Injury of Bacteria by Sanitizerst

Transcription

1 APPLID MICROBIOLOGY, Jan. 1971, p Vol. 21, No. 1 Copyright 1971 American Society for Microbiology Printed in U.S.A. Injury of Bacteria by Sanitierst D. L. SCHUSNR,2 F. F. BUSTA,3 AND M. L. SPCK Department of Food Science, North Carolina State University, Raleigh, North Carolina 2767 Received for publication 27 August 197 Injury of test cultures was quantitated by differences in colony counts obtained with a complete medium and those obtained on conventional selective media. Staphylococcus aureus, Streptococcus faecalis, and several strains of scherichia coli were injured when exposed to the quaternary ammonium methylalkyltrimethyl ammonium chloride. Representative hypochlorite sanitiers also caused injury of. coli ML3. Sanitier concentration appeared to be the main factor in the cause of death and injury, a higher concentration being needed to cause death. Increases in temperature did not result in substantial increases in injury; however, the lethal effect was greater at higher temperatures. Varying the cell concentration from 17 to 19 cells per ml did not change the fraction of cell population killed or injured. The inability or failure of common selective media to detect injured bacteria in food could have serious public health consequences. Injury of bacteria can be interpreted as an inability to obtain nutrients from inorganic versus organic sources or an increased sensitivity to otherwise uninhibitory components in growth media (e.g., NaCl). Injured bacteria have been detected after exposure to freeing temperatures (9-11, 16), heat (4, 5), irradiation (12), and chemicals such as phenol (7). Injury to microorganisms by environmental stresses has attracted increasing attention as a possible problem of extensive public health interest. Information on the injury of bacteria by chemical sanitiers is limited. Metabolically injured bacteria are detected by comparison of counts from a complete medium and a minimal medium (8, 16). The presence of selective agents in media may contribute added stress to injured cells (2). If sanitiers, which are commonly used in the food industry, injure bacteria that serve as indicators of contamination, these bacteria may not be detected with the selective media commonly employed. The purpose of this study was to evaluate the injury of such indicator bacteria by commonly used sanitiing s and to determine the effect of such injury on the enumeration of these bacteria by selective media. A preliminary report of these findings has been presented (D. L. Scheusner and F. F. Busta, Bacteriol. Proc., p. 17, 1968). 1 Paper no of the Journal Series of the North Carolina State University Agricultural xperiment Station, Raleigh, N.C. 2 Present address: Department of Food Science, Michigan State University, ast Lansing, Mich Present address: Department of Food Science and Industries, University of Minnesota, St. Paul, Minn MATRIALS AND MTHODS Cultures. scherichia coli strains 3 A, 5 A, NCSM, and ML3, Staphylococcus aureus MF31, and Streptococcus faecalis 979 were obtained from the departmental stock culture collection. All except S. faecalis were incubated for 24 hr at 35 C on Trypticase Soy Agar (TSA; Difco) slants and stored at 5 C. Cultures were transferred monthly. S. faecalis was maintained in litmus milk; incubation was for 24 to 28 hr at 35 C and was followed by storage at 5 C. Media. Trypticase Soy Broth (TSB), TSA, Mannitol Salt Agar, and Violet Red Bile Agar () were obtained in the dehydrated form (BBL) and rehydrated as directed. Aide Dextrose Agar contained: beef extract (Difco), 4.5 g; tryptone (Difco), 15. g; dextrose, 7.5 g; NaCl, 7.5 g; sodium aide (Fisher Scientific Co., Pittsburgh, Pa.),.2 g; agar (Difco), 15. g; water, 1, ml. The media were rehydrated, dispensed in about 1-ml quantities, and autoclaved 15 min at 121 C (except the which preferably is not autoclaved). Media that were not used immediately were stored at room temperature until needed, at which time they were melted in flowing steam for 25 min. Minimal agar (MA) was prepared as three separate solutions. Solution A consisted of K2HPO4, 7. g; KHaPO4, 3. g; sodium citrate-2h2,o.1 g; MgSO4-7H2,.1 g; (NH4)SO4, 1. g; and water, 4 ml. Solution B consisted of 2. g of glucose and 1 ml of water. Solution C consisted of 15 g of agar (Difco) and 5 ml of water. The solutions were autoclaved for 15 min at 121 C and stored at room temperature. Just prior to use, solution C was melted, and solutions A, B, and C (4:1:5) were tempered and combined. Buffer. Standard phosphate buffer was prepared as directed in Standard Methods for the xamination of Dairy Products (1). 41

