DETECTION AND INACTIVATION OF ENTEROBACTER SAKAZAKII (CRONOBACTER) IN POWDERED INFANT MILK FORMULA

M.M. DEEB, AZZA

doi:10.21608/avmj.2010.174237

DETECTION AND INACTIVATION OF ENTEROBACTER SAKAZAKII (CRONOBACTER) IN POWDERED INFANT MILK FORMULA

Author

AZZA M.M. DEEB

Dept. of Food Control, Fac. of Vet. Med., Kafr El-Sheikh University, Kafr El-Sheikh 33516, Egypt.

10.21608/avmj.2010.174237

Abstract

E. sakazakii is considered as an opportunistic bacterium in elderly people and infants. Epidemiological studies implicate dried infant formula as the primary source of transmissionof this pathogen. A total of 177 powdered infant milk formula (PIMF) [39 low birth weight formula from 1 day: 6 months (LBWF), 63 infant milk formula for infant below 6 months (IMF), 55 follow-on formula from 6:12 months and 20 growing children formula for age 1:4 years). The samples were collected from different pharmacies in Kafr El-Sheikh Governorate, Egypt, within the accurate shelf life period, and then transferred to the laboratory in their packages to be tested for detection of E. sakazakii and detection of the efficacy of Lysozyme and thermally modified lysozyme for inactivating E. sakazakii in reconstituted infant formula at different storage temperatures. The results revealed that E. sakazakii could be detected in 5.1, 6.3 and 5% of examined LBWF, IMF and growing children formula respectively with total percentage of 3.95%, but the organism could not be detected in follow-on formula. The results indicated that thermally modified lysozyme, was more effective than lysozyme in inhibition of E. sakasakii growth at 4 ºC (p <0.001), thermally modified lysozyme had more inhibitory effect on E. sakasakii at 4 ºC than at 25and 37 ºC. In conclusion the combined efforts of public health and regulatory officials, as well as manufacturers, were considered important aspects of the management of risks associated with disease causing E. sakazakii in PIMF, also the uses of thermally modified lysozyme can exert a significant inhibitory activity against this organism in reconstituted milk formula specially when kept at refrigeration temperature.

Keywords

Full Text

Dept. of Food Control,

Fac. of Vet. Med., Kafr El-Sheikh University,

Kafr El-Sheikh 33516, Egypt.

DETECTION AND INACTIVATION OF ENTEROBACTER SAKAZAKII (CRONOBACTER) IN POWDERED INFANT MILK FORMULA

(With 5 Tables and 4 Figures)

AZZA M.M. DEEB

(Received at 15/9/2010)

الکشف عن ميکروب الإنتيروباکتر ساکازاکى وإضعافه فى ألبان الأطفال الجافة

عزة مرغني محمد ديب

يعد ميکروبالإنتيروباکتر ساکازاکى من الميکروبات الممرضة التى تهدد حياة الأطفال حديثى الولادة وحتى الشهر السادس وکذلک الأطفال ناقصى الوزن والمناعة لانه يسبب الإلتهاب السحائى وإلتهاب الأمعاء المتنکرز ولذلک أجريت هذه الدراسة للکشف عن هذا الميکروب فى ألبان الأطفال الجافة لأنها الغذاء البديل للبن الأم فى هذه المرحلة العمرية وکذلک استخدام انزيم الليسوزيم والليسوزيم المعدل بالحرارة لمعرفة مدى ملائمة استخدام هذا الإنزيم الطبيعى فى إضعاف الميکروب فى لبن الأطفال0 وقد أظهرت النتائج أن هذا الميکروب قد تم عزله بنسبة 1‚5 ؛ 3‚6 و 5 % من ألبان الأطفال ناقصى الوزن ، الألبان المخصصة للأطفال أقل من 6 شهور وترکيبة النمو للأطفال من عمر سنة إلى أربع سنوات بنسبة کلية 95‚3% ؛ بينما لم يتم عزله من ترکيبة حليب لمتابعة الرضاعة (من سن 6 أشهر)0 وقد أثبتت التجربة أن استخدام إنزيم الليسوزيم قد أثبت فاعلية لإضعاف الميکروب فى اللبن المعاد حله والمحفوظ فى الثلاجة لمدة ثلاث ساعات فقط؛ بينما إنزيم الليسوزيم المعدل بالحرارة قد أثبت فاعلية فى إضعاف الميکروب فى اللبن المعاد حله فى درجات حرارة 4، 25 و 37 درجة مئوية بنسب متفاوتة وکان أقوى تأثير مضعف للميکروب عند درجة حرارة 4 درجة مئوية0 ولهذا لابد من تضافر جهود منظمات الصحة مع مصانع انتاج ألبان الأطفال لتجنب الأخطار الناجمة عن وجود مثل هذا الميکروب وکذلک التوصية بإستخدام مثبطات الميکروبات الطبيعية مثل انزيم الليسوزيم المعدل بالحرارة لما له من فاعلية فى إضعاف ميکروب الإنتيروباکتر ساکازاکى0

