Dept. of Food Hygiene,

Fac. of Vet. Med., Assiut Uni., Egypt. 

INCIDENCE AND SURVIVALOF Enterobacter sakazakii IN INFANTS POWDERED MILK-BASED FORMULAE USED BEFORE AND AFTER WEANING

 (With 5 Tables and 5 Figures)

By

A.A-H. Ahmed; Enas El-Prince; Eman Korashy* and Marwa M.N. Al-Gendi*

* Animal Health Research Institute, Assiut

(Received at 14/5/2008)

مدي تواجد ميکروب Enterobacter sakazakii في توليفات أغذية الأطفال الجافة المحتوية علي اللبن المستخدمة قبل وبعد الفطام

  أحمد عبد الحميد ، إيناس البرنس ، إيمان قرشي ، مروة الجندي

تعد کل من الألبان الجافة والأغذية الجافة المحتوية علي اللبن من الأغذية واسعة التدوال لدي الکبـار والأطفال، وتلوثها بالميکروبات الضارة من الأمور التي تستوجب الأهتمام والدراسة. لذلک تضمنت هذه الدراسة فحص عدد 250 عينة عشوائية من أغذية الأطفال اللبنية الجافة بواقع 70 عينة من لبن البودرة للأطفال حديثي الولادة، 90 عينة من کل من أغذية الفطام الجافة المحتوية علي خلاصة الحبوب واللبن الجاف، وکانت صالحة للاستهلاک حيث تمتد فترة صلاحيتها لمدة لا تقل عن عام من تاريخ الإنتاج، وتم تجميع هذه العينات من العديد من المحال التجارية والصيدليات في مدينة وقري أسيوط لمعرفة مدي تلوثها بميکروبE. sakazakii   لما له من خطورة کبيرة علي صحة الأطفال. وأظهرت النتائج تواجدE. sakazakii بالنسب الآتية : صفر، 6.7 و%1.1 من عينات ألبان الأطفال، أغذية الأطفال الجافة االمحتوية علي اللبن واللبن الجاف علي التوالي. وهذا يدل علي أن أغذية الأطفال الجافة االمحتوية علي اللبن تعتبر الأسوأ من حيث تلوثها بميکروبsakazakii E.. وکذلک تم دراسة قدرة هذا الميکروب علي النمو والبقاء في أغذية الأطفال الجافة االمحتوية علي اللبن متأثرا بالسائل الذي يستخدم في تحضيره (عصير التفاح والماء) ودرجة الحرارة التي يتم التخزين فيها (درجة حرارة الغرفة 16±2°م ودرجة حرارة الثلاجة 4 ±1°م) وذلک باستخدام ميکروب E. sakazakiiالذي تم عزله من أغذية الأطفال المحتوية علي اللبن وتصنيفه معمليا باستخدام API ثم حقنه في أحد أنواع أغذية الأطفال المحضرة معمليا. ولقد تبين من الفحص أن الميکروب لم ينمو باستخدام عصير التفاح عند درجتي الحرارة المستخدمة، وکذلک أيضا في حالة استخدام الماء عند درجة الحرارة (4 1± °م). أما في حالة حفظ الوجبة المحضرة باستخدام الماء في درجة حرارة الغرفة ( 16± 2 °م)، فقد وجد أن الميکروب بدأ بالزيادة بعد ساعة واحدة من حفظه وأخذ في الزيادة حتي وصل الي أعلي عدد بعد 4 ساعات وأنه کان هناک أيضا انخفاضا في العدد بعد 8 ساعات حتي وصل الي مستوي أقل من 100 ميکروب/جرام. ومن هذه النتائج ينصح بعدم ترک الوجبة أکثر من ساعة في درجة حرارة الغرفة وحفظها في الثلاجة، کما يفضل استخدام عصير التفاح في تحضير هذه الوجبات. هذا وقد نوقشت الأهمية الصحية لميکروب  E. sakazakiiوالشروط الواجب إتباعها لمنع تلوث ألبان وأغذية الأطفال المختلفة به.

