PBS) was used, PBS contained a higher concentration of solute than PBDW, (c) cells were harvested from a Tryptose broth culture by centrifugation, then washed and suspended using PBDW as described previously, (d) the suspension was prepared as in (c) except PBS was used instead of PBDW. Each of these suspensions was treated separately with the same concentration of available chlorine (ca. 2 ppm) under the same experimental conditions. The arsenious oxide titration method was used to determine available chlorine. Length of treatment and repeatability. The fate of L. monocytogenes that was exposed to 1 ppm available chlorine for 30 s to 4 h was investigated. The chlorine electrode was used to determine the concentration of available chlorine when test solutions were prepared. Concentration of available chlorine. Effectiveness of 0.5, 1, 2, 5, and 10 ppm available chlorine (ph 7 and 25 C) against L. monocytogenes was investigated. The chlorine electrode was used to determine the concentration of available chlorine when these solutions were prepared. Consumption of chlorine during treatment. Consumption of chlorine from solutions containing 0.5, 1, and 2 ppm available chlorine was determined at intervals of 1,2,3,4,5,10,20, and 30 min after adding the suspension of L. monocytogenes cells. The hypochlorite solution and suspension of cells were prepared as described previously. One ml of cell suspension was added to 99 ml of hypochlorite solution. After a given exposure time, the concentration of available chlorine was determined using the chlorine electrode. The control was prepared by adding 1 ml of sterile PBS instead of the cell-suspension. Presence of organic compounds. The effect of 0.05 or 0.1% peptone in hypochlorite solutions (10 or 20 ppm available chlorine) on stability of this solution was determined. Hypochlorite solutions were prepared as described previously and 100 ml of each solution (the volume required to measure the strength of solution by the electrode) was dispensed into each flask. Peptone to yield the desired percentage was added to each flask and dissolved immediately by gentle shaking. The concentration of available chlorine was determined 1, 2, 3 and 4 min after adding peptone to the hypochlorite solution. A separate flask was used for each time interval. Solutions containing 19 ppm available chlorine and 0.05,0.1, 0.5,1.0 or 2.0% lactose or glucose were prepared as described for making the chlorine-peptone solutions. Available chlorine was determined 1 min after adding sugar. In another trial, available chlorine was monitored in a chlorine-lactose solution (2.0 ppm available chlorine +1% lactose) at intervals between 1 min and 7.0 h. A third trial was done using 0.1% glucose and 12.7 ppm available chlorine with an exposure of 1 min to 1 h. RESULTS AND DISCUSSION Age of culture Data in Fig. 1 show the number of survivors when 24- and 48-h-old cells of L. monocytogenes were exposed to a if) O > 10 '* 10 "' O 10 CL 10 -*? 10 "" i i i i i i i i i i i i i i i i i i t i i i i i i i i i i i i i i i i i i TIME(MIN) Figure 1. Inactivation of 24- or 48-h-old culture of L. monocytogenes strain Scott-A using 1 ppm available chlorine at ph7and25 C.( o o ) 24-h-oId culture, ( a a ) 48-hold culture.

3 522 EL-KEST AND MARTH TIME (MINI) TIME (MIN) Figure 2. Effect ofculturing and washing procedures on inactivation o/listeria monocytogenes using sodium hypochlorite (ca. 2 ppm available chlorine). ( o o-' Cells from Tryptose agar slant were washed and suspended using PDDW. (,.[] - ) Cells from Tryptose agar slant were washed and suspended using PBS. ( A A ) Cells from Tryptose broth culture were washed and suspended using PBDW. ( x -x ) Cells from Tryptose broth culture were washed and suspended using PBS. sodium hypochlorite solution (same strength of available chlorine for cells of both ages). The initial CFU (ca ) of cells from the 24-h-old culture decreased by 2.65, 2.88, and 3.79 orders of magnitude after 30 s, 10 min, and 4 h of exposure to chlorine, respectively; the decreases were 4.61, 4.27, and 4.50 orders of magnitude, respectively, when cells from a 48-h-old culture were used. When the cells were exposed to chlorine for 30 s, 10 min or 4 h, the difference in number of survivors from cultures of these two ages was 1.96, 1.41 or 0.72 order of magnitude, respectively. Accordingly, L. monocytogenes from a 24-h-old culture was somewhat more resistant to chlorine than were the cells from the older culture. Cells from the younger culture needed a longer exposure to chlorine than did cells from the older culture for an equivalent degree of inactivation. The reason for this observation may be related to the difference between young and old cells in the structure of the cell wall. Young cells of L. monocytogenes have a relatively thicker cell wall than do older cells. The cell wall from a young culture consists of three apparent layers. In older cells, these layers have a diffuse appearance (14). This structural difference may have an effect on the amount of HOC1 that penetrates into the cell and the speed with which this happens. Culturing and washing procedures Figure 2 shows results of tests on inactivation of four different cell suspensions of L. monocytogenes. These sus- Figure 3. Behavior ofl. monocytogenes during three independent trials in a solution containing 1 ppm available chlorine (ph 7 and 25 C). pensions were prepared from broth or slant cultures and the cells were washed and suspended using PBS (20 mm) or PBDW (0.312 mm). The cells from a broth culture that were washed and suspended using PBS were more resistant to chlorine than the other cells. The liquid medium is better suited for bacterial growth (8). The solution in which cells of L. monocytogenes were suspended was more protective to the bacterium when the solution contained a higher rather