2 42 SCHUSNR, BUSTA, AND SPCK APPL. MICROBIOL. Sanitiers. Product A, a quatemary ammonium (QAC), was methylalkylbenyltrimethyl ammonium chloride; the alkyl constituent was a saturated carbon chain containing from 9 to 15 carbon atoms. The 12 carbon (dodecyl or lauryl) chain formed 8% of the mixture. Product B was n-alkyl (5% C12, 3% C14, 17% C16, 3% C,8)dimethylbenyl ammonium chloride. Product C was a commercial detergent-sanitier which contained 1% n-alkyl (4% C12, 5% C14, 1% C16)dimethylbenyl ammonium chloride and undefined amounts of tetrapotassium pyrophosphate, ethylenediaminetetraacetic acid, sodiumcarbonate, and a nonionic surfactant. The phenolic sanitier (product D) was a commercial preparation of various phenolic s and contained 62.51% active ingredients listed as isopropanol, sodium dodecylbenenesulfonate, sodium xylenesulfonate, sodium o-benyl-p-chlorophenate, sodium p-tertiary amylphenate, sodium o-phenylphenate, sodium 4-chloro-2-phenylphenate, sodium 2-chloro-4-phenylphenate, tetrasodium ethylenediaminetetraacetate, sodium 6-chloro-2-phenylphenate. Product was dioctyl sodium sulfosuccinate and is an anionic surface-active. One hypochlorite preparation (product F) was a commercial product containing 5.25% sodium hypochlorite. The other hypochlorite preparation (product G) was a commercial preparation containing 3.25% sodium hypochlorite, 91.71% sodium phosphate,.1% potassium permanganate, and.4% sodium lauryl sulfate. All sanitier solutions were diluted to the desired concentrations in the standard phosphate buffer and were at ph 7.2 in the test concentrations. Neutraliers. The hypochlorites were neutralied by dilution into TSB. Before plating, the other sanitiers were neutralied with the neutralier suggested in the Official Methods of Analysis of the Association of Official Agricultural Chemists (6). Preparation of cell suspension. After the stored cultures had been serially transferred at least three times through TSB, a.1-ml inoculum was placed into 99 ml of TSB and incubated for 12 hr in a water bath at 35 C. [Preliminary studies showed that equal colony counts of. coli ML3 were obtained on TSA, MA, and after 8 hr of incubation (13)]. To minimie the influence of physiological differences on the response of the cells to sanitiers, cultures were examined after the stationary phase (12 hr) was attained. The cells were aseptically harvested by centrifugation for 15 min at 5, X g at 2 C. The supernatant liquid was decanted, the pellet of cells was resuspended in 99 ml of standard phosphate buffer, and, after centrifuging again, the pellet was resuspended in 1 ml of the phosphate buffer. The second pellet of S. faecalis was resuspended in 5 ml of standard phosphate buffer and blended for 2 min at low speed on a Waring Blendor to minimie chain length of S. faecalis. Cell suspensions were stored briefly in an ice bath until tested. xposure of bacteria to sanitier. Sanitiers were diluted to the desired concentration with standard phosphate buffer and tempered to the test temperature. A 9.-ml quantity of sanitier was dispensed rapidly with a 12-ml sterile syringe (Roehr monoject 512) into a screw-cap tube containing 1. ml of cell suspension at the test temperature. This cell-sanitier mixture was mixed vigorously. After the selected exposure time, a 1.-ml sample was neutralied. A 9.- ml quantity of sterile standard phosphate buffer was substituted for the sanitier mixture as the control. Neutraliation of sanitier. A 1.-ml quantity of the cell-sanitier mixture or control cell suspension was added to 9. ml of neutralier in a sterile screw-cap tube at test temperature and mixed thoroughly. After 6 sec, 1. ml was removed, diluted, and plated. Plating methods. Dilutions were made by using standard phosphate buffer tempered in an ice bath, and all platings were performed in duplicate. With the S. faecalis culture and with the. coli cultures, 1.