Summary

A total of two hundred and fifty random samples of infant's milk powder for babies after birth (70 samples), milk-based cereal weaning food (90 samples) and dried milk powder (90 samples) were purchased from different shops and pharmacies in Assiut city and villages around the city. The samples were still valid for consumption as shelf life is at least to be more than one year from production time and they were transferred to the laboratory in their packages to be examined for prevalence of Enterobacter sakazakii which could be isolated from 6/90 milk-based cereal baby food samples and from 1/90 dried milk powder samples, however, failed to be detected in infant milk formulae which considered as non sterile products. The survival and growth of E. sakazakiiin milk-based cereal weaning food using different reconstituted liquids (apple juice and water) stored at different temperatures (room temperature      16 ± 2°C and refrigerated temperature 4±1°C) were carried out. The results revealed that the growth did not occur in cereal reconstituted with apple juice, regardless of storage temperature, or in cereal reconstituted with water stored at 4±1°C. Upon reaching maximum populations of 4 log10 cfu/ g, in some instances populations decreased to nondetectable values during subsequent storage which was concurrent with decrease in pH values. E. sakazakii initially at very low populations can rapidly grow in infant cereal reconstituted with water. The public health hazards of E.sakazakii and the suggestive measures for improving the quality of infants' food were discussed.

Key words: E.sakazakii, Incidence, Survival, Infants milk powder, Milk-based cereal weaning food, Dried milk powder.

Introduction

Infants should beexclusively breast fed for thefirst 6 months oflife and those, whoare not, should beprovided with a suitablebreast milk substitute. The reconstitutionof powdered infant formula (PIF) shouldbe undertaken by caregiversusing good hygienic measuresand in accordance withthe product manufacturer's foodsafety guidelines (Drudy et al., 2006). However, consumption of contaminated PIF has been epidemiologically linked with cases of infection. Contamination can occur during the manufacturing process or during post manufacture reconstitution of formula which is not a sterile product (Mullane et al., 2007). E. sakazakii is an opportunistic pathogen and the etiological agent in rare but life-threatening cases of meningitis, necrotizing enterocolitis, and sepsis in infants. Among infants, those at greatest risk are neonates (<28 days), particularly those born prematurely or of low birth weight (<2500 g). It was recorded that infant mortality for E. sakazakii meningitis is 40–80%, with death often occurring within hours of infection (Arseni et al., 1987). Although there is no epidemiological evidence for the amount of an infectious dose for E. sakazakii, it would seem reasonable to use a detection limit <1 cell in 25 g of infant food formula, equivalent to that of detection of Salmonellae in milk powder (Mansfield and Forsythe, 2000).  

Moreover, there have been fewreports recording E. sakazakii infectionin adults, and itis not usually lifethreatening as they had seriousunderlying diseases, such asmalignancies. However, E. sakazakii is a rare cause of invasive disease when meningitis occurs, severe neurological complications, including cerebral abscess formation, are common, and death occurs in 33-80% of cases (Nazarowec-White  and  Farber, 1997a and Lai, 2001). The most frequently recorded method of entry of Enterobacter in patients with bacteremia was ingestion of contaminated foods or food ingredients like cereals, fruits and vegetables, legume products, herbs and spices as well as from animal food sources like milk, cheese, meat and fish and their products both raw and processed (Weischer and Kolmos, 1992 and Friedemann, 2007). It was hypothesized that the reservoir for E. sakazakii, in addition to other coliforms (Klebsiella oxytoca, K. pneumoniae, E. cloacae, and Citrobacter spp.) may be from primarily environmental and from plant materials (Mossel and Struijk, 1995).

Many authors could isolate E. sakazakii from infant milk formulae as Postupa and Aldová (1984); Simmons et al. (1989); Nazarowec-White and Farber (1997a); Nazarowec-White and Farber (1997c); Van Acker et al. (2001); MMWR (2002); Leuscher et al. (2004); Baiguini (2005); Jarvis (2005); Estuningsih et al. (2006); Iversen and Forsythe (2007); Shaker et al. (2007)and Torres-Chavolla et al. (2007). Moreover, Restaino et al. (2006), El-Prince et al. (2007) and Shaker et al. (2007) could isolate E. sakazakii from dried infant foods samples. Also, the survival and growth of E. sakazakii in infant cereals as affected by composition, reconstitution liquid, and storage temperature were measured by many investigators (Richards et al., 2005, Gurtler and Beuchat, 2007 and Lin and Beuchat, 2007).

