Radiation Physics and Chemistry 81 (2012) 82–85

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Radiation Physics and Chemistry

0969-80

doi:10.1

n Corr

E-m

journal homepage: www.elsevier.com/locate/radphyschem

Effectiveness of radiation processing in elimination of Campylobacter frompoultry meat

Amol D. Raut a, Ravindranath Shashidhar b, Jayant R. Bandekar b,n, Balu P. Kapadnis a

a Department of Microbiology, University of Pune, Pune, Ganeshkhind 411007, Indiab Food Technology Division, Bhabha Atomic Research Centre, Mumbai 400085, India

a r t i c l e i n f o

Article history:

Received 30 June 2011

Accepted 2 September 2011Available online 8 September 2011

Keywords:

Campylobacter

Gamma radiation

Decimal reduction dose

Local poultry meat

Indian meat

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016/j.radphyschem.2011.09.003

esponding author. Tel.: þ91 022 25593961;

ail address: jrb@barc.gov.in (J.R. Bandekar).

a b s t r a c t

Campylobacter, a common poultry intestine commensal, is a well known cause of human gastric

illnesses across the globe. Consumption of contaminated poultry meat is a major cause of Campylo-

bacter related infections. In the present study, radiation sensitivity of indigenous strains of C. jejuni and

C. coli isolated from poultry was evaluated. The decimal reduction dose (D10) values of different

Campylobacter isolates at 0–4 1C in saline and blood broth were in the range of 0.120–0.210 kGy and

0.170–0.234 kGy, respectively. D10 values in chicken meat homogenate for Campylobacter were in the

range of 0.110–0.190 kGy. Chicken meat samples were inoculated with C. jejuni and exposed to gamma

radiation to study the effectiveness of radiation treatment in elimination of Campylobacter. Radiation

treatment with a dose of 1 kGy could achieve complete elimination of 105 CFU of Campylobacter/g in

poultry meat samples. No recovery of Campylobacter was observed, even after enrichment and selective

plating in 1 kGy treated chicken meat samples stored at 4 1C up to 7 days. Present study shows that

irradiation of poultry meat with 1 kGy can ensure safety of poultry meat.

& 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Campylobacter, a gram negative, non-sporulating, motile bac-terium, is commonly isolated as a pathogen associated withdiarrhea in many industrialized countries (Stanley and Jones,2003). It is known to be a major cause of gastroenteric infectionsin the USA and UK, with infection rates much higher than thosecaused by Salmonella (Franco and Williams, 2001; Zhao et al.,2001). Chickens are major reservoirs of and are frequentlycolonized by pathogenic Campylobacter species like C. jejuni andC. coli (Corry and Atabay, 2001). Prevalence of Campylobacter inraw poultry meat products as well as processed meat productshas been reported extensively from developed countries (Zhaoet al., 2001). Intestines of poultry birds are known to harbor veryhigh levels of Campylobacter spp. Reports indicate that all birds ina flock can be colonized with Campylobacter when the flock isinfected (Corry and Atabay, 2001). The poultry has been consid-ered as a major vehicle for spread of this pathogen (Jacob-Reitsma, 1997). Moreover, an increase in antimicrobial resistanceof Campylobacter, particularly to tetracycline, fluoroquinolonesand erythromycin has been reported in many countries (Pratset al., 2000). Thus, antimicrobial resistance in Campylobacter

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strains has become a major public health concern in bothdeveloped as well as developing countries in recent years.

Reports indicate high prevalence of Campylobacter in Indianpoultry meat (Chowdhary et al., 1984; Varma et al., 2000). More-over, it has been observed that serotypes common in humanclinical isolates are prevalent among poultry birds (Bhadra et al.,1989), indicating potential threat to the consumers. Irradiation is asafe, efficient, environmentally clean and energy efficient processfor decontamination of food. Irradiation is particularly valuable asan end product decontamination procedure (Farkas, 1998).

Data available on radiation sensitivity of Campylobacter iso-lates in Indian foods and poultry meat products is very scanty.The objectives of the present study were to (i) determine D10

values of gamma radiation for Campylobacter isolates from poul-try in saline, blood broth and chicken meat homogenate, (ii)determine the radiation dose required for a 5 log CFU eliminationof Campylobacter from meat samples, (iii) study the survival andrecovery, if any, of Campylobacter in inoculated and radiation-treated poultry meat samples, during storage at 4 1C.

