Dept. of Food Hygiene,

Animal Health Research Institute, Dokki.

Effect of Irradiation on Prevalence

of E.coli O157: H7 in Raw Milk

and Some Milk Products

(With 6 Tables)

By

R.H. Abdel Dayem and Sahar R. Mohamed*

* Bacteriology Dept., Animal Health Research Institute, Dokki.

(Received at 24/12/2005)

تأثير التشعيع على تواجد الميکروب القولونى H7: O157 

فى الألبان وبعض منتجاتها

ربيع حماد عبدالدايم ، سحر رشدى محمد

تم جمع (130) عينة عشوائية من المحلات والسوبر مارکت من عينات الألبان وبعض منتجاتها. ثلاثون عينة من الزبادى وخمسون عينة من کل من اللبن والجبن. تم نقل هذه العينات إلى المعمل مبـاشرة حيث تم فحصها للميکروب القولونىH7:O157. تم عزل الميکروب القولونى H7:O157 من عينة واحدة (2%) من کل من اللبن والجبن. ولم يتم عزل الميکروب من أى عينات الزبادى. تم اختبار ضراوة الميکروب على حيوانات التجارب ووضح تأثيره فى إحداث الانيميا النزيفية والوفاه وکذلک إفرازه للسموم المعوية. وفى الجزء التجريبى من البحث تم حقن عينات من الألبان ومنتجاتها (الزبادى والجبن) التى ثبت خلوها من الميکروب موضوع الدراسة بعترة من الميکروب القولونى H7:O157 إلى جرعات مختلفة من اشعة جاما. وبعد ذلک تم حفظ العينات فى الثلاجة عند درجة حــرارة 5°م لمدة ثلاثة أيام وعند فحص العينات وجد أن الميکروب القولونى H7:O157 فقد قدرته على إحداث الوفاه أو إفراز السموم المعوية.

SUMMARY

A total of one hundred and thirty random milk and some milk products samples were collected from different markets, and were examined for the presence of Escherichia coli O157:H7. The organism could be isolated from one sample (2%) of each of milk and cheese samples, but it failed to detect in yoghurt samples. An important cause of haemolytic anaemia syndrome and lethal effect on mice was detected, while enterotoxin of strain gave maximum secretion in rabbit ligated ileal loop.

Raw milk and milk products samples (cheese and yoghourt) free from  E. coli O157:H7 were inoculated by strain of E. coli O157:H7 obtained from a Bacteriology Veterinary Lab. The effects of irradiation on the inoculated milk and milk products stored at 8°C and 5°C were determined. As survivor level of E. coli O157:H7 following gamma irradiation appeared completely discarded in refrigerator on 1, 2 & 3 days of incubation at 5°C. The cytotoxin production was completely stopped by irradiation at 0.25-0.53 kgy.

Key words: Irradiation, E. coli 0157:H7, milk, milk products

INTRODUCTION

            The increasing number of foodborne bacteria and the worldwide outbreaks of bacteria, particularly E. coli, have increased interest in irradiated food, which is a promising technique for the elimination of this bacterium (Dussa et al., 2004). The unpasteurized milk was first recognized as a vehicle of transmission of E. coli O157:H7 by Chapman et al. (1993).

            The bovine intestinal tract is a known reservoir of E. coli O157:H7; hence, feces may contaminate milk by the organism (Montenegro et al., 1990) Furthermore, enteropathogenic E. coli O157: H7 has been incriminated in many cases of foodborne disease outbreaks, travelers diarrhea and colibacillosis in adults. Verocytotoxin producing E. coli O157:H7 was subsequently found to be common cause of diarrhea haemorrhagic colitis, haemolytic uramic syndrome (Yamamoto et al., 2003 and Vernozy. et al, 2005).

Rediation is the process of emission of energy by exited atoms. loizing radiation is capable of converting atoms and molecules to ions via the removal of electrons, also radiation can be energetic charged particles such as electrons or high energy photons such as X-Rays or Gamma Rays. (Thayer 1995).Also, at has been stated that irradiation is an excellent method for the reduction and/ or elimination of pathogenic food borne microorganisms (Monk et al., 1995).

            Therefore, this work was carried out to isolate E. coli O157:H7 as well as to study the effect of the irradiation on this bacterium on this bacterium.

MATERIALS and METHODS

Survey of E. coli O157:H7 in milk and some milk products:

            One hundred and thirty samples of milk and milk products were collected from different markets; including 30 samples of yoghourt, and 100 samples of raw milk and cheese, (50 each) and the samples were examined for the presence of E.coli O157:H7 according to the method recommended by Adesiyun et al. (1997).

