Response of Campylobacter jejuni to Sodium Chloride

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1982, p Vol. 43, No /82/ $02.00/0 Response of Campylobacter jejuni to Sodium Chloride MICHAEL P. DOYLE* AND DEBRA J. ROMAN Food Research Institute, University of Wisconsin-Madison, Madison, Wisconsin Received 10 July 1981/Accepted 28 October 1981 Studies were done to provide more comprehensive information on the response of Campylobacterjejuni and nalidixic acid-resistant, thermophilic Campylobacter (NARTC) to sodium chloride at 4, 25, and 42 C. Three strains of C. jejuni were studied, and all could grow at 42 C in the presence of 1.5% NaCl, but not 2.0% NaCl. At the same temperature, NARTC could grow in 2.0% NaCl and was substantially more tolerant to 2.5 and 4.5% NaCl than was C. jejuni. Both C. jejuni and NARTC grew poorly in the absence of added NaCl and grew best in the presence of 0.5% NaCl at 42 C. At 25 C, NaCl concentrations of 1.0 to 2.5% were protective to NARTC, but the same concentrations of salt generally enhanced the rate of death of C. jejuni. At 4 C, both C. jejuni and NARTC were sensitive to 1.0% or more NaCl; however, the rate of death at this temperature was substantially less than that which occurred at 25C. A 3 log10 decrease of cells occurred in 4.5% NaCl after 1.2 to 2.1 days at 25 C, and a similar reduction in cells took approximately 2 weeks at the same salt concentration and 4 C. Although C. jejuni grows best in the presence of 0.5% NaCl, the presence of NaCl at concentrations as low as 1.0% may retard growth or increase rate of death; hence, it is advisable that growth media used for recovering or enumerating this organism contain 0.5% NaCl, but not 1.0% or more NaCl. Campylobacter jejuni is now recognized as a leading cause of acute bacterial gastroenteritis in humans (1). Several studies have found that its rate of isolation from stools of hospitalized, diarrhetic patients is comparable to that of Salmonella. A variety offoods, including raw milk, poultry and pork, have been implicated as vehicles responsible for transmitting campylobacters to susceptible individuals (1). Foods of animal origin are of particular concern because many animals contain C. jejuni as part of their normal intestinal flora (9). Since this organism may be present on meats that are ultimately consumed (4, 5, 10, 13, 14), it is important to know how campylobacters will respond to conditions that are used to preserve and store such foods. Sodium chloride is one of the most important food adjuncts in food preservation. It is commonly used in conjunction with drying, but may be added to food as a brine or may be pumped into tissues. Although investigators have reported that C. jejuni wiu not grow in brucella medium to which 3.5% sodium chloride has been added (3, 8), it is not known how the organism would respond to different concentrations of salt or in the presence of salt during storage at room or refrigeration temperatures. The purpose of this study was to define these relationships. 561 MATERIALS AND METHODS Organisms. Four strains of Campylobacter from three different sources were studied. Two strains, FRI-CF6 and FRI-CF8, were isolated from infected humans and were obtained from A. Helstad (Wisconsin State Laboratory of Hygiene, Madison, Wis.). One strain, FRI-CF74C, was isolated from an apparently healthy laying hen. Both human strains and the avian isolate were identified as C. jejuni biotype 1 according to the scheme of Skirrow and Benjamin (6). The fourth strain, FRI-CF31P, was isolated from the cecal contents of an apparently healthy, freshly slaughtered pig. This organism was identified as a nalidixic acid-resistant, thermophilic Campylobacter (NARTC) (7). Cultural conditions. Organisms were cultured under conditions described previously (2). Cultures were grown on blood agar plates (brucella agar