- or.1-ml quantities were pipetted into each of duplicate plates. With the. coli cultures an overlay of about 4 ml of the appropriate medium was poured onto the solidified agar. For enumeration of S. aureus,.1 ml of the diluted culture was pipetted onto the solidified agar medium and spread with a sterile, bent glass rod. The plates were inverted and incubated at 35 C for 48 hr. All visible colonies were counted. Calculations. The number of dead bacteria was determined from the difference of counts on TSA before and after exposure to sanitiers. Metabolic injury was determined by plating on MA, and the number of injured bacteria was the difference in counts on MA and TSA. Injury as measured by the inability to grow on a selective medium was determined by plating on the medium appropriate for the particular culture (Table 2). TABL 1. Death and injury of scherichia coli ML3 after treatment with various sanitiers Per cent San- Active Per cent of it- Type san- sur- surier itier vivorsg v (pg/mi) ors in- Juredb A Quaternary ammonium 1 1 A Quaternary ammonium B Quaternary ammonium C Quaternary ammonium C Quaternary ammonium D Phenolic 1, 42 Anionic surfactant 1, 1 F Hypochlorite G Hypochlorite Treatment of about 19 cells per ml at C for 6 sec. b Injury detected by using Violet Red Bile Agar.

3 VOL. 21, 1971 BACTRIAL INJURY TABL 2. Response on complete and selective media of bacteria exposed to a QAC (product A) F. scherichia coli ML3. coli 3A. coli NCSM. coli 5A Staphylococcus aureus MF31 Streptococcus faecalis 979. cn _ U) -4 Cs O MSA AZD a Treatment of about 19 cells per ml at C for 3 sec., Violet Red Bile Agar; MSA, Mannitol Salt Agar; AZD, Aide Dextrose Agar. c No sanitier treatment; indicates reduction in count observed on selective medium as compared to complete medium. Ct) - IL i I x19f 8xiO 8ka 6xIO8 L 5xlO 4xIO8 3xI8 2x8 - -TRYPTICAS SOY AGAR A-MINIMAL AGAR O-VIOLT RD BIL AGAR X I8 L XPOSUR TIM (sec) FiG. 1. ffect of exposure time on death and itnjury of scherichia coll ML3 exposed to 3,u QAC (product A) at C. RSULTS Injury of. coli ML3 by sanitiers. Injury detectable on selective media was caused by all the sanitiers except products D and when. coli ML3 was the test organism (Table 1). Sanitier D caused some death, but none of the survivors showed injury. Product, which is an 56 anionic, caused no injury or death of the test organism. Injury of several types of bacteria by a quaternary ammonium sanitier. The response of several microorganisms after exposure to product A was tested on appropriate selective media (Table 2). In some cases, death was observed as measured on the complete medium, TSA. No metabolic injury of treated bacteria was detected on the minimal medium (data not presented); however, injury was detected on the selective medium. ven with no sanitier treatment, some test strains did not grow equally well on the complete and selective media. This was taken into account in the determination of per cent of injury on selective media. In no case was the count on the selective medium higher than on the complete medium. xposure time. The action of QAC (product A) on cells of. coli ML3 was most rapid during the initial 3 sec of exposure, although additional injury occurred during exposure up to 12 sec (Fig. 1). Injury was detected only on the selective medium and not on the minimal medium. xperiments with. coli 3 A and. coli 5 A showed results similar to those obtained with. coli ML3. xposure temperature. An increase in the temperature of exposure of cells to the QAC resulted in large increases in the number of cells dying but did not result in major changes in the percentage of survivors which showed injury detected on a selective medium (Fig. 2). The counts on I x19 - -O x18 5 xli 8 3xlo 2x 18 I xlos L Z ~~~~~7 u- 5xIOT g/ml 3 x 17 77' 2 x I'_ 7~ ~ ~ - TRYPTI CAS VIOLT RD SOY AGAR BIL AGAR ----NO TRATMNT- - - *- PRODUCTA. A *_ TMPRATUR (C) FIG. 2. ffect of exposure temperature on death and injury of scherichia coli ML3 exposed for 6 sec to 3,ug/ml QAC (product A).