SUMMARY

Key words: Powdered infant milk formula, E. sakazakii, lysozyme, thermally modified lysozyme

INTRODUCTION

Powdered infant milk formula (PIMF) constitutes the majority of infant formula fed to infants' worldwide (Drudy et al., 2006). This product is formulated to mimic the nutritional profile of human breast milk (Breeuwer et al., 2003). PIMF is not a sterile product and can act as a potential source of harmful pathogens. In addition, infants and young children do not have a well developed immune system and hence are more vulnerable to food-borne infections. Therefore the microbiological safety of the infant and follow-up formula is critical. To assure the microbiological safety of PIMF, several microbiological tests are recommended and compared with the microbiological criteria set by the Codex Alimentarius Commission (CAC, 1979). The specific microbes commonly tested include Staphylococcus aureus, Bacillus cereus, Enterobacter sakazakii (E. sakasakii), other Enterobacteriaceae and Salmonellae (Forsythe, 2005). Among the specific microbes tested for presence in infant milk formula, E. sakazakii is placed under category A by FAO–WHO (FAO/WHO, 2004) and is considered to be a potential agent for causing neonatal infections.

E. sakazakii, is a motile, non-sporeforming Gram-negative facultative anaerobe. It is considered as an opportunistic bacterium in elderly people and infants. While infections in adults are often underreported, in neonates and infants it is often occur as severe infection with a reported case fatality rate of 40–80% (Bowen and Braden, 2006). It is causing a rare, but life threatening form of neonatal meningitis, bacteremia, necrotizing colitis and meningo-encephalitis (Nazarowec-White and Farber, 1997a; Sanders and Sanders, 1997; Van Acker et al., 2001). In addition to the high fatality rate of E. sakazakii infections, it may result in severe neurological sequelae such as hydrocephalus, quadriplegia and retarded neural development in survivors (Forsythe, 2005). Although the environmental source of E. sakazakii is not clearly understood, epidemiological studies implicate PIMF as the primary source of transmission (Van Acker et al., 2001; Weir, 2002).

The bacterium has been isolated from PIMF by numerous investigators (Biering et al., 1989; Simmons et al., 1989; Muytjens and Kollee, 1990). Moreover; there were many recalls of E. sakazakii-contaminated infant formula in the United States. In November 2002, a nationwide recall of more than 1.5 million cans of dry infant formula contaminated with E. sakazakii was reported (FSNET, 2002). On April 12, 2002, the United States Food and Drug Administration (FDA) issued an alert to U.S. health care professionals regarding the risk associated with E. sakazakii infections among neonates fed PIMF (FDA, 2002). In addition, the International Commission on Microbiological Specification for Foods (ICMSF, 2002) has ranked E. sakazakii as ‘Severe hazard for restricted populations, life-threatening or substantial chronic sequelae of long duration’. The FAO/WHO (2004; 2006 and 2008) recommended that research should be promoted to gain a better understanding of ecology, taxonomy, virulence and other characteristics of Cronobacter.

Being a nutrient-rich medium, reconstituted PIMF can support bacterial growth when favorable conditions of water availability, time and temperature are provided. Therefore once rehydrated the only limiting conditions for bacterial growth and infection are storage time and temperature. In this regard, E. sakazakii possesses several characteristics that enable it to be a successful infant formula-borne pathogen, Breeuwer et al. (2003) revealed that E. sakazakii has a high tolerance to osmotic stress and desiccation. E. sakazakii can grow at temperatures as low as 5.5 °C (Nazarowec-White and Farber, 1997b), which has been reported to be the temperature of many home refrigerators. Improper storage of reconstituted formula may permit its substantial growth. Therefore, incorporation of an effective antimicrobial barrier may potentially reduce the likelihood of outbreaks of E. sakazakii infection in infants through ingestion of contaminated reconstituted infant formula.