As E. sakazakii was classified as a severe hazard for restricted populations, causing life-threatening or substantial chronic sequelae or illness of long duration (ICMSF, 2002), therefore, its incidence in infants powdered milk-based formulae used before and after weaning was studied. Also, its survival in cereals using different reconstitution liquids at various temperatures was investigated.

Materials and Methods

Collection of samples:

A total of two hundred and fifty random samples of infant's milk powder for babies after birth (70 samples), milk-based cereal weaning food and dried milk powder (90 samples each) were purchased from different shops and pharmacies in Assiut city and villages around the city. The samples were still valid for consumption as their shelf life is at least to be more than one year from the production time and they were transferred to the laboratory in their packages to be examined for the prevalence of E. sakazakii.

I- Isolation and identification of E. sakazakii (FDA., 2002):

Each sample was aseptically opened and 10 g were weighed and homogenized in 90 ml of sterile distilled water, shacked by hand until the powder was uniformly suspended, incubated overnight at 36°C. 10ml of the incubated sample were transferred to 90 ml ofEnterobacteriaceae enrichment broth (EE) and incubated overnight at 36°C. Loopfuls were streaked onto violet red bile agar (VRBL) and incubated overnight at 36°C.

Isolation procedures:

From each VRBL plate, all colonies were purified on Tryptone Soy Agar (TSA). The TSA plates were incubated for 24 to 72 h in daylight at room temperature (about 25°C). The yellow pigmentation on TSA is a characteristic feature of E. sakazakii. 

Identification of isolates:

Isolates were identified using biochemical tests including Triple Sugar Iron (TSI), Urease test, Sugar fermentation tests, IMViC tests, catalase test then oxidase test. Oxidase-negative isolates were further identified using API 20E biochemical identification test system (bioMerieux SA, France). API 20E systems have been used for presumptive-positive confirmations of E. sakazakii via biochemical characteristics (Kandhai et al., 2004 b).

II- Survival and growth of E. sakazakii in infant cereals using different reconstitution liquids at different storage temperatures.

      Culture preparation:

E. sakazakii strain used in the present study was previously isolated and identified from the examined dried milk-based baby foods. The organism was propagated in EE broth at 37°C for 24 h. One ml of the culture was serially diluted in 0.1% peptone water to attain the desired inoculum level.    

Preparation and inoculation of dried milk-based baby food samples:

Six portions of dried milk-based baby food obtained from Assiut markets, previously examined and found to be free from E. sakazakii, were reconstituted with sterile apple juice and sterile water as recommended by manufactures instructions (50 g of dried milk-based baby food reconstituted with 150 ml of reconstituted liquid). The prepared samples were inoculated with enough amount of broth culture to yield approximately 2 x 10, 2 x 10² and 2 x 10³ cfu/ml of E. sakazakii and control samples were prepared for each treatment, without addition of the organism. Each sample was divided into 2 equal portions, one was stored at room temperature (16±2°C) and the other at refrigeration temperature (4±1°C) with their control.

Preparation of dried milk-based baby food samples for examination:

Samples were prepared following the procedures described by American Public Health Association (A.P.H.A., 1992).

Enumeration of E. sakazakii in the inoculated samples:

From the inoculated samples and their control, 10 fold serial dilutions were prepared using 0.1 % peptone water for determination of E. sakazakii count, using direct spreading method. Duplicate TSA plates were inoculated with each dilution by spreading 0.1 ml evenly onto the surface of each plate with a sterile glass spreading rod. Inoculated plates were incubated at 30°C for 48-72 h. The cfu/g was calculated and recorded. Samples and their control stored at room temperature (16±2°C) were examined for E. sakazakii at 0, 1, 4, 8 and 12 h, while those stored at refrigerating temperature (4±1°C) were tested every 2, 4, 8 12 and 48 h.

Results

The obtained results are recorded in Tables 1-5 and Figures 1-5

Table 1: Incidence of E. sakazakii in the examined samples of powdered milk-based formulae.

Fig. 1: Incidence of E. sakazakii in the examined samples of powdered milk-based formulae.

Table 2: Survival of E. sakazakii in infant cereal milk-based food using water as reconstitution liquid and stored at room temperature (16±2 °C).

      * Not detected on the plate (<100), but the organisms could be isolated from the examined samples.