Assay for cytotoxin:

            Assay for cytotoxin was performed by modification of the method of Klipstein et al. (1983). Fresh trypsinized cells were diluted 1: 5 in eagle minimal essential medium plus 2% foetal calf serum. One hundred ml samples were placed in 96 well microtiter plates to which 100 ml bacterial free supernatant were added to each well. The plates were incubated for 18 hrs at 36ºC in 5% CO₂ and these were fixed with methanol, then stained with Giemsa stain. The results are reported as the last dilution that showed > 50% rounded cells.

Rabbit ligated ileal loop:

            The technique recommended by Klipstein et al. (1983)was applied. Toxins preparation in 250 ml of buffer were placed for 16 hrs into single 10 cm ligated ileal loops of fasting rabbit weighting about one kg.

            The results reported for each data point were the mean ± the standard error of the mean for the volume/ length ratio V/ L into rabbit of each strain.

Mouse lethality:

The applied method stated by Ratman et al. (1980)was done as follows:

Mouse lethality assayed by intraperitoneal injection of toxin diluted in normal saline into 15- 20 g BAL B/ C mice death between 2^nd and 7^th days.

Screening raw milk for E. coli O157: H7 to be used in irradiation experiment:

            The method reported by Massa et al. (1999) was applied. One hundred raw milk samples were collected from dairy farms. The samples were taken and maintained at 4ºC until analysis within 5 hrs of sampling by direct plating method. 25 ml of each sample were separately homogenized in 0.1% peptone water. Serial diluations were made. One hundred microliters of appropriate dilutions were spread on sorbitol MacConkey agar plates. The inoculated plates were incubated at 37ºC. After 18- 24 hrs, the plates were examined for non sorbitol fermenting presumptive colonies which were confirmed by latex agglutination using E. coli latex kit (Oxoid, DR 320). The milk products samples were manufactured from pasteurized milk.

Irradiation protocol:

            The technique of irradiation recommended by Lucht et al. (1997) was done. For gamma irradiation, sterile scrow cap test tubes (16 X 125 mm) containing 0.5 ml culture were immersed in 2- L beaker containing crushed ice. An aluminum disc assembly with holes at the circumference was used to position of the test tubes for electron beam irradiation, the samples (0.5 ml, 1 X 10⁸ C.F.U./ ml) in screw- cap. Test tubes were horizontally placed in a plastic tray on a layer of crushed ice.

Damage in E. coli:

            Damage in E. coli was immediately examined following the gamma irradiation (0.44 and 0.76 kgy) survivors were serially diluted and surface spread (0.1 ml) onto plates containing yeast extract agar (BYEA).

This medium described by Lucht et al. (1997):

The series was initially incubated at 18ºC for 0, 1, 2, 4, 5, 24, 28 and 48 hrs.

The ratio of CFU was obtained.

Survival of E. coli O157: H7 strain in raw irradiated milk and milk products (Massa et al., 1999):

            Whole milk was collected from a local dairy farm, stored at 4ºC and used within 2 hrs of collection. The used strain of E.coli O157: H7 in this study was obtained from Bacteriological Veterinary Laboratory. Tryptone soya broth was used for the preparation of the working cultures by growing the strains over night at 37ºC with shaking. The strain was diluted for working cultures in saline (0.85%) and then 1ml of the culture was inoculated into 100 ml of raw milk and some milk products (yoghurt and cheese) at final concentration between about 10³ and 10⁶ (CFU/ ml^-1). Milk was held at 8ºC and 5ºC and then aerobic plate count was determined.

            Escherichia coli O157:H7 as counted by serially diluting (1: 10) milk and milk products in 0.1% peptone water and then surface plating. The plates were incubated at 37ºC for 18- 24 hrs and selected colonies were confirmed to be E.coli O157:H7.

RESULTS

A total of 100 milk samples were assayed for E. coli O157:H7 and cytotoxins. The organism and cystotoxins were not found in any samples. The behaviour of E.coli in milk and milk products held at 8ºC as in Table 2 essentially showed no changes in viable population by direct plating method. The colony forming units ranged from 10⁴-10⁶ CFU/ml^-1. The initial pH of the milk was 6.7, which is the normal value of freshly drawn milk and had declined to 4.8.

Challenged rabbit with enterotoxin in ileal loop, cytotoxicity was detectable more often in the proximal and distal small intestine, it is also found to mice cytotoxin as lethal.

Irradiation at 0.15, 0.53, 0.77 and 0.85 KGY caused a damage in minimal time. Furthermore, the damage was increased with increase in time as in Table 5. In Table 6, irradiated products, refrigerated at 8ºC and 5ºC, showed no growth of E.coli O157:H7 for 3 days.

Table 1: Serovars of pathogenic E.coli in raw milk and milk products.

Table 2: Survival of Escherichia coli in raw milk and milk products after inoculation by 1 ml of E.coli O157: H7 at 8ºC.

Table 3: Biological proportion of toxin extract from E.coli O157: H7 in mouse.

Table 4: Pathogenic properties of E.coli O157: H7.