2. Material and methods

2.1. Bacterial strains

C. coli MTCC 1126 (Microbial Type Culture Collection, Chandi-garh, India) was used as a reference strain. Twelve C. jejuni

A.D. Raut et al. / Radiation Physics and Chemistry 81 (2012) 82–85 83

isolates (no. 1–12) and 12 C. coli isolates (no. 13–24) obtainedfrom poultry were selected for studies (unpublished data). Allstrains were maintained routinely at �70 1C in glycerol broth(20% glycerol and Brucella broth 80%).

2.2. Chemicals and media

Microbiological media were obtained from Hi Media Labora-tories, Mumbai, India. Horse blood was obtained from HaffkineBiopharmaceutical Ltd., Pune, India.

2.3. Irradiation

Irradiation at low doses (0.1–0.5 kGy) was done in a cobalt-60irradiator (Gamma cell 220, Atomic Energy of Canada Ltd.,Ontario, Canada) at a dose rate of 0.011 kGy/min. Irradiation athigh doses (1–5.0 kGy) was done in a cobalt-60 irradiator(Gamma cell 5000, Board of Radioisotope and Technology, Mum-bai, India) with a dose rate of 0.083 kGy/min. Dose rate of all theradiation sources was measured using the Fricke method (ASTM,E 1026, 2004).

2.4. Determination of D10 values in saline and blood broth

Campylobacter cultures were grown on blood agar (BA) con-taining 5% horse blood for 48 h at 42 1C, in microaerobic environ-ment (5% CO2, 10% CO2 and 85% N2). Inoculum was prepared byadding 5 ml phosphate buffered saline to BA plates and scrappingoff growth with a sterile glass rod. Cells were centrifuged toobtain pellet and washed thrice with sterile saline to removemedia components and suspended in sterile saline and bloodbroth (BB) to obtain cell density of 108 CFU/ml. Aliquots of 1.2 mlof suspensions prepared in saline and BB were dispersed inmicrofuge tubes. The tubes were placed in ice and irradiated at0.1, 0.2, 0.3, 0.4 and 0.5 kGy at 0–4 1C in Gamma cell 220. Totalviable counts (TVC) after irradiation were determined by platingthe aliquots after appropriate dilutions on BA plates. The plateswere incubated at 42 1C for 48 h under microaerobic conditionsand colonies were counted. The logarithm of the bacterial countwas plotted against radiation dose, individual regression lineswere plotted and D10 values were obtained as the reciprocal of theslope (Patterson, 1995).

2.5. Determination of D10 values in chicken meat homogenate

Five Campylobacter cultures (C. jejuni no. 11 and 12, C. coli no.18 and 23, C. coli MTCC 1126) showing highest radiation resis-tance in BB were checked for radiation sensitivity in 10% chickenmeat homogenate (w/v). Homogenate was prepared by homo-genizing boneless chicken meat (10 g) in D/W (90 ml) and waskept in ice and decontaminated by exposure to 5 kGy dose inGamma cell 5000. Campylobacter suspensions were prepared asmentioned earlier and suspended in 1.2 ml homogenate so as toobtain a cell density of 108 CFU/ml. D10 values of gamma radiationfor the five cultures were determined as mentioned earlier.

2.6. Decontamination of chicken meat

Chicken meat samples (25 g) were packed in low densitypolyethylene bags (LDPE) of 35 mm thickness (ACE packagingLtd., Mumbai, India) and sealed using heat sealer (Sevana,Mumbai, India). The meat samples were kept in ice and radiationprocessing (5 kGy) for decontamination was carried at 0–4 1C inGamma cell 5000. The decontamination of the chicken sampleswas checked by homogenizing 25 g of the meat sample in 225 mlsterile 0.1% peptone water using a stomacher (Stomacher lab

blender, model 400, Seward, London, UK) for 1 min at 100 rpm.The homogenate liquid was appropriately diluted and aliquotswere plated on BA plates. The plates were observed for growthafter incubation at 42 1C for 72–96 h. Absence of growth evenafter 96 h incubation indicated decontamination of sample.

