Abstract: Major purpose of this study was to investigate survival of thermophilic Campylobacter spp. isolates from environmental samples in sterile chicken extract and milk and to determine sensitivity of them to food preservatives. Six thermophilic Campylobacter isolates were subjected to determine their survival and the decimal reduction time in sterile chicken extract and milk at -15, 4, 32°C and 55, 60, 65°C, respectively. Minimal Inhibitory Concentrations (MICs) of food preservatives against the Campylobacter isolates were determined by E-test. The results obtained indicated that Campylobacter isolates survived relatively longer in the milk. The decimal reduction time for the isolates was approximately 1 min at 55°C and less than 1 min at 60°C. Most of the isolates were rapidly inactivated at 65°C. Although, most of the Campylobacter isolates were sensitive to food preservatives, MIC values of lactic and acetic acid against the isolates were relatively low. Overall, the thermal sensitivity of Campylobacters would not allow them to survive in the food even with moderate cooking. Hence, heat, lactic and acetic acids could be considered as functional physical agent and food preservatives against Campylobacter.
Introduction
Thermophilic campylobacters are widespread in the environment, where they are a sign of recent contamination of the environment with animal and avian feces (Jones, 2001). Gastroenteritis caused by Campylobacter spp. has been recognized as one of the important food borne disease in the developed countries (Chaveerach et al., 2002). The most important pathogenic species belong to the group of so-called thermophilic campylobacters. Camp. jejuni, Camp. coli and Camp. lari are most important pathogenic Camprlobacter (Skirrow, 1994). Transmission of thermophilic Campylobacter spp. from environment to human could be often through contaminated food as well as contaminated water. Several report have revealed that 30-100% of poultry, 40% of cattle and 60-80% of swine carry Campylobacters in their intestinal tract. For this reason, the organism is principally associated with foods of animal origin (Doyle, 1984). Household pets with diarrhea have been shown to be the source of infection for man (Frost, 2001). The infectious dose of Campylobacter required to cause foodborne disease is very low. It means only a few hundred cells can produce illness in babies; young children and debilitated people. Symptoms of the infection vary from mild to severe with bloody diarrhoea as the most characteristic symptom (Ketlety, 1997). However, treatment of infection using antibiotics is always final remedy. But prevention of infection must be considered most important than that of treatment. Prevention of campylobacter enteritis depends largely on interrupting the transmission of the organism to humans from farm and domestic animals, food of animal origin, or contaminated water. Individuals can reduce the risk of campylobacter enteritis by using properly cooked and stored meat and dairy products, (Tosh et al., 1981; Dawkins et al., 1984; Diker et al., 1987; Spacciapoli et al., 2001). In view of above background and in order to reduce campylobacter enteritis, the present study was undertaken to determine survival of thermophilic Campylobacter isolates in the sterile foods, the decimal reduction time (D-values) for the isolates at different temperatures and to evaluate the effect of food preservatives on the thermophilic Campylobacter isolates from environment.
Materials and Methods
Islation and Identification of Thermophilic Campylobacter
Twenty-five strains of Campylobacter spp. were isolated from different
sources viz., animal feces, poultry and meat using KB device and preT- KB methods
(Baserisalehi et al., 2004a, b). All suspected colonies have grown on
the KB medium were confirmed by typical morphology, darting motility, Gram staining,
oxidase and catalase tests. The isolates exhibiting characteristics typical
of Campylobacter were characterized using standard Campylobacter
phenotypic identification tests recommended by Atabay and Corry (1997). These
tests included, H2S lead acetate strip, nitrate reduction, growth
in 1% glycine and 3.5% NaCl, growth at different temperatures, viz., 25, 37
and 42°C and resistance to nalidixic acid (30 μg disc) and cephalothin
(30 μg disc). All thermophilic campylobacters isolated from each sample
were confirmed using hippurate hydrolysis, Indoxyl acetate and Urease tests.
