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|Year : 2004 | Volume
| Issue : 1 | Page : 39--43
Effect of gamma radiation on survival of campylobacters in various food samples
SP Bhavsar, M Baserisalehi, BP Kapadnis
Department of Microbiology, University of Pune, Pune - 411 007, Maharashtra, India
B P Kapadnis
Department of Microbiology, University of Pune, Pune - 411 007, Maharashtra
PURPOSE: Campylobacter spp. is a major food borne pathogen and shows resistance towards gamma radiation. In the present study, effect of gamma radiation was assessed on the indigenous strains of Campylobacter spp. inoculated in food and water samples. METHODS: Campylobacter spp. were isolated from river water and faeces of various birds and animals. The growth rate was studied for these isolates by propagating them in Kapadnis-Baseri medium. The survival of Campylobacter spp. inoculated in food and water samples was tested after exposing them to gamma radiation. RESULTS: The isolates survived well in meat and milk samples and were sensitive to 1.8 KGy dose of gamma radiation, which lies with in the FDA limit. The effect of radiation on Campylobacter spp. varied with the species and the type of food. CONCLUSIONS: The results obtained suggest that the dose of gamma radiation should be standardized depending on the Campylobacter spp. and the type of food that is being processed.
|How to cite this article:|
Bhavsar S P, Baserisalehi M, Kapadnis B P. Effect of gamma radiation on survival of campylobacters in various food samples.Indian J Med Microbiol 2004;22:39-43
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Bhavsar S P, Baserisalehi M, Kapadnis B P. Effect of gamma radiation on survival of campylobacters in various food samples. Indian J Med Microbiol [serial online] 2004 [cited 2019 Oct 18 ];22:39-43
Available from: http://www.ijmm.org/text.asp?2004/22/1/39/8060
Campylobacter spp. are recognized worldwide as an important food borne pathogen. Human gastroenteritis caused by these organisms is frequently associated with consumption of red meat and poultry meat. Infective dose of Campylobacter ranges from 500-10,000 cells, depending on the strain, environmental stress and susceptibility of the host. These infections are manifested as meningitis, pneumonia, miscarriage and a severe form of Guillian Barre's Syndrome.
Campylobacters are very small, curved, thin, gram negative rods and motile with a corkscrew motility. They belong to the family Campylobacteriaceae. They are nonspore forming rods that may form spherical or coccoid bodies in old cultures or cultures exposed for prolonged periods to air.,  They are microaerophilic in nature and survive for a longer time outside the host. The organisms are susceptible to drying, high oxygen conditions and low pH. The effect of gamma radiation depends on the type of food. Irradiation is less effective on frozen foods than on refrigerated or the food stored at room temperature. They produce cytotoxin, enterotoxin or both. Cytolethal distending toxin activity causes certain cell types to become slowly distended, progressing to death. These microorganisms have the enzymatic defences against the toxic oxygen metabolites. Formate dehydrogenase is one of the enzymes in C. sputorum, C. jejuni and C. mucosalis.
Campylobacter spp. are widespread in the environment, where they are a sign of recent contamination with animal and avian faeces, agricultural run off and sewage effluent. Intestinal carriage of Campylobacter spp. is ubiquitous in livestock, domestic animals, wild birds and poultry and depends on the environmental factors. They contaminate the milk bottles by pecking at the top of the bottles. The presence of campylobacters in the migratory birds is the indication of the large distances that campylobacters can be transferred.
In India so far, Campylobacter spp. have been studied only for their clinical aspect. In the present work Campylobacter spp. have been studied for their survival in different food and water samples, suspected to be the reservoirs. The radiation susceptibility of indigenous strains of campylobacters in different samples was studied in order to standardize the dose of gamma radiation for preservation, which is unique part in preservation of Campylobacter spp. contaminated foods.
Materials and Methods
Sampling and sample processing
Faecal samples were collected from different animals and birds like buffalo, ox, cow, goat, sheep and sea gulls. Each faecal sample was emulsified in 0.1 M phosphate buffered saline (pH-7) to give 10% suspension. Water samples were obtained from different sites along the rivers and examined within one hour of collection. 100 mL of each water sample was filtered through sterile 0.45mm membrane filter, which was suspended into 10 mL sterile saline and vortexed on cyclomixer. Each suspension was diluted in saline.