than a lower concentration of solute. This protection may be related to membrane stabilization (1). Length of treatment and repeatability When L. monocytogenes was exposed to 1 ppm of available chlorine, the number of surviving cells decreased rapidly (2.65 orders of magnitude) during the first 30 s of treatment. This was followed by a slow decrease (1.14 order of magnitude) during the rest of the 4-h treatment time (Fig. 3). Butterfield et al. (4) observed the same trend during treatment of enteric pathogens with chlorine. In our study, the action of chlorine against L. monocytogenes occurred primarily during the first 30 s of exposure. Charlton and Levine (6) mentioned that destruction of vegetative cells by chlorine compounds is so rapid that accurate determination of survivors is subject to some errors. Rapid inactivation of L. monocytogenes by chlorine is important since utensils sanitized by chemicals often will be exposed for no more than 120 s (17). Survival of L. monocytogenes in three trials varied among 0.073, 0.31, or 0.9 order of magnitude after 30 s, 10 min, or 4 h of exposure, respectively. The coefficient of variation in per cent (CV%) among these three trials was 11.8, 33.7 and 93.5, respectively, for 30 s, 10 min, and 4h of exposure. Accordingly, behavior of L. monocytogenes during treatment with chlorine was more repeatable after 30 s exposure than after longer exposures.

4 CHLORINE INACTIVATES LISTER/A TIME (MINI) AVAILABLE CHLORINE (PPM) 0.0 Figure 4. Inactivation ofl. monocytogenes strain Scott-A using different concentrations of available chlorine at ph 7 and 25 C. Symbols representing different concentrations of available chlorine are as follows: (o o)05,(o a)l, ( A.~~A)2,( X- X )5,and( +- + ) 10 ppm. Concentration of chlorine and inactivation ofh. monocytogenes Data in Fig. 4 indicate that the larger the amount of available chlorine that was used, the smaller the number of cells of L. monocytogenes that survived after a given exposure time. When L. monocytogenes was exposed to 5 ppm available chlorine for 30 min, the number of survivors decreased by 5.7 orders of magnitude. Survival after 1 h of exposure, if it occurred, was below the detection level (33 CFU/ml of treated solution) of our method. When L. monocytogenes was exposed to 10 ppm available chlorine for 5 min, the number of survivors decreased by 6.4 orders of magnitude. Again, survival, if it occurred after 10 min of treatment, was below the detection level (22 CFU/ml of treated solution) of our method. During the first 30 s of exposure of cells to.5, 1, 2, 5, and 10 ppm available chlorine, the D-values were 61.7, 11.3, 6.7, 4.9, and 4.7 s, respectively. The higher the concentration, the lower was the D-value. Figure 5 is a plot of D-values versus concentrations of available chlorine. This figure indicates that the rate of the change in D-value decreased by increasing the concentration of available chlorine. Escherichia coli is equally or more resistant to chlorine than are many other vegetative bacteria, including some pathogens (Staphylococcus aureus, Streptococcus hemolysis, Streptococcus faecalis, and others). Chlorine solutions ( ppm) caused complete death of 1 x X 10 2 CFUs of E. coli/ml in 15 s (75). In that study, residual chlorine was not neutralized after the exposure time and thus may have continued to inactivate the bacterium after the indicated time. Another study showed that ppm chlorine was effective against a suspension containing 2 x 10 3 cells of E. coli/ml. In that study, however, the chlorine determination was based on inaccurate quantitative tests (4) and the cells Figure 5. D-values calculatedfrom data obtained during the first 30 s of exposure ofh. monocytogenes strain Scott-A to different concentrations of available chlorine. of bacteria were not washed during preparation of the suspension. Presence of organic matter in the suspension may have affected the effectiveness of chlorine against the organism. The resistance of L. monocytogenes to chlorine in our study can not be compared with the resistance of E. coli to chlorine in those studies because we used a larger number (2.5- to 4-fold) of L. monocytogenes /ml than was used in the other studies. The higher the number of organisms/ml of chlorine solution, the greater will be the resistance of the mass of cells to inactivation by chlorine (12,15). However, resistance of L. monocytogenes to chlorine is similar to that of other nonsporeforming pathogens. In one study (13), 100 ppm available chlorine reduced the population of L. monocytogenes by 5 orders of magnitude. In another study, a chlorine concentration of less than 50 ppm showed no antimicrobial effect against L. monocytogenes strain Scott-A (3). Our results differ from those of previous reports in that they show L. monocytogenes to be more sensitive to chlorine than suggested by the other authors. During our study, chlorine concentration was measured by an accurate method (chlorine electrode) and loss of chlorine from prepared solutions was controlled. Dark-colored flasks were used to protect chlorine solutions from light. Chlorine consumption by organic matter that might be present in the cell-suspension was avoided by washing cells three times during preparation of the suspension. Chlorine consumption through the chlorine demand of glassware was eliminated by following Chambers' method and sterilizing the glassware overnight in a hot air oven at 175 C. Chlorine decomposition caused by exposure to high temperature was controlled by using a constant 25 C during treatment of L. monocytogenes with chlorine. Decomposition and loss of chlorine caused by a low ph was avoided by using a ph 7 to achieve stability of the chlorine solution. Since all these precautions were taken, we believe that our data truly reflect the sensitivity of L. monocytogenes to chlorine.