Six thermophilic Campylobacter belonging to Camp. jejuni, Camp. coli and Camp. lari randomly were selected for future study. Camp. jejuni P1, Camp. lari P2 and Camp. lari P3, isolated from poultry, Camp. jejuni M1 and Camp. coli M2 isolated from beef and Camp. coli F5 isolated from animal feces. All the isolates were maintained at -15°C in the Brucella broth with 15% glycerol.
Food Samples and Their Processing
Fresh raw milk (buffalo’s milk) obtained from animal husbandry in Pune
city was immediately sterilized upon arrival in the laboratory, at 121°C
for 15 min. Chicken meat was obtained from retail market in Pune city. The sterile
chicken extract was prepared by adding 50 mL distillated water to 250 g ground
meat and sterilized at 121°C for 15 min.
Effect of Temperature on the Survival of Thermophilic Campylobacter Isolates
in Sterile Chicken Extract and Milk
Six sets of chicken extract and milk tubes, each with three tubes of chicken
extract and three tubes of milk were made inoculated with six different isolates
of campylobacter separately. Three subsets of each with one tube of chicken
extract and one tube of milk, of each set were made and incubated at three different
temperatures (-15, 4 and 32°C). At different time intervals (6, 12, 18,
24, …h) viable count of each isolate was determined by standard plate count
technique using Luria Bertani agar.
Sensitivity of the Thermophilic Campylobacter Isolates to Heat as a Function
of Time
The decimal reduction time (D-value) is a measure of sensitivity of microorganisms
to heat, was determined for six isolates belonging to Camp. jejuni, Camp.
coli and Camp. lari at varied temperatures (55, 60 and 65°C).
To determine the D-value at each temperature, test tubes (5x1/2 inch), containing
1 mL of food products (sterile chicken extract or milk) were placed in the water
bath to required temperature. After 10 min, 0.1 mL culture of one of the Campylobacter
isolates (1.5x108 cells mL-1) was inoculated into each
tube in the water bath. After every 20 sec 2 tubes one each of milk and chicken
extract were placed in an ice bath for 10 min. Then the suspension was suitably
diluted in sterile distillated water and plated onto the Luria Bertani agar
(LB agar) for viable count (Gill and Harris, 1982). This procedure was repeated
for remaining isolates of Campylobacter at the different temperatures.
The D-value was calculated as the time in minutes required for reducing 90%
of microbial population.
| LogN0 | = | Log. Campylobacter population at time t1 |
| LogN | = | Log. Campylobacter population at time t2 |
| t1 | = | Initial incubation time. |
| t2 | = | Final incubation time. |
Minimal inhibitory concentrations of food preservatives for thermophilic
Campylobacter isolates
Six isolates of Campylobacter belonging to Camp. jejuni,
Camp. coli and Camp. lari were grown in 5 mL Nutrient Broth (NB)
in 15x2 cm tubes at 37°C for overnight. The number of cells per mL of culture
was determined in terms of turbidity (Mcfarland scale No. 0.5 1.5x108 cfu
mL-1). The food preservatives were diluted (0.05, 0.1, 0.25, 0.5,
1, 2 and 3%) in the sterile distilled water and sterilized using membrane filter
(0.45 μm pore size).
Minimal inhibitory concentration of food preservatives for theremophilic Campylobacter isolates was determined by E-test (Baker, 1992). E-test strip for each food preservative was made using a piece of filter paper (Whatman No. 10, size 1x7 cm). Each strip was divided into seven squares (1x1 cm). The squares were labeled by respective concentration in increasing order. The diluted food preservatives were dropped (10 μL) at the respective square of the strip and allowed to dry.
To perform the E-test, each culture was spread inoculated onto Mueller-Hinton agar plate separately. Then two strips of two food preservatives were applied on each plate. The plates were incubated at 37°C for 48 h under microaerophilic conditions and inhibitory concentration of each preservative was read at the point where the elliptical zone of inhibition intersected the E-test strip.