In recent years, there has been an increasing interest in the use of natural antimicrobial substances due to concerns regarding the safety of synthetic compounds (Abee et al., 1995). This is especially significant when selecting antimicrobials for use in infant foods. Lysozyme is part of the innate immune system, however, reduced lysozyme levels have been associated with broncho-pulmonary dysplasia in newborns (Revenis and Kaliner, 1992). Children fed infant formula lacking lysozyme in their diet have three times the rate of diarrheal disease. Since lysozyme is a natural form of protection from pathogens like Salmonella, E.coli, and Pseudomonas, a deficiency due to infant formula feeding can lead to increased incidence of disease (Lonnerdal, 2003). Lysozyme found in egg white, tears, and other secretions. It is responsible for breaking down the polysaccharide walls of many kinds of bacteria thus it provides some protection against infection. Investigations conducted by Ibrahim et al. (1996) indicated a possibility of extending the range of lysozyme activity to include Gram-negative bacteria, using thermal modification. It has also been found that heat denaturation of lysozyme caused by increasing temperatures results in the progressive loss of enzymatic activity, while its antimicrobial action against Gram-negative bacteria is greatly enhanced.

The aim of this work was planned to detect E. sakazakii in PIMF and the efficacy of lysozyme and thermally modified lysozyme for inactivating E. sakazakii in reconstituted infant formula at different storage temperatures.

MATERIALS and METHODS

1. Isolation and identification of E. sakazakii

1.1. Collection of samples

One hundred and seventy seven PIMF [39 low birth weight formula from 1 day: 6 months (LBWF), 63 IMF for infant below 6 months, 55 follow-on formula from 6:12 months and 20 growing children formula for age 1:4 years). The samples were collected from different pharmacies in Kafr El-Sheikh Governorate, Egypt, within the accurate shelf life period, and then transferred to the laboratory in their packages to be tested for detection of E. sakazakii.

1.2. Preparation of samples

Twenty-five grams of each sample after sterilization and opening of the cans were homogenized for 1 min at medium speed in a Seward Stomacher (Seward, Thetford, UK) in 225 ml buffered peptone-water (CM 509 Oxoid Ltd.) and incubated overnight at 37°C (ISO 8261, 2001).

1.3. Isolation of E. sakazakii procedure (ISO/TS 22964, 2006)

To 10 ml Cronobacter Screening Broth (CSB) (CM1121 Oxoid Ltd.) 0.1 ml of pre-enrichment BPW was added and incubated at 41.5 °C for 24 h. From yellow colored broth tube 10 µl were streaked onto the surface of Brilliance Enterobacter sakazakii Agar (DFI) (CM 1055 Oxoid Ltd.) and colony morphology observed after incubation at 44°C for 24 h. Blue green colonies were picked off and streaked on Tryptone soy agar (TSA) (CM131 Oxoid Ltd.). Colonies that produced yellow pigment after incubation at 25°C for 48–72 h were termed presumptive E. sakazakii.

1.4. Identification of presumptive E. sakazakii

Biochemical identification of presumptive E. sakazakii was done according to Farmer and Kelly (1992).

2. Inactivation of E. sakazakii

2.1. Bacterial strain preparation

E. sakazakii isolate was cultured in 10 ml of sterile Tryptic soy broth (TSB) at 37°C for 20 h. The bacterial population was determined by pour plate technique after preparation of serial dilutions according to APHA (1992). Loopfuls from each dilution were streaked on previously prepared TSA plate and then the plates were incubated at 37ºC for 24 h, the colonies forming unite / ml was calculated.

2.2. Preparation of antimicrobials

Egg white lysozyme (BioShop, Canada Inc.) stock solution of 1mg/ml in potassium phosphate buffer (10 mM, pH 7.0) was prepared (Barbara et al., 2000) and divided into two parts, one part was kept frozen at -20 ºC and the other part modified thermally by heating at 80 ºC for 20 min ( Ibrahim et al., 1996).