Fig. 2: Survival of E. sakazakii in infant cereal milk-based food using water as reconstitution liquid and stored at room temperature (16±2 °C).

Table 3: Survival of E. sakazakii in infant cereal milk-based food using apple juice as reconstitution liquid and stored at room temperature (16±2 °C).

     * Not detected on the plate (<100) but could be isolated.  ND = E. sakazakii could not be isolated in 1, 10 and 25 g of infant cereal milk-based food.  

Fig. 3: Survival of E. sakazakii in infant cereal milk-based food using apple juice as reconstitution liquid and stored at room temperature (16±2 °C).

Table 4: Survival of E. sakazakii in infant cereal milk-based food water as reconstitution liquid and stored at refrigerated temperature (4±1°C).

   * Not detected on the plate ( count < 100) but the organisms could be isolated.

Fig. 4: Survival of E. sakazakii in infant cereal milk-based food using water as reconstitution liquid and stored at refrigerated temperature (4 ±1°C).

Table 5: Survival of E. sakazakii in infant cereal milk-based food apple juice as reconstitution liquid and stored at refrigerated temperature (4±1°C).     

* Could not be detected on the plate (count < 100) but the organisms could be isolated.

   ND = E. sakazakii could not be isolated in 1, 10 and 25 g of infant cereal milk-based food. 

Fig. 5: Survival of E. sakazakii in infant cereal milk-based food using                apple juice as reconstitution liquid and stored at refrigerated temperature (4±1 °C).

Discussion

The results achieved in Table 1 & Fig. 1, indicated that IMF samples were found to be free from E. sakazakii. However, many investigators could isolate E. sakazakii from IMF as Postupa and Aldová (1984); Simmons et al. (1989); Nazarowec-White and Farber (1997a); Nazarowec-White and Farber (1997c); Van Acker et al. (2001); MMWR (2002); Leuscher et al. (2004); Baiguini (2005); Jarvis (2005); Estuningsih et al. (2006); Iversen and Forsythe (2007) and Shaker et al. (2007). Moreover, higher incidence of E. sakazakii could be detected by Torres-Chavolla et al. (2007). Also, E. sakazakii could be isolated from 6 (6.7%) of the examined milk-based cereal weaning food samples. Somewhat similar percentage was obtained by El-Prince et al. (2007) who could isolate E. sakazakii from 1 out of 30 (3.33%), of examined dried infant foods samples. While, higher percentages were detected by Restaino et al. (2006) and Shaker et al. (2007). Moreover, 1 strain (1.1%) of E. sakazakii was recovered from the examined samples of dried milk powder, however it failed to be detected as postulated by    El-Prince et al. (2007) and Shaker et al. (2007). While, slightly higher percentage was estimated by Postupa and Aldová (1984) who could isolate 4 strains of E. sakazakii from milk powder in Czechoslovakia. Nazarowec-White and Farber (1997b) stated that microbial pathogens could gain access to the milk powder from the environment or from the addition of ingredients at the powder stage.

According to Commission Regulation (EC) (2005) on the microbiological criteria for foodstuffs, E. sakazakii is considered a microorganism of greatest concern in infant formulae and follow-on formulae. E. sakazakii is included between “safety criteria” and it must be absent in 10 g of dried infant formulae and dried dietary foods for special medical purposes intended for infants below 6 months of age (Conte and Passantino, 2007). The infectious dose can vary according to the organism's history (stress response factors), host state (healthy or immunocompromised) and food matrix. Several physiological characteristics of E. sakazakii appear to play an important role in this transmission route: (i) it can grow in a broad range of temperatures of  6–45 °C (Iversen et al., 2004 a); (ii) it can survive longer than other Enterobacteriaceae under conditions of dry or osmotic stress, which might provide a selection pressure in dried milk products (Breeuwer      et al., 2003); (iii) it can efficiently form biofilms on various materials, including the frequently used polycarbonate material from infant-feeding bottles (Iversen et al., 2004 b and Lehner and Stephan, 2004); (iv) in reconstituted infant formula, E. sakazakii seems to be more heat-resistant than other species of Enterobacteriaceae (Nazarowec-White and Farber, 1997 b).        