2.7. Inoculated pack studies for determination of dose required to

eliminate 5-log CFU of C. jejuni/g of chicken meat

C. jejuni (isolate No. 12), showing highest D10 value in homo-genate, was selected for the inoculated pack studies. Test strainwas grown on BA plates and the inoculum was prepared asdescribed in Section 2.4. The decontaminated chicken samples(25 g), in triplicate were inoculated with the test strain (105 CFU/gof meat). The inoculated packs were irradiated in melting iceconditions with 1.0, 3.0 and 5.0 kGy in Gamma cell 5000. Sampleswere homogenized aseptically for 1 min in a sterile stomacherbag (Stomacher lab blender, model 400, Seward, London, UK)containing 225 ml of sterile saline. The surviving population ofCampylobacter was determined by plating serial dilutions ofhomogenized samples on mCCDA plates after incubation at42 1C for 48 h under microaerobic conditions. Enrichment usingPreston Enrichment (PE) broth (with 5% v/v blood) and plating onBA were carried out to confirm the complete elimination of thispathogen. Each experiment was repeated three times.

2.8. Storage studies of gamma-irradiated chicken samples

inoculated with C. jejuni isolate

The inoculated chicken meat packs were prepared as describedin Section 2.7 and irradiated in melting ice conditions with dosesof 1.0, 3.0 and 5.0 kGy in Gamma cell 5000 and stored at 4 1C. Thesamples were analyzed on the 1st, 3rd, 5th and 7th day forCampylobacter as described in Section 2.7. Enrichment using PEbroth (with 5% v/v blood) and plating on BA were carried out toconfirm elimination of this pathogen. Each experiment wasrepeated three times.

2.9. Statistical analysis

All the data for D10 values of Campylobacter strains in saline,blood broth and chicken meat homogenate were analyzed statis-tically using Microsoft Excel software, version 2000, USA. Sig-nificant differences in D10 values between different isolates wereanalyzed by one-way ANOVA and paired t-test.

3. Results and discussion

3.1. D10 values of Campylobacter isolates in saline and blood broth

All Campylobacter isolates were found to be highly sensitive togamma radiation. The D10 values for all isolates ranged from0.170 to 0.234 kGy in BB and from 0.120 to 0.210 kGy in saline(Table 1). Campylobacter isolates are known to be more sensitiveto gamma radiation than other food-borne pathogens like Salmo-

nella and Listeria (Monk et al., 1995; Patterson, 1995). Sensitivityto gamma radiation was more in saline than in BB, and significantdifferences were found in the D10 values (Po0.05). Patterson(1995) reported that increase in total solids in the suspendingmedium inversely affects the irradiation efficacy. Thus, higher D10

values in BB than in saline may be due to the media componentsthat confer protection to the Campylobacter cells duringirradiation.

No significant differences (Po0.05) were observed in the D10

values of C. jejuni and C. coli isolates; however, there was

Table 1D10 values of gamma radiation for C. jejuni and C. coli isolates irradiated in saline

and blood broth.

Campylobacterisolate no.a

D10 values (kGy)

Bloodbroth

Saline

1 0.189 0.120

2 0.174 0.134

3 0.190 0.158

4 0.205 0.165

5 0.220 0.198

6 0.215 0.175

7 0.187 0.147

8 0.170 0.122

9 0.201 0.169

10 0.213 0.185

11 0.225 0.200

12 0.234 0.210

13 0.185 0.164

14 0.196 0.178

15 0.178 0.136

16 0.185 0.154

17 0.198 0.169

18 0.226 0.201

19 0.201 0.188

20 0.182 0.176

21 0.188 0.172

22 0.212 0.184

23 0.221 0.191

24 0.218 0.205

C. coli (MTCC) 0.212 0.187

a Isolate no.: 1–12, C. jejuni; 13–24, C. coli.

Table 2D10 values of gamma radiation for Campylobacter isolates irradiated in chicken

meat homogenate and blood broth.

Campylobacterisolate

Isolatenumber

D10 in 10% chickenhomogenate (kGy)

D10 in bloodbroth (kGy)

C. jejuni 12 0.110 0.234

C. jejuni 11 0.190 0.225

C. coli 18 0.165 0.226

C. coli 23 0.142 0.221

C. coli MTCC

1126

0.180 0.212

A.D. Raut et al. / Radiation Physics and Chemistry 81 (2012) 82–8584

significant difference (P40.05) in the radiation sensitivity ofdifferent Campylobacter isolates. Amongst all C. jejuni isolatestested, isolate no. 12 was found to be the most resistant to gammaradiation, both in BB and saline with D10 values of 0.234 and0.210 kGy, respectively. Whereas, amongst C. coli isolates, isolateno. 18 was most resistant in BB (0.226 kGy) and isolate 24 wasfound to be most resistant in saline (0.205 kGy).