Results
Effect of Temperature on Survival of Thermophilic Campylobacter Isolates
in Sterile Chicken Extract and Milk
The population of Campylobacter increased initially upto 2-4 days
at 32°C. The rate of decline on subsequent incubation was significantly
high indicating relatively less survival of all Campylobacters at 32°C.
The results from survival of thermophilic Campylobacter isolates in the
foods indicated that the population of Campylobacter isolates during
storage at -15 and 4°C declined. However, the rate of decline was relatively
less than that at 32°C. The rate of decline of Campylobacter at -15°C
was relatively more than that 4°C. In general, survival of the Campylobacter
isolates was relatively greater in the milk however, survival of Camp. coli
M2 and Camp. jejuni M1 was relatively greater in the chicken extract.
However, the population of Camp. jejuni P1, Camp. coli F5, Camp.
lari P2 and P3 inoculated in milk decreased. But rate of decline was relatively
less in milk. The population of Camp. jejuni M1, Camp. coli
M2 inoculated in milk decreased more than that in chicken extract (Fig.
1).
Sensitivity of the Thermophilic Campylobacter to Heat as a Function of Time
D-values of thermophilic Campylobacter isolates in sterile chicken
extract and milk are given in the Table 1. As seen in this
table, thermophilic Campylobacter isolates were inactivated when incubated
at 55, 60 and 65°C. Virtually the D-value for all isolates was 1 min at
55°C and less than 1 min at 60°C. D-value for thermophilic Campylobacter
isolates at 65°C was not determined because, most of them were rapidly inactivated
at 65°C.
| Fig. 1: | Effect of temperature on survival of Campylobacter isolation in food products |
| Table 1: | D-values of Campylobacter isolates in food products at different temperatures |
| Table 2: | MIC-values of food preservatives* by E-test** against Campylobacter isolates |
| * LA lactic acid, CA citric acid, AA acetic acid, PA propionic acid, SA sodium sorbate, BA sodium benzoate, NaNO3 sodium nitrite, NaCl sodium chloride. ** On Mueller- Hinton agar at 37°C for 48 h under microaerophilic conditions | |
These results indicated that D-values of the isolates in milk and chicken extract were similar. Therefore, there is no significant correlation between resistance of the Campylobacter isolates to heat and type of the foods. Although, D-value for Camp. jejuni M1was relatively high and for Camp. coli F5 it was relatively low, rest of the isolates had similar D-value.
Minimal Inhibitory Concentrations of Food Preservatives for Thermophilic
Campylobacter Isolates
MIC values of six weak acid preservatives and two salt preservatives were
determined against thermophilic Campylobacter isolates. As seen in the
Table 2, MIC values of weak acid preservatives ranged from
0.1% for lactic acid to 2% for citric acid.
Although, Camp. coli and Camp. jejuni isolates showed similar behavior against organic acids tested, Camp. lari isolates were relatively resistant. Lowest MIC values of lactic acid was against Camp. jejuni isolates, citric acid against Camp. jejuni strain P1, acetic acid against Camp. jejuni P1 and Camp. coli M2, propionic acid against Camp. jejuni isolates and Camp. coli M2, sorbic acid was against Camp. jejuni M1, Camp. lari P2 and Camp. coli isolates and benzoic acid was against Camp. jejuni M1. The results of MIC of salts against Campylobacter isolates indicated varied MIC values of sodium nitrate and sodium chloride. However, the MIC values of sodium nitrite against the isolates were significantly less than that of sodium chloride.