2.3. PIMF preparation

PIMF was reconstituted as per the manufacturer's instructions on the label. Briefly, 135 g of the formula were reconstituted in 900 ml of sterile distilled water; 100 ml volumes were dispensed into screw capped bottles (9) and pasteurized at 63ºC for 30 min.

2.4. Inoculation, incubation and determination of antibacterial activity

To the bottles 2, 5 and 8 lysozyme solution was added, and to bottles 3,6 and 9 thermally modified lysozyme (denaturated) solution was added in final concentration 50µg/ml (1000 U) of reconstituted IMF. Bottles 1, of 4 and 7 were control (devoid of any antibacterial agent). E. sakazakii was added to the nine bottles in final count 2x10⁶/ ml of reconstituted IMF. Bottles 1, 2 and 3 were incubated at 4ºC, while bottles 4, 5 and 6 were incubated at 25ºC and bottles 7, 8 and 9 were incubated at 37ºC for 0, 3, 6, 9, 12 and 24 h. The surviving populations of the pathogen were enumerated by plating after serial dilutions (1:10) on triplicate TSA after incubation at 37ºC for 24 h.

3. Statistical analysis

Data were analyzed using General Linear Models procedures after log transformation. Least square means were computed for each treatment and group differences were tested using Bonferroni test. All experiments and analyses were replicated 3 times (SAS Institute, 1999).

RESULTS

Table 1: Incidence of E. sakazakii in examined powdered infant milk formula.

Types of samples	No. of examined samples	E. sakazakii positive samples
Types of samples	No. of examined samples	No	%
Low birth weight formula	39	2	5.1
PIMF for infant below 6 months	63	4	6.3
Follow-on formula from 6:12 months	55	0	0
Growing formula for age 1:4 years	20	1	5
Total	177	7	3.95

Table 2: Effect of lysozyme and thermally modified lysozyme on E. sakazakii count incubated at 4ºC.

Groups	Mean log count +SE
Groups	0^*	3	6	9	12	24
Control	14.26+ 0.15^a	14.41+0.32 ^a	17.12+0.56^a	18.32+0.32^a	19.17+0.17^a	20.02+0.11^a
Lysozyme	13.60+0.51 ^a	11.74+0.46^b	13.73+0,26^b	14.12+0.31^b	15.40+0.17^b	16.52+0.07^b
Thermally modified lysozyme	13.76+0.43 ^a	2.95+0.37^c	5.90+0.32^c	6.58+0.65^c	9.08+0.39^c	11.51+0.06^c

Means in the same column without a common letter differ significantly (p<0.01).

*No significance difference p = 0.41

Table 3: Effect of lysozyme and thermally modified lysozyme on E. sakazakii count incubated at 25ºC.

Groups	Mean log count +SE
Groups	0^*	3	6	9	12	24
Control	14.11+ 0.62^a	15.91+0.51 ^a	17.70+0.43^a	20.00+0.025^a	24.93+0.15^a	28.27+0.38^a
Lysozyme	13.41+1.10 ^a	16.00+0.34^ab	17.18+0.20^ab	18.37+0.05^b	20.49+0.46^b	25.23+0.32^b
Thermally modified lysozyme	13.78+0.77 ^a	9.11+0.32^c	12.99+0.07^c	13.57+0.15^c	17.67+0.25^c	22.81+0.06^c

Means in the same column without a common letter differ significantly (p<0.01).

*No significance difference p = 0.85

Table 4: Effect of lysozyme and thermally modified lysozyme on E. sakazakii count incubated at 37ºC.

Groups	Mean log count +SE
Groups	0^*	3	6	9	12	24
Control	14.06+0.63^a	16.65+1.13 ^a	18.13+0.55^a	20.03+1.01^a	23.85+1.60^a	29.71+0.07^a
Lysozyme	13.29+0.89 ^a	17.06+0.63^ab	17.99+0,63^ab	18.71+0.41^ab	22.59+0.63^ab	27.34+0.55^b
Thermally modified lysozyme	13.34+0.99 ^a	11.65+0.52^c	13.45+0.37^c	13.95+0.52^c	18.18+0.15^b	22.81+0.33^c

Means in the same column without a common letter differ significantly (p<0.01).

*No significance difference p = 0.78

Table 5: Effect of lysozyme and thermally modified lysozyme on E. sakazakii count at different incubation temperature

throughout the experiment.