Amongst all C. coli isolates tested, isolate no. 15 was found tobe most sensitive to gamma radiation, both in BB and saline withD10 values of 0.178 and 0.136 kGy, respectively. C. jejuni isolateno. 8 was found to be most sensitive in BB (0.170 kGy) and isolate15 was most sensitive in saline (0.120 kGy). The differences in theD10 values observed for various isolates in the present study maybe due to the different Campylobacter strains. Five Campylobacter

cultures (C. jejuni no. 11 and 12, C. coli no. 18 and 23, C. coli 1126,MTCC) showing highest radiation resistance in BB were checkedfor radiation sensitivity in chicken meat homogenate (10% w/v).

3.2. D10 values in chicken meat homogenate

D10 values in chicken homogenate for all isolates were lowerthan those observed in BB (Table 2). Although reports indicatedecrease in irradiation efficacy due to increase in total solids inthe irradiation medium (Patterson, 1995), our results contradictthis observation. The chicken meat homogenate, inspite of being acomplex suspending medium with more solids than BB, conferredless protection to Campylobacter cells. The difference in chemicalcomposition may be responsible for the differences in D10 valuesin blood broth and chicken homogenate.

3.3. Dose required for elimination of 5-log CFU/g of inoculated cells

from chicken meat homogenate

Campylobacter counts of control samples decreased duringstorage at 4 1C— 5.1070.23 (1st day), 3.4170.15(3rd day),

2.7870.18 (5th day) and 1.8270.14 log CFU/g (7th day).Campylobacter counts in all chicken meat samples irradiated at1 kGy were reduced from 5-log CFU/g to undetectable counts.Enrichment of samples in PE broth was carried out in order tocheck the possibility of resuscitation of Campylobacter cellsinjured during irradiation; however, no Campylobacter cells wererecovered. This indicates that even a dose as low as 1 kGy can beused to ensure safety of chicken meat.

Radiation treatment at doses of 2–7 kGy, depending on thecondition of irradiation and food, are known to effectivelyeliminate pathogenic bacteria such as Campylobacter from foodproducts without affecting sensory, nutritional and technicalqualities (Farkas, 1998). Thus, Campylobacter can be eliminatedfrom the chicken meat using low gamma radiation doses such as1 kGy without losing the organoleptic qualities of the meatproducts.

3.4. Storage studies of irradiated chicken samples inoculated with C.

jejuni isolate

No viable counts were detected in 1, 3 and 5 kGy treated meatsamples during storage for seven days at 4 1C even after enrich-ment of samples in PE broth. This indicates that 1 kGy doseof gamma radiation can be used to eliminate Campylobacter

completely from meat samples without chances of recovery ofinjured cells.

Chickens are major reservoirs of and are frequently colonizedby pathogenic Campylobacter species like C. jejuni and C. coli

(Corry and Atabay, 2001). Counts of Campylobacter present inwhole bird carcasses may range from less than 2.6 log CFU percarcass to more than 5.8 log CFU per carcass (Chrystal et al.,2008). Such contamination rates are alarming, as even a numberas low as 500 cells of Campylobacter can cause infection inhumans (Robinson, 1981). 5 log reductions in Campylobacter

counts of poultry meat samples achieved by irradiation with adose of 1 kGy can provide near complete elimination of Campy-

lobacter from the meat samples.

4. Conclusions

Campylobacter isolates from chicken marketed in India werefound to be sensitive to gamma radiation. A low radiation dose of1 kGy is effective in achieving 5-log reduction in Campylobacter

populations in chicken meat. No growth of Campylobacter wasobserved in irradiated meat products during storage for sevendays at 4 1C. This is the first report of radiation sensitivity andinoculated pack studies of Indian Campylobacter isolates irra-diated in chicken meat. Food laws in India permit radiationprocessing of meat and meat products with 2.5–4 kGy dose forelimination of Salmonella. Radiation processing for elimination ofSalmonella can also ensure safety from Campylobacter.

A.D. Raut et al. / Radiation Physics and Chemistry 81 (2012) 82–85 85

Acknowledgments

We thank Mr. Vandan Nagar for his assistance in writing themanuscript.

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