4 44 SCHUSNR, BUSTA, AND SPCK APPL. MICROBIOL. TABL 3. ffect of cell concentration on death and injury of scherichia coli ML3 after exposure to QAC Dilution Count Per Count P~er cent of cell ontsab cent ont injury suspension' death on None 6.8 X X :1 7. X X : X X a Suspension contained about 19 cells per ml; the indicated dilution was exposed to 3 jag of product A per ml for 6 sec at C. b Trypticase Soy Agar. c Violet Red Bile Agar. TSA and on MA did not differ significantly (data not shown). The data in Fig. 2 also indicate that death and injury of cells occurred in the absence of QAC at the higher test temperatures. Concentration of cell suspension. The percentage of death and injury did not significantly change when the original cell suspension (19 cells per ml) was diluted 1:1 before addition of the sanitier (Table 3). When the cells were diluted 1: 14 before addition of the sanitier, decreased percentages of death and injury were observed. Sanitier concentration. Low concentrations of QAC (25,g/ml) caused injury detectable on selective media, whereas higher concentrations were needed to cause a significant amount of death (Fig. 3). The counts on MA were essentially the same as on TSA. Results with other strains of. coli showed a similar relationship at QAC concentrations as low as 1 Ag/ml of product A. DISCUSSION Injury of microorganisms can be detected as a result of various stresses. Injury usually is recognied as an increased nutritional requirement by the affected cells (8, 16). Increasingly injury is also being used to describe any debilitated condition of cells which increases their lability to restrictive cultivation procedures (2, 3, 5). Since injury increases the difficulty of customary microbiological detection procedures, it is important to recognie the consequences of different environmental stresses placed upon cells and to describe the resultant limitations on analytical microbiology. The injury now demonstrated as a result of the action of chemical sanitiers adds to factors known to cause sublethal impairment. Significantly, the injury was evident when concentrations of sanitiers were much lower than those recommended for sanitiing treatments. This emphasies the need for monitoring the sanitier treatments to insure the proper concentrations to effect death; otherwise, only apparent death may result. The inability of selective media to detect injured cells reduces the effectiveness of many tests for pathogens and indicator bacteria, particularly since the tests normally employ media which contain components that add to the inability of injured cells to grow. The seriousness may be gauged by the results in Table 1 in which as many as 99% of the survivors were injured. In addition, the relationship of bacterial injury to pathogenicity or toxigenicity is not well understood, but the implications are serious owing to the report that injury of Salmonella gallinarum by freeing had no significant effect on pathogenicity (14). The amount of injury or death caused by sanitiers was dependent upon the bacterial strains. Gram-positive test organisms were more resistant to injury by QAC than the gram-negative strains tested. This, however, appears to have been the result of the inhibitory activity of the selective agent in the medium rather than a result of a difference in sensitivity to QAC activity (13). The lethal action of QAC on the bacterial cells appeared to be complete within 3 sec which is in agreement with a previous report (15). The results suggested that timing, if kept constant, had little effect on the amount of injury, especially since the technique did not permit an exposure time of less than 3 sec. As has been noted by others (17), the bactericidal effectiveness of QAC was greater at higher temperatures. The capacity of QAC to injure the bacterial cell appeared to be relatively unchanged over the temperature range tested, but 1 X19 7xlo8 5x18-3x 18 CD) 2X18_\ 8 lx18 7xl >- 5x7 O 3x17 C. - TRYPTICAS SOY AGAR IlX QAC CONCNTRATION (pg/mi) FIG. 3. ffect of QAC (product A) concentration on death and injury of scherichia coli ML3 after exposure for 6 sec at C. o