Groups	Mean log count +SE
Groups	4º C	25º C	37º C
Lysozyme	14.19±0.38^a	18.45±0.92^c	19.50±1.10^c
Thermally modified lysozyme	8.30±0.88^b	14.99±1.05^d	15.56±0.94^{e d}

Means in the same column without a common letter differ significantly (p = 0.03).

Means in the same row without a common letter differ significantly (p = 0.02).

C: control

L: Lysozyme

TML: Thermally modified lysozyme

DISCUSSION

In Europe and the United State, several studies have reported cases of E. sakazakii infection in infants taking a PIMF, raising an important issue. This study was focused on PIMF in response to the FAO/WHO (2008) call for appropriate microbiological data. Results given in Table 1 pointed out that the rate of contamination with E. sakazakii was 5.1, 6.3 and 5% of the examined LBWF, IMF (for age < 6 months) and growing formula, respectively with a total percentage of 3.95%, while the organisms could not be detected in follow-on formula. Concerning prevalence in powdered formulae, some reported prevalence figures in positive batches were about 1 and 3% (FAO/WHO, 2008), and nearly similar results were reported by Yoo et al. (2005); Chap et al. (2009) and Oonaka et al. (2010). Presence of E. sakazakii in PIMF may be attributed to inadequate hygiene in factory or contamination of raw materials.

Cronobacter spp. infections have been reported for all age groups. Worldwide there have been 120 reported cases in infants and children < 3 years in age, of which 8 were cases aged between 6 and 35 months. In the UK between 1999 and 2007, 15/570 laboratory reported infections were from infants (< 12 months in age), and 16/570 were from children (1–4 years in age) (Food and Agriculture Organization/World Health Organization (FAO/WHO), 2008 Food and Agriculture Organization/World Health Organization (FAO/WHO), Enterobacter sakazakii (Cronobacter spp.) in powdered follow-up formulae. MRA series (2008) Available at http://www.who.int/foodsafety/publications/micro/MRA_followup.pdf. Date last accessed 03/09/09.FAO/WHO, 2008). Risk for infection might depend on several factors, including the number of bacteria present in the product, handling after preparation, and underlying patient characteristics (e.g., immune-suppression, prematurity, or low birth weight). Because powdered formula is not sterile and can provide a good medium for growth, prolonged periods of storage or administration at room temperature might amplify the amount of bacteria already present.

FDA (2002) recommended that powdered infant formulas not be used in neonatal intensive care settings unless there is no alternative available. If the only option available to address the nutritional needs of a particular infant is a powdered formula, risks of infection can be reduced by preparing only a small amount of reconstituted formula for each feeding to reduce the quantity and time that formula is held at room temperature for consumption; minimizing the holding time, whether at room temperature or while under refrigeration, before a reconstituted formula is fed; and minimizing the “hang-time” (i.e., the amount of time a formula is at room temperature in the feeding bag and accompanying lines during enteral tube feeding), with no “hang-time” exceeding 4 hours. Longer times should be avoided because of the potential for significant microbial growth in reconstituted infant formula.

Natural antimicrobials have gained attention because of the demand for preservative-free food products (Payne et al., 1990). Included as natural antimicrobial is lysozyme enzymes that can inhibit the growth of various intestinal pathogens and which can protect children against gastroenteritis (Lonnerdal, 2003).

The growth of E. sakazakii was evaluated after treatment with lysozyme and thermally modified lysozyme in reconstituted milk formula and incubated at 4, 25 and 37 ºC. The Effect of lysozyme and thermally modified lysozyme on E. sakasakii count in reconstituted milk formula incubated at 4ºC throughout the experiment was presented in Table 2 and Figure 1. The results declared that there was no significant difference between control, lysozyme and thermally modified lysozyme treated samples at zero time (p = 0.41), while there were significant difference between the three treatments from 3h to 24h (p <0.01), at 3h of incubation the population of E. sakazakii was reduced to 11.74±0.46 log base CFU/ml by lysozyme and to 2.95±0.37 log base CFU/ml by thermally modified lysozyme. At the end of the 24 h, the count of E. sakasakii in the sample containing lysozyme reached to 16.52±0.07 log base CFU/ml which is 1.2 times than the initial count whereas that containing thermally modified lysozyme had 11.51±0.06 log base CFU/ml which is less than the initial count (13.76±0.43 log base CFU/ml). In the control sample devoid of neither lysozyme nor thermally modified lysozyme, the pathogen grew, reaching a final population of 20.02±0.11 log base CFU/ml. These results indicate that thermally modified lysozyme, was more effective than lysozyme in inhibition of E. sakazakii growth at 4 ºC (p <0.001).