containing 5% defibrinated sheep blood), transferred to brucella broth containing 0.1% agar, and incubated at 42 C for 24 h. Each culture was then transferred into 500-ml side-arm filter flasks, each containing 100 ml of brucella broth supplemented with 0.3% sodium succinate and 0.01% cysteine hydrochloride. The atmosphere in each flask was replaced with a gas mixture of 5% 02-10% C02-85% N2, and then flasks were shaken at 100 gyrations per min for 16 to 18 h at 42 C in a gyratory water bath (New Brunswick Scientific Co., New Brunswick, N.J.; model G76). These cells, which were in the late logarithmic phase of growth, were used as inocula for the salt challenge studies. Survival and growth studies. Studies were done with a series of screw-cap test tubes (16 by 125 mm)

2 562 DOYLE AND ROMAN En 106 LAl -j V 05%/NaCl -j I o 1.0%/o NaCI co * 1.5% NaCl z A 2.0%/ NaCa > 015 A 2/5% NaCI 81o4 0tD 45%NaCI 0 z % AS/ 20% HOURS AT 420C FIG. 1. Growth or inactivation at 42 C of C. jejuni FRI-CF8 in brucella broth containing 0.1% agar and different amounts of NaCl. containing 1.9 ml of brucella broth (Difco Laboratories, Detroit, Mich.) (ph 6.9) supplemented with 0.1% agar and 0, 0.5, 1.0, 1.5, 2.0,4.0, or 6.0% (wt/vol) sodium chloride. Before supplementation, the brucella broth contained 0.5% sodium chloride. For studies comparing the response of campylobacters in a saltfree environment with a 0.5% NaCl environment, brucella medium was specially formulated as follows: (grams per liter) tryptone (Difco), 10; peptone (Difco), 10; yeast extract (Difco), 2.0; dextrose, 1.0; sodium bisulfite, 0.1; and sodium chloride, as required (O or 0.5%). Cells of the test strain were diluted in 0.1% peptone water, and 0.1 ml of the appropriate dilution was added to each tube of brucella medium. The tubes were incubated at 4, 25, or 42 C in an atmosphere of 5% 02-10% C02-85% N2. At selected times, the contents of the tubes were serially diluted in 0.1% peptone and plated onto blood agar plates for enumeration of cells. Blood agar plates were incubated at 42 C for 48 h in the presence of the gas mixture described above. Duplicate determinations were done for each treatment studied. The lines of best fit for data of survivor curves exhibiting straight-line relationships were determined by using linear regression analysis (11). RESULTS Survival and growth at 42 C. Doyle and Ro- APPL. ENVIRON. MICROBIOL. TABLE 1. Rates of growth or inactivation of Campylobacter in brucella broth plus 0.1% agar supplemented with different amounts of sodium chloride and incubated at 42 C NaCl concn Doubling time or D-value (h) (%) FRI-CF6 FRI-CF8 FRI-CF74C FRI-CF31P o.oa 14.5b,c 7.20b.c 2.94b,c NGd 0.5a 1.20b 1.18b 0.97b 1.oob b 1.28b 1.03b 0.86b b 1.32b 1.45b 1.10b b 1.74b 3.94b 1.28b e 4.91e 5.72e 2.86bjf e 3.28e 2.35e NGd e 1.78e 1.54e 6.66e a Medium specially formulated using individual ingredients; not prepared commercially. b Doubling time. I Growth after 12-h lag. d NG, No growth or death up to 30 h. e D-value. f Growth after 6-h lag. man (2) have shown previously that selected strains of C. jejuni grow optimally at 42 to 450C in brucella broth containing 0.1% agar. Figure 1 illustrates the response of strain FRI-CF8 to salt concentrations ranging from 0.5 to 4.5% at 420C. It is evident that growth can occur in the presence of up to 1.5% NaCl, but not when the concentration of salt is 2.0% or greater. Furthermore, the graph illustrates that, in the presence of 2.0, 2.5, or 4.5% NaCl, the rate of death of the organism follows a logarithmic, straight-line relationship. Hence, rates of death can be reported in terms of D-values or times required to inactivate 90% of the population. A comparison of the rates of growth or inactivation of the four strains at 42 C in the presence of different concentrations of NaCl is shown in Table 1. The ability of the campylobacters