Isolation and identification
The campylobacters from above suspensions were isolated by spread plate method on selective and differential Kapadnis-Baseri medium (KB medium) at 370C in candle extinction jars. Typical colonies were subcultured and maintained on KB slants. All the isolates were examined for typical darting motility and spiral shape by wet mount under phase contrast microscope. Presumptive campylobacters were subjected to different tests like production of oxidase, catalase, and urease, temperature tolerance, H2S production, glucose utilization, hippurate hydrolysis, utilization of amino acids, and sensitivity to antibiotics such as nalidixic acid and cephalothin to confirm as Campylobacter spp. and identify their species.
Growth rate of Campylobacter spp.
C. coli and C. sputorum respectively from river water and faeces of buffalo, selected for further studies, were studied for their growth rate. Each culture (200mL of 108 cells/mL) was inoculated separately into 100mL KB broth in 250mL conical flask and incubated as mentioned above. Total viable count (TVC) of both the cultures was monitored after every four hours using KB medium. Log of cfu/mL vs. incubation period in hrs. were plotted and growth rates determined.
Survival of Campylobacter spp. in various food samples and water
Survival studies were conducted in sterile pond water, milk (fresh cow milk), chicken meat extract [one gram fresh chicken meat (equal parts of neck, chest and liver) in 9 mL of 0.1% peptone water, blended well and heat sterilized] and mushroom extract (fresh oyster mushrooms were selected and blended), by taking 15mL of each sample in 50 mL conical flask and inoculating with Campylobacter spp. separately at the rate of 108 cells/mL at 37°C. The viable count of Campylobacter spp. in each sample was monitored as described above after every four hours for 48-72 hrs.
Sensitivity of campylobacters to gamma radiation
For this purpose, milk and meat samples were selected based on survival studies. Sensitivity of campylobacters in food samples to gamma radiation was determined based on decimal reduction values (D10) of gamma radiation (Co source) for each Campylobacter spp. The dose of gamma radiation to eliminate campylobacters from each food was standardized based on the D10 value for respective Campylobacter spp. Milk and chicken meat extract was taken separately into two bumper tubes and inoculated with 108 cells/mL and incubated at 37°C for 48 hours under microaerophilic conditions. Each sample was then transferred separately to sterile petriplates which were allowed to move down in the radiation chamber and exposed to 0.6KGy, 1.2KGy, 1.8KGy and 2.4 KGy dose of gamma radiation. Unirradated plates of each sample was included as control. Viable count of Campylobacter spp. in each plate was determined as described above. D10 values were determined based on this data.
Isolation and identification of Campylobacter spp.
Isolation of Campylobacter spp. was done from the river water and the faeces of healthy domestic animals like buffaloes, cows and goats. Sample collection and isolation of campylobacters was conducted during the period from August through December, 2002. All the presumptive campylobacters had flat, reddish pink, circular colonies with smooth consistency and darting motility with spiral shape. From 263 isolates, two isolates were selected on the basis of their growth rate, catalase and oxidase activity. These were positive for oxidase, and were gram negative with spiral shape. These were further confirmed to belong to genus Campylobacter spp. by catalase test, urease test, glucose utilization test and nitrate test. They were urease negative and so were differentiated from Helicobacter. The Campylobacter spp. isolated from the buffalo faeces was identified as C.coli while the one isolated from river water as C.sputorum and this was confirmed by doing the temperature sensitivity tests, hippurate hydrolysis test and sensitivity to various antibiotics. The results are as in [Table:1].
Growth rate of Campylobacter spp.
Growth curves were established for both the Campylobacter isolates and it was found that C.coli grew faster with the growth rate of 0.4 generations/hour and C.sputorum was a slow grower with the growth rate of 0.25 generations/hour.
Survival of Campylobacter spp. in food samples and water
C.coli and C.sputorum could survive well in milk and the chicken extract. Both of these isolates could not survive in the mushroom extract. The relative survival curve is given in the [Figure:1] and [Figure:2].
Sensitivity of campylobacters in different types of food to gamma radiation
Milk and chicken extract artificially inoculated with C.coli and C.sputorum were exposed to doses of gamma radiation varying from 0.6 to 2.4 KGy and the D10 values (dose required to kill 90% of the bacteria) were determined for both. The D10 values depended on the type of food. The D10 value for C.sputorum was 2KGy when present in milk while 0.5-0.6KGy when present in meat. The D10 value of C.coli was 0.2-0.4KGy when present in meat while 1.17KGy when present in milk. The results are given in [Table:2].