5 VOL. 21, 1971 BACTRIAL INJURY 45 in the absence of death the larger numbers of injured cells were obtained at and 2 C. Since sanitiing agents which are used in the food industry were capable of causing injury to. coli ML3, the possibility of food contamination by injured bacteria does exist. The QAC and hypochlorite tested caused injury at concentrations far below the concentration recommended for sanitiation. At higher concentrations there was more death, but also a larger percentage of the survivors were injured. This suggests the necessity of maintaining the sanitier concentration at a level which is definitely lethal to most of the bacteria present. ACKNOWLDGMNTS This investigation was supported by Public Health Service training grant S-61 from the Division of nvironmental Health Sciences, and by Public Health Service research grant no. FD 85 from the Food and Drug Administration. LITRATUR CITD 1. American Public Health Association Standard methods for the examination of dairy products, 11th ed. American Public Health Association, New York. 2. Busta, F. F., and J. J. Jeeski ffect of sodium chloride concentration in an agar medium on growth of heatshocked Staphylococcus aureus. Appl. Microbiol. 11: Clark, Carol W., and Z. John Ordal Thermal injury and recovery of Salmonella typhimurium and its effect on enumeration procedures. Appl. Microbiol. 18: dwards, J. L., F. F. Busta, and M. L. Speck Heat injury of Bacillus subtilis spores at ultrahigh temperatures. Appl. Microbiol. 13: Greenberg, R. A., and J. H. Silliker vidence for heat injury in enterococci. J. Food Sci. 26: Horwit, W. (ed.) Official methods of analysis of the association of official agricultural chemists. Association of Official Agricultural Chemists, Washington, D. C. 7. Jacobs, S.., and N. D. Harris The effect of modification in the counting medium on the viability and growth of bacteria damaged by phenols. J. Appl. Bacteriol. 24: Moss, C. W., and M. L. Speck Injury and death of Streptococcus lactis due to freeing and froen storage. Appl. Microbiol. 11: Moss, C. W., and M. L. Speck Identification of nutritional components in trypticase responsible for recovery of scherichia coli injured by freeing. J. Bacteriol. 91: Nakamura, M. and D. A. Dawson Role of suspending and recovery media in the survivial of froen Shigella sonnei. Appl. Microbiol. 1: Postgate, J. R., and J. R. Hunter Metabolic injury in froen bacteria. J. Appl. Bacteriol. 26: Roepke, R. R., and F.. Mercer Lethal and sublethal effects of X-rays on scherichia coli as related to the yield of biochemical mutants. J. Bacteriol. 54: Scheusner, D. L., F. F. Busta, and M. L. Speck Inhibition of injured scherichia coli by several selective agents. Appl. Microbiol. 21: Sorrells, K. M., M. L. Speck, and J. A. Warren Pathogenicity of Salmonella gallinarum after metabolic injury by freeing. Appl. Microbiol. 19: Stedman, R. L.,. Kravit, and J. D. King Studies on cell surface-germicide and enyme-germicide reactions and their contributions to the lethal effect. J. Bacteriol. 73: Straka, R. P., and J. L. Stokes Metabolic injury to bacteria at low temperatures. J. Bacteriol. 78: Weber, G. R Steriliation of dishes and utensils in eating establishments. J. Milk Food Technol. 11: , 351.