to grow in the presence of or tolerate NaCl was strain dependent. The human and chicken isolates could grow in medium containing up to 1.5% NaCl, but died in medium containing 2.0% NaCl. In the presence of 1.5% salt, the growth of FRI-CF6 was substantially slower than that of the other three strains. The porcine isolate was more tolerant to NaCl than the others, as it grew in the presence of 2.0% salt; it neither grew nor died after 30 h in the presence of 2.5% salt, and it died, but at a rate much slower than the others, in 4.5% NaCl. In the presence of 0.5% salt, the porcine isolate could grow more rapidly than any of the others. At 420C, all strains grew best in 0.5% NaCl and grew poorly after an extended lag in medium containing no added NaCl. Survival at 25 C. When C. jejuni and NARTC were held in the presence of different concentrations of NaCl at 250C, the rates of death again

3 VOL. 43, 1982 TABLE 2. Rates of inactivation of Campylobacter in brucella broth plus 0.1% agar supplemented with different amounts of sodium chloride and incubated at 25 C NaCi concn D-value (h) (%) FRI-CF6 FRI-CF8 FRI-CF74C FRI-CF31P O.Oa a amedium specially formulated using individual ingredients; not prepared commercially. occurred as logarithmic, straight-line relationships. Hence, D-values can be calculated from these data. Shown in Table 2 are the D-values calculated for the four strains in the presence (and absence) of added NaCl at 25 C. In the presence of comparable concentrations of salt, all strains survived substantially longer at 25C than at 42 C. Furthermore, contrary to what was observed at 42 C, increasing the concentration of NaCl did not always increase the rate of death. For example, for three of the four strains, cells survived longer in the presence of 4.5% NaCl than in 2.5% NaCl. In addition, the presence of 2.0% NaCl was protective of FRI-CF31P as this strain survived almost twice as long in 2.0% salt as it did in medium that contained no or 0.5% NaCl. Unlike the NARTC strain, optimal survival of C. jejuni at 25 C was in the presence of 0.5% NaCl. Survival at 4 C. Figure 2 illustrates the response of FRI-CF8 to different concentrations of NaCl when held at 4 C. With the possible exception of the trials in which no (unpublished data) or 0.5% salt was present, the rates of death do not follow the same straight-line, logarithmic relationships that were observed for the other temperatures evaluated. Hence, these data could not be presented in terms of D-values. However, it is clearly evident that the organism survives substantially longer in the presence of salt at 4 C than at 25 C. Results from tests done in specially formulated media containing no or 0.5% NaCl indicated that there were no appreciable differences in the rates at which each strain was inactivated in either medium at 4 C (unpublished data). The sensitivity of the other three strains to the same concentrations of NaCl at 4 C followed trends similar to those observed for FRI-CF8. An example is shown in Fig. 3 which represents strain FRI-CF31P. Although data are not pre- SALT SENSITIVITY OF CAMPYLOBACTER 107F o\5.5% > z 16.5%/ V 05%NaCl o 1.0/o NaCl 102 * 1.5% NaCI A 2.5% NaCI o 4.5% NaCI * 6.5%NaCA DAYS AT 4 C FIG. 2. Inactivation at 4 C of C. jejuni FRI-CF8 in brucella broth containing 0.1% agar and different amounts of NaCl. sented, survival of FRI-CF6 under such conditions was intermediate to that of FRI-CF8 and FRI-CF31P, and survival of FRI-CF74C was comparable to that of FRI-CF31P. Both the porcine and avian isolates were more sensitive to storage at 4 C than was FRI-CF8, as an approximate 0.5 log1o decrease of viable cells occurred in medium containing 0.5% salt after 14 days for CF8, whereas under the same conditions strains CF31P and CF74C experienced a 2 loglo decrease during the same period of time. As might be anticipated, as the concentration