Table 3 and Figure 2 show the effect of lysozyme and thermally modified lysozyme on E. sakasakii count in reconstituted milk formula incubated at 25ºC throughout the experiment and revealed that there was significant difference between lysozyme and thermally modified lysozyme treated samples (p <0.01), lysozyme had no antibacterial effect on E. sakazakii throughout the experiment. While at 3h and 6h thermally modified lysozyme reduced the pathogen count by 9.11±0.32 and 12.99±0.07 log base CFU/ml. At 9h of incubation the count reach approximately the initial count (13.57±0.15 log base CFU/ml), at 24 h of incubation the count reached 22.81±0.06 log base CFU/ml which is 1.7 times than the original count. In the control sample devoid of neither lysozyme nor thermally modified lysozyme, the pathogen grew, reaching a final population of 28.27±0.38 log base CFU/ml. These results indicate that thermally modified lysozyme, was effective in inhibition of E. sakazakii growth at 25 ºC till 6 h of storage.

The Effect of lysozyme and thermally modified lysozyme on E. sakazakii count in reconstituted milk formula incubated at 37ºC throughout the experiment is presented in Table 4 and Figure 3 and revealed that there was significant difference between lysozyme and thermally modified lysozyme treated samples (p <0.01), lysozyme had no antibacterial effect on E. sakazakii throughout the experiment. While at 3h thermally modified lysozyme reduced the pathogen count by 11.65±0.52 log base CFU/ml. At 6h of incubation the count reach approximately the initial count (13.45±0.37 log base CFU/ml), at 24 h of incubation the count reached 22.81±0.33 log base CFU/ml which is 1.7 times than the original count. In the control sample devoid of neither lysozyme nor thermally modified lysozyme, the pathogen grew, reaching a final population of 29.71±0.07 log base CFU/ml. These results indicate that thermally modified lysozyme, was effective in inhibition of E. sakazakii growth at 37 ºC till 3 h of storage.

There were significant differences between lysozyme and thermally modified lysozyme treated samples at different incubation temperatures (p <0.03). There were significant differences between lysozyme treated samples incubated at 4 ºC and lysozyme treated samples incubated at both 25 and 37 ºC, also there were significant differences between thermally modified lysozyme treated samples incubated at 4 ºC and thermally modified lysozyme treated samples incubated at both 25 and 37 ºC (Table 5 and Figure 4). The results indicated that the thermally modified lysozyme had more inhibitory effect on E. sakazakii at 4 ºC than at 25 and 37 ºC.

Heat denaturation of lysozyme resulted in the progressive loss of enzymatic activity, but a greatly improved antimicrobial action towards Gram-negative bacteria through membrane perturbation (Ibrahim, 1998). The possibility to extend the range of lysozyme activity to include Gram-negative bacteria i.e. E. coli, is offered by the thermal and chemical-thermal modification, which leads to the formation of an enzyme preparation with increased content of polymeric forms (Lesnierowski et al., 2004).

The antimicrobial action of lysozyme was due to structural factors. Specific bactericidal domain may be involved in the antimicrobial action of lysozyme (Düring et al., 1999; Ibrahim, 1998 and Ibrahim, 2003).

The inhibitory effect of thermally modified lysozyme on E. sakazakii at 4 ºC was more than at 25and 37 ºC this may be attributed to the long generation time of the pathogen at refrigeration temperature (4.98h) than at room temperature (40 min.) and at 37 ºC (24 min) (Nazarowec-White and Farber, 1997a; Pagotto and Farber, 2009).

The results of this study indicate that despite the fact that formulas are exposed to heat treatment during processing E. sakazakii was still isolated from these products. The combined efforts of public health and regulatory officials, as well as manufacturers, were considered important aspects of the management of risks associated with disease causing E. sakazakii in PIMF, also the uses of thermally modified lysozyme can exert a significant inhibitory activity against this organism in reconstituted milk formula specially when kept at refrigeration temperature.

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References