V/ L Volume length ratio of Challenged Rabbits legated Leal loop by enterotoxin

Table 5: Efficacy of gamma radiation doses on O157:H7 in milk and milk product.

Table 6: Shelf life of refrigerated irradiated raw milk and some milk products.

DISCUSSION

            From the obtained data, it could be concluded that the cheese is the most contaminated product with pathogenic E. coli. This product may be subjected to contamination during its preparation. The results of the present study are in agreement with those reported by Ansay and Kaspar. (1997). However, Adesiyun et al. (1997) found that 17 (18.5%) of 94 strains of E. coli isolated from bulk milk products.

            A total of 100 milk samples were assayed for E. coli O157: H7 and cytotoxins. The organism and cytotoxins were not found in any samples. The behaviour of E. coli in milk and milk products held at 8ºC as in Table 2 essentially showed no changes in viable population by direct plating method. The colony forming unit ranged from 10⁴ - 10⁶ CFU/ ml¹. The initial pH of the milk was 6.7, which is the normal value of freshly drawn milk and had declined to 4.8. Challenged rabbit with enterotoxin in ileal loop, cytotoxicity was detectable more often in the proximal and distal small intestine, it is also found lethal to mice. Following irradiation at 0.15, 0.53, 0.77 and 0.85 at 0.7ºC, it caused damage in minimal time and further damage with increase in time occurred as in Table 5. In Table 6, refrigerated and irradiated products at 8ºC and 5ºC showed no growth of E.coli O157:H7 for 3 days.

            Verototoxin producing Escherichia coli O157:H7 is now recognized as an important enteric pathogen for humans and has been implicated as the causative agent of several food associated outbreaks of disease (Doyle, 1991).

            Raw milk was first recognized as a vehicle of transmission of E.coli O157:H7 (Anonanys, 1994). E.coli O157:H7 organisms are reinforcing the observation that they are not very common, the results of the present study are in agreement with those reported by Hancock et al., (1994).

            The behaviour of E.coli in milk and some milk products held at 8ºC as shown in Table 2, reveal that the initial colony forming units ranged from 10³- 10⁶CFU ml^-1. The results obtained in the study confirmed the ability of E.coli O157:H7 to grow or maintain themselves at both low temperature and pH.

            In this respect, Palumbo et al. (1995) found that haemorrhagic strain of E.coli O157: H7 grew in brain heart infusion broth at 8ºC and produced cytotoxin at 10ºC. On the other hand, Sabra (2000) examined 50 local human and animal isolates of Enteropathogenic E.coli for production of verotoxin, the highest verotoxin producers were E.coli of bovine origin.

            The present study has revealed that cytotoxins have lethal effect to mice and LD₅₀ varied from 60 to 75 mg protein in different cytotoxins produced from the isolated E.coli. This agrees with Ratman et al. (1980) who recorded that LD₅₀ of cytotoxin in mice varied from 660 to 90 mg protein.

            The obtained results in Table 4 showed that the toxins of E. coli O157: H7 isolated from milk and milk products had distinct cytopathic effect with titre 12. These findings are supported by results of Massa     et al. (1999) who stated that the cytoxin producing E. coli is recognized as an important enteric pathogen for human and has been implicated as a causative agent of several food associated outbreaks.

            The obtained results in Table 4 showed that E. coli enterotoxins isolated from milk, yoghurt and cheese showed a maximum secretion in ligated ileal loop. This result is similar to that recorded by Waker et al. (1988) who found that all E. coli proved to be toxin positive and caused a fluid accumulation in ligated ileal loop.

            The rate and total amount of repair in injured microorganisms are dependent on various environmental factors, including freezing, drying, acid or salt treatment has been shown to repair within 30 min, in contrast, heat injured cell have been reported to take from 3 to 4 hrs (Ray, 1989).

            However, the observation that optimal repair of E. coli following irradiation occurred when initially maintained at suboptimal temperature over via of data presented by Ray (1989). Moreover, Monk et al. (1995) suggested that the ability of irradiated E.coli to survive treatment as dependent on to counter- current metabolic processes one process, involved in synthesis was thought to promate repair and operated at temperatures up to 18ºC. Also Hammad et al. (1998) found that irradiation dose at 4 KGY greatly reduced the initial microbial counts.

            The other process was promoted damage and or permanent injury leading to death and operated at temperatures higher than 1ºC.

            Although the exact nature of this destructive process is thought to promote thermolability of various cellular enzyme mediated physiological reaction and/ or structural components. Also Chapman     et al. (1993) confirmed that the pathogen is unable to multiply or produce cytotoxins if the milk or milk product kept refrigerated at the correct temperature (5ºC).

            In conclusion, potentially lethal damage to E.coli O157:H7 appeared by gamma irradiation regarding cytotoxicity was susceptible to irradiation.

            The findings suggests that, irradiation is an effective means to kill the bacteria also irradiation differentially inactivates some activities of cytotoxin.

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