of NaCl was increased, the number of surviving cells of CF8 decreased. However, erratic results were observed for the avian and porcine isolates, as cells of CF74C were almost equally sensitive to 2.5 and 4.5% NaCl and those of CF31P were equally sensitive to 1.0 and 1.5% and to 2.5 and 4.5% NaCl. All strains died most rapidly in the presence of 6.5% NaCl, exhibiting a 3.5 to 4.0 loglo decrease in cells after 14 days at 4 C. Contrary to results obtained at 25 C, at which CF31P was substantially more resistant to inactivation in the presence of 1.0 to 2.5% NaCl, at 4 C this strain died more rapidly when salt was present in concentrations of 1.0% or more. DISCUSSION Although data obtained from these studies cannot be extrapolated directly to foods, such information is useful in defining how C. jejuni

4 564 DOYLE AND ROMAN -J C-) E CD v 0.%QC ou1.%n.5% 2~~~*1.5%NaCt~.5 * 2.05% NaCl o 4.5% NaCa * 6.5% NoCa DAYS AT 40C FIG. 3. Inactivation at 4 C of NARTC FRI-CF31P in brucella broth containing 0.1% agar and different amounts of NaCl. and NARTC will respond to different temperatures in environments containing sodium chloride. All three strains of C. jejuni could grow at 42 C in the presence of 1.5% NaCl, but not 2.0% NaCl. In contrast, Skirrow and Benjamin (7) reported that most of the strains of C. jejunilcoli they evaluated could not grow on 1.5% NaCl agar at 37 C. The reason for this difference is not known; however, 42 C is within the range of optimum temperatures for growth of C. jejuni, and 37 C is not (2). It may be that C. jejuni can tolerate higher concentrations of NaCl at its optimum temperature for growth. As the concentration of NaCl in the growth medium was increased above 2.0%, the rate at which the organism died at 42 C markedly increased. Decreasing the temperature to normal room temperature (25 C) greatly increased the organisms' ability to tolerate salt. For example, decreasing the temperature of incubation from 42 to 25 C increased the tolerance of C. jejuni to 4.5% NaCl five- to eightfold. Under such conditions the rates of death were reduced from 1 loglo per 1.5 to 2 h at 42 C to 1 log1o per 10 to 13 h at 25 C. Although C. jejuni is sensitive to NaCl, when large numbers of the organism were initially present (106 to 107/ml) in 6.5% salt and held at 4 C, viable cells could still be recovered after more than 3 weeks (unpublished data). Hence, at refrigeration temperature C. jejuni may survive in the presence of 4.5 to 6.5% salt for an APPL. ENVIRON. MICROBIOL. extended period of time. Thus, salt would not be an effective means for eliminating this organism from refrigerated foods. However, at room temperature (25 C) and in the presence of 4.5% NaCl, a comparable (106 to 10'/ml) population of C. jejuni may survive for only 3 to 5 days. In Germany, bowels from pigs are often used for making sausages (15). In preparing the bowels for stuffing, butchers soak the tissues in salt, using a concentration that can only be guessed at. After overnight soaking the bowels are washed and ready for sausage making. Sticht- Groh (15) assayed several such pig bowels for Campylobacter spp. after overnight soaking in salt; based on the results of our study, it is not surprising that 30% of the samples contained campylobacters. Although C. jejuni is sensitive to NaCl, unless the temperature is unusually warm, a simple overnight soaking in salt would not be sufficient to eliminate large numbers of the organism from pig bowels. Sticht-Groh (15) speculated that in countries where much pork sausage and related products are consumed, this may be an additional cause of digestive tract infection in humans. The NARTC strain was generally more tolerant to NaCl than were the strains of C. jejuni. At 42 C, this organism could grow in the presence of 2.0% NaCl and was substantially more tolerant to 2.5 and 4.5% NaCl than the three