Discussion
In general, survival of the most thermophilic Campylobacter isolates was greater in milk compared to chicken extract. The population of Campylobacter increased initially up to 2-4 days at 32°C then declined on subsequent incubation. Although, the population of the isolates declined at 4 and -15°C, the rate of decline was relatively high at -15°C. Parallel to our finding, Stern and Kotula (1982) reported that the levels of Camp. jejuni inoculated in ground meat decreased during storage at -15°C, while it remained constant during storage at 4°C. Solow et al. (2003) reported that the population of thermophilic Campylobacter isolates remained constant in the foods during storage at 4°C. On the other hands, based on our observations most of the isolates were survived relatively longer in the milk however, in contrary of our data Doyle and Roman (1982) reported that the combination of lactoperoxidase with H2O2 and SCN- in raw milk elicited to produce metabolites with bactericidal property and the survival of Gram-negative bacteria was less in raw milk. As an interpretation, it should be noted that the sterilization of the milk-culminated in the inactivation of its antimicrobial compounds, therefore, antimicrobial property of sterile milk is different with raw milk. Hence Campylobacter could survive greater in the sterile milk compared to chicken extract. The results indicated that the survival of some Campylobacter isolates viz., Camp. coli M2 and Camp. jejuni M1 was greater in the chicken extract than in milk. Probably the source of isolates must be considered as reason for the results obtained. It is because Camp. coli M2 and Camp. jejuni M1 were isolated from meat therefore their survival in the meat is relatively more due to their previous adaptation. In this case, Lior et al. (1981) reported that Campylobacter jejuni strains of the same serogroup could be isolated from a variety of different animals. Hence, similar strains of the same serogroup may be isolated from both poultry and cattle. Therefore, it could be concluded that probably due to previous adaptation of these isolates to meat, the survival of Camp. coli M2 and Camp. jejuni M1 was greater in chicken extract compared to milk.
The results obtained from decimal reduction time (D-values) for thermophilic Campylobacter isolates indicated that D55 and D60 values for thermophilic Campylobacter isolates were 1 min and less than 1 min, respectively. While all the isolates were rapidly inactive at 65°C. A number of publications indicated that campylobacters are more sensitive to heat than other Gram negative pathogens (ICMSF, 1996). Doyle and Roman (1981) found D-values for five strains of Camp. jejuni in milk ranging from 1.56 to 1.95 min at 53°C and 0.74 to 1 min at 55°C. They opined that “ the times and temperatures used to pasteurize milk should be sufficient to free milk of even unusually large numbers of viable cells of Camp. fetus subsp. jejuni. Then it has been interpreted that nonhomogenous fat may serve to create microenvironments to protect microorganisms against heat therefore protection of the microorganisms by nonhomogenous fat must be considered for adjusting temperature to make milk free from the microorganisms. Blankenship and Craven (1982) found D-value for Camp. jejuni ranging from 8.8 min at 51°C to 0.8 min at 57°C in ground chicken meat. Gill et al. (1981) reported that the high temperature, short time process (71.7, 15 sec) is sufficient for inactivating campylobacters. Therefore, it can be concluded that thermal sensitivity of these bacteria would not allow the organism to survive in the food even with moderate cooking.
The results obtained from minimal inhibitory concentration of food preservatives for thermophilic Campylobacter isolates indicated that all of the isolates tested were sensitive to organic acids with varied MIC values. Probably these results are due to varied responses of species as well as different strains belonging to a species. Several studies reported that organic acids, such as formic, acetic, ascorbic and lactic acids, rapidly inhibited the growth of Campylobacter species (Chaveerach et al., 2002; Cudjoe and Kapperud, 1991; Waterman and Small, 1998; Cuk et al., 1987; Fletcher et al., 1983), while, on the basis of our observations lactic and acetic acids inhibited growth of the Campylobacter isolates relatively with high efficiency.
However, most of the Campylobacter isolates were sensitive to 2% NaCl. But Camp. lari P2 and Camp. coli F5 were resistant to 2% NaCl. Therefore, it can be concluded that most of the isolates were sensitive to NaCl however; MIC values of sodium chloride against the isolates were relatively high. Although, sodium nitrite eliminated growth of all the Campylobacter isolates, MIC values of this salt against them were relatively low.
In conclusion, the people working in kitchen and those who are in contact with cooking should take necessary precautions regarding personal hygiene and cleanliness. In fact the present study illustrated that thermal sensitivity of Campylobacters would not allow the organism to survive in the food even with moderate cooking. Therefore, heat and some food preservatives such as lactic and acetic acids can be considered as functional physical and chemical agents against Campylobacter.