5 524 EL-KEST AND MARTH TIME (MIN) 30 TIME (HOUR) Figure 6. Chlorine consumption during treatment of L. monocytogenes (ca. 10 s CFUlml) in sodium hypochlorite solutions that initially contained different concentrations of available chlorine. ( o o) 05, ( D ) ;, and (A -A) 2 ppm available chlorine present initially. Consumption of chlorine during treatment ofl. monocytogenes The concentration of available chlorine decreased rapidly initially during treatment of cells of L. monocytogenes, and then continued to decrease slowly. This pattern of chlorine consumption is similar to the pattern of death of L. monocytogenes when it was exposed to chlorine. Data in Fig. 4 and 6 show that after 30 s of exposure of L. monocytogenes to 2.0 ppm chlorine, there was 1.4 ppm available chlorine in contact with 4.5 X 10 3 CFUs of L. monocytogenes/ml solution. The activity of chlorine against this population was very slow. It is likely that during treatment we selected for mutants of L. monocytogenes with greater resistance to chlorine than that of the major portion of the population. Neutralization of chlorine by organic compounds Neutralization by peptone. Data in Fig. 7 show that presence of low concentrations of peptone caused a large and rapid decrease in concentration of the available chlorine in solution. The higher the amount of peptone present, the greater was the decrease in concentration of available chlorine. For this reason, in some experimental conditions peptone can serve as a neutralizer for chlorine (4). Activity of chlorine is markedly reduced in the presence of protein (2). Chlorination and oxidation reactions between the amino group and chlorine likely are responsible for this effect (11). Since peptone (nitrogenous compound) causes this effect, it is important to remove such compounds (e.g. proteins) before using chlorine as a disinfectant. A higher concentration of chlorine needs to be used to compensate for the presence of these compounds in or on materials being sanitized. Figure 7. Neutraliiation of chlorine in sodium hypochlorite solutions (initially contained different concentrations of available chlorine) at intervals after adding different amounts of peptone. ( o o ) 10 ppm available chlorine % peptone. ( a- D-) 10 ppm available chlorine + 0.1% peptone. C A A) 20 ppm available chlorine % peptone. ( x x) 20 ppm available chlorine + 0.1% peptone. Neutralization by sugars. There was no effect by either lactose or glucose on the strength of chlorine solutions (data are not shown). This result agrees with those of other studies (7,11). Oxidation and chlorination reactions between sugar and chlorine seem to be minimal. CONCLUSIONS The effectiveness of a sodium hypochlorite solution against L. monocytogenes was controlled by three factors that we studied. These are: (a) concentration of available chlorine (the higher the concentration, the greater the germicidal activity), (b) presence of proteins (the higher the concentration, the greater the amount of chlorine that was neutralized), and (c) resistance of L. monocytogenes to chlorine. Four factors had an impact on resistance of L. monocytogenes to chlorine. These were: (a) age of culture (24-h-old culture was more resistant to chlorine than the 48- h-old culture), (b) medium used to grow the bacterium before treatment (cells of L. monocytogenes from a broth culture were more resistant than those from a slant culture), (c) time of exposure of L. monocytogenes to chlorine (resistant mutants were selected by the first 30 s of exposure), and (d) the solute concentration around cells of L. monocytogenes (L. monocytogenes was more resistant to chlorine when the cells were suspended in a higher rather than lower concentration of phosphate buffer). From a practical viewpoint, it is evident that L. monocytogenes is not particularly resistant to chlorine. Hence, its control in the food industry through a good program of cleaning and sanitizing should be no more difficult than control of any other non-sporeforming bacterium, regardless of whether it is a pathogen or a cause of spoilage. con't. on p. 530

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