isolates of C. jejuni. At 25 C, NaCl was protective to the porcine isolate at concentrations of 1.0 to 2.5% and most protective at 1.5 to 2.0%. The ability of the NARTC strain to grow in the presence of 2.0% NaCl, but not 2.5% NaCl, conforms with the observations of Skirrow and Benjamin (7), who reported that of 27 NARTC strains tested, all grew on medium containing 1.5% NaCl, but none grew on medium containing 3.5% NaCI. Interestingly, the NARTC strains isolated and studied by Skirrow and Benjamin (7) were primarily associated with wild birds, such as seagulls, and to a lesser extent with dogs, but not pigs. Since our NARTC isolate did originate from a pig, it appears that swine are also a source of this organism. These studies indicate that a number of strains of C. jejuni are sensitive to NaCl at concentrations of 1.0% or more. Along these lines, Steele and McDermott (12) reported that the use of saline as a suspending medium in making wet mounts for microscopic observation of such organisms is unsatisfactory, as their motility appears to be inhibited. Since the presence of NaCl at concentrations as low as 1.0% may retard growth or increase the rate of death, depending on temperature, it would be advisable to avoid using NaCl at these concentrations in growth media or diluents used for recovering or enumerating C. jejuni. However, all strains grew

5 VOL. 43, 1982 SALT SENSITIVITY OF CAMPYLOBACTER 565 best in the presence of 0.5% added NaCl and grew poorly and only after extended time lags when no NaCl was added. Hence, the presence of 0.5% NaCl in media used for growing C. jejuni is suggested. ACKNOWLEDGMENTS We thank A. Helstad, Wisconsin State Laboratory of Hygiene, Madison, Wis., for some of the cultures used in this study. This work was supported by the College of Agricultural and Life Sciences, University of Wisconsin-Madison, and by contributions to the Food Research Institute. LITERATURE CITED 1. Doyle, M. P Campylobacterfetus subsp. jejuni: an old pathogen of new concern. J. Food Prot. 44: Doyle, M. P., and D. J. Roman Growth and survival of Campylobacter fetus subsp. jejuni as a function of temperature and ph. J. Food Prot. 44: Holdeman, L. V., E. P. Cato, and W. E. C. Moore (ed.) Anaerobe laboratory manual, 4th ed., p Virginia Polytechnic Institute Anaerobe Laboratory, Virginia Polytechnic Institute and State University, Blacksburg, Va. 4. Luechtefeld, N. W., and W.-L. L. Wang Campylobacterfetus subsp. jejuni in a turkey processing plant. J. Clin. Microbiol. 13: Simmons, N. A., and F. J. Gibbs Campylobacter spp. in oven-ready poultry. J. Infect. 1: Sklirrow, M. B., and J. BenJamin Differentiation of enteropathogenic campylobacter. J. Clin. Pathol. 33: Sklrrow, M. B., and J. Benjamin '1001' campylobacters: culture characteristics of intestinal campylobacters from man and animals. J. Hyg. 85: Smibert, R. M Vibrio fetus var. intestinalis isolated from fecal and intestinal contents of clinically normal sheep: biochemical and cultural characteristics of microaerophilic vibrios isolated from the intestinal contents of sheep. Am. J. Vet. Res. 26: Smibert, R. M The genus Campylobacter. Annu. Rev. Microbiol. 32: Smith, M. V., I, and P. J. Muldoon Campylobacterfetus subspeciesjejuni (Vibriofetus) from commercially processed poultry. Appl. Microbiol. 27: Steele, R. G. D., and J. H. Torri Principles and procedures of statistics. McGraw-Hill Book Co., New York. 12. Steele, T. W., and S. McDermott Campylobacters isolated from hospital patients. Med. J. Austr. 2: Stern, N. J Campylobacter fetus subsp. jejuni: recovery methodology and isolation from lamb carcasses. J. Food Sci. 46: Stern, N. J Recovery rate of Campylobacter fetus subsp. jejuni on eviscerated pork, lamb and beef carcasses. J. Food Sci. 46:1291, Sticht-Groh, V Campylobacters in pig faeces. Vet. Record 108:42. Downloaded from on March 27, 2019 by guest