The habitat of campylobacters is the intestinal tract of domestic and wild animals and it can be transmitted to the environment through the faeces of these animals. Population of campylobacters isolated from faeces of domestic animals, can be considered as rate of occurrence of Campylobacter through fecal contamination in that particular geographical area. Further, it can be transmitted from these environmental sources to humans through meat, poultry products, milk and water which can then culminate into gastroenteritis in humans. In the present work, strains were selected on the basis of the catalase test and growth rate. Mainly, C.coli and C.sputorum were the strains chosen in this study because information about these strains is less in India as compared to C.jejuni. These isolates were identified by the standard procedures that could discriminate between Helicobacter and Campylobacter spp. Helicobacter pylori shows a strong urease test. These Campylobacter isolates were identified by recommended tests and found to be negative for urease., Growth curves were established for both the isolates and it was found that C.coli entered quickly in the exponential phase with the growth rate of 0.4 generations/hour. C.sputorum takes time to adapt to the new surrounding and grows slow with the growth rate of 0.25 generations/hour. It was necessary to know the growth pattern of the natural Campylobacter isolates from different origin in various food samples before it gets transmitted to humans.
Campylobacter spp. are important human enteropathogens. In England and Wales in 1999, over 50,000 cases of infection were confirmed. A recent study on incidence of infectious intestinal disease suggests that the true figure may be higher. The human pathogens C.jejuni and C.coli are causative agents of acute human enterocolitis and are the most common cause of food borne diarrhoea in many industries. On the basis of catalase test, campylobacters are divided into two groups. Catalase positive campylobacters have been reported as pathogenic campylobacters however catalase negative campylobacters had been reported as non pathogenic Campylobacter but recently catalase negative campylobacters were reported as a cause of disease in humans.,,,, It has been reported that C.sputorum (catalase negative Campylobacter) is a cause of bacteraemia and abscess formation in humans. C.sputorum is found in the enteric and reproductive tracts of various production animals and it is recently reported as a pathogenic Campylobacter for human. C.sputorum biovar sputorum can be found in the oral cavity and the gastrointestinal tract of humans. In this study, we recovered this organism from faecal samples of patients with diarrheoa. One of the patients showed nausea, vomiting, chills and in later stages knee abscess followed by severe chest infection. C.sputorum biovar sputorum is associated with lung, axillary, scrotal, groin abscesses and enteritis in humans. Taking into consideration these reports on pathogenesis, C.sputorum was selected for survival studies.
Organic matter is present in abundance in pond water as compared to river water. When C.coli was inoculated in pond water it could utilize the high concentration of organic matter due to the presence of the enzymes needed for degradation. When the C.sputorum isolated from river water was inoculated in the pond water it could not utilize the high concentration of organic matter. C.sputorum survives well in river water because there are plenty of competitive micro-organisms present. It utilizes the trace amount of organic matter that remains for it, thus it is not able to tolerate the high concentration of organic matter. These results are similar to those reported earlier, which conclude that predation and competition affects the survival of Campylobacter species in aquatic environment. C.sputorum grew well in milk because it could utilize the abundantly available amino acids as a source of carbon and nitrogen in milk and the C.coli isolated from buffalo grew well in meat because presence of amino acids, traces of blood, and tissue fractions in the meat, are favourable for its growth., Both the isolates could not grow in mushroom probably because of its antimicrobial activity.
The effect of gamma radiation was checked on these isolates and it was found that the effect depended upon the medium in which the campylobacters were present, for example, the D10 value for C.sputorum was 2KGy when it was present in milk while 0.5-0.6 KGy when present in meat. The D10 value of C.coli was 0.2-0.4KGy when present in meat while 1.17KGy when present in milk. From the above results we can conclude that the effect of gamma radiation on the Campylobacter spp. varied with the type of food. These results are in concordance with those reported by Patterson who checked the sensitivity of Campylobacter spp. to irradiation in poultry meat. Both the Campylobacter spp. isolates were sensitive to 2.4 KGy which lies in the FDA limit of 3 KGy.
The basic objective of this work was to study the behaviour of naturally isolated campylobacters in various food samples that could lead to campylobacteriosis in humans on ingestion. Taking into consideration their ill effects on humans as a major food borne pathogen, this work also deals with the elimination of campylobacters. Some species of Campylobacter jejuni show resistance towards gamma radiation which is the preferred physical method of preservation of food by most food industries. This study recommends that the industries should make an attempt to standardize the gamma radiation dose.
Thanks are due to the Director, National Centre for Cell Science (NCCS), for providing gamma radiation source.
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