Hygiene mismanagement due to incorrect temperature and time factors as well as due to the contact transmission of microorganisms via hands, insects, small vertebrates and equipment should be avoided during production, preparation and storage of food and drink.                E. sakazakii may be associated with food spoilage, but, the detection of the ubiquitous E. sakazakii in food is not always an indicator for hygiene mismanagement.                       

It is obvious from the aforementioned results that milk-based cereal weaning food samples were the highest in contamination with     E. sakazakii. Its presence in the examined samples probably originated from factories producing milk powder, cereals, chocolate, potato flour and pasta as well as in households strongly indicates that the organisms are widespread. Also, the high tolerance of E. sakazakii to desiccation provides a competitive advantage in dry environments thereby increases the risk of post-pasteurization contamination of the finished product (Breeuwer et al., 2003 and Kandhai et al.,2004 a).

The survival of E. sakazakii in infant cereal milk-based food using water as reconstitution liquid and stored at room temperature (16±2 °C) was recorded in Table 2 & Fig.2. It is evident by using 3 different inoculums (3 x 10, 3 x 10² and 2 x 10³) that, the numbers of    E. sakazakii increased to 1 x 10², 4 x 10² and 3 x 10³ cfu/g after 1 h of inoculation and reach the peak after 4 h to be 3 x 10⁴, 1 x 10³ and 4 x 10³ cfu/ g, respectively. The organisms began to decline rapidly  after 8 h to reach 1 x 10², 8 x 10², 6 x 10² cfu / g, respectively, then reached to undetectable numbers after 12 h of storage (<100/g). However, it was detected by enrichment of one g of each treatment. From the previous results, it is clear that E. sakazakii initially at very low populations can rapidly grow in infant cereal reconstituted with water. These results are harmony with those obtained by Richards et al. (2005), Gurtler and Beuchat (2007) and Lin and Beuchat (2007).

From the data recorded in Table 3 & Fig. 3, it is apparent that    E. sakazakii failed to be counted (<100) in the 3 different inoculated samples after one hour of inoculation when apple juice was used as a reconstituted liquid stored at room temperature (16±2 °C). After 12 h, E. sakazakii could not be isolated from 1, 10, 25 g of examined samples using E E broth. Absence of E. sakazakii in examined samples reconstituted with apple juice coincided with decreases in pH and an increase in the population of lactic acid bacteria. These results go parallel with the findings obtained by Weir (2002), Richards et al. (2005) and Lin and Beuchat (2007).

On the other hand, E.sakazakii did not grow in infant cereal milk-based food reconstituted either with water or apple juice and stored at 4±1 °C. E.sakazakii failed to be detectable in both water and apple juice reconstituted samples after 2 h of storage and thereafter. However, the organisms could be isolated by enrichment procedures from water reconstituted samples after 4, 8 and 12 h and failed to be isolated from 1, 10 and 25 g after 12 h of storage in apple juice reconstituted samples (Tables 4 & 5 and Fig. 4 & 5). Our results were in accordance with those obtained by Farmer et al. (1980), Iversen et al. (2004 b), Richards et al. (2005), Gurtler and Beuchat (2007) and Lin and Beuchat (2007). 

E.sakazakii is a pathogen of great concern to food industry, especially in foods normally stored under refrigeration conditions, unlike most food borne pathogens; it is able to multiply at refrigerator temperature (6°C). These findings confirm the importance of proper refrigeration, consequently, refrigeration should not be relied upon as the sole method for control of E. sakazakii, but should be incorporated with other means of preservation. Simmons et al. (1989) recommended the using of refrigeration and limiting the hang time to prevent or retard the growth of E. sakazakii. Nazarowec-White and Farber (1997a) found that           E. sakazakii did not grow at 4°C and began to die off during storage at this temperature and warned improper preparation and storage of reconstituted dried infant formulae at ambient temperature. Moreover, trained personnel should prepare dried milk-based products under aseptic techniques and conditions in a designated area following the manufacturer’s instructions. The composition of infant cereals did not markedly affect the survival or growth of E. sakazakii in reconstituted cereals (Lin and Beuchat, 2007).

It is clear from the present study that reconstituted infant cereal can support luxuriant growth of E. sakazakii and when it is not immediately consumed should be discarded or stored at a temperature at which it cannot grow for only 24 h. Also, the reconstituted infant cereals stored at 30 ºC should be discarded within 1 h after preparation.

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