Open Veterinary Journal, (2023), Vol. 13(1): 108–113

Original Research

10.5455/OVJ.2023.v13.i1.11

Thermal tolerance of Cronobacter sakazakii and Cronobacter pulveris in reconstituted infant milk formula

Aboubaker M. Garbaj¹, Samira A. Farag¹, Jihan A. Sherif¹, Aml F. Lawila², Hanan L. Eshamah¹, Salah M. Azwai³, Fatim T. Gammoudi³, Hesham T. Naas¹, Allaaeddin A. El Salabi⁴ and Ibrahim M. Eldaghayes^3*

¹Department of Food Hygiene and Control, Faculty of Veterinary Medicine, University of Tripoli, Tripoli, Libya

²Food and Drug Control Center, Tripoli, Libya

³Department of Microbiology and Parasitology, Faculty of Veterinary Medicine, University of Tripoli, Tripoli, Libya

⁴Department of Environmental Health, Faculty of Public Health, University of Benghazi, Benghazi, Libya

Submitted: 11/10/2022 Accepted: 25/12/2022 Published: 21/01/2023

*Corresponding Author: Ibrahim Eldaghayes. Department of Microbiology and Parasitology, Faculty of Veterinary Medicine, University of Tripoli, P.O. Box 13662, Tripoli, Libya. Email: ibrahim.eldaghayes [at] vetmed.edu.ly

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Abstract

Background: Cronobacter sspecies are the most significant foodborne pathogen in infant milk formula (IMF). These pathogens have been incriminated in severe forms of neonatal meningitis, sepsis, and necrotizing enterocolitis with a high mortality rate.

Aim: This study was performed to elucidate the effect of heat stress on Cronobacter spp. (C. sakazakii and C. pulveris) in reconstituted IMF (RIMF).

Methods: The reconstituted formula was inoculated with five C. sakazakii isolates and four C. pulveris isolates separately. The nine isolates of Cronobacter spp. were heated in RIMF at 48°C, 52°C, 56°C, 60°C, 64°C, and 66°C. The D- and z-values were determined by using linear regression analysis.

Results: The D˗values of all isolates of C. sakazakii (CS1, CS3, CS4, CS5, and CS6) at 48°C, 52°C, 56°C, 60°C, 64°C, and 66°C were in the ranges 7.29–23.47, 2.77–15.50, 0.62–1.04, 0.62–1.02, 0.62–1.00, 0.62–1.00 minutes, respectively; while, the z˗values extended from 2.50°C to 4.28°C. The D˗ values of C. pulveris isolates (CP1, CP2, CP3, CP4) were in the ranges 7.60–22.32, 1.42–8.45, 0.62–1.08, 0.62–0.78, 0.62–0.78, 0.62–0.79 minutes at 48°C, 52°C, 56°C, 60°C, 64°C, 66°C, respectively and the calculated z-values ranged from 3.33°C to 4.89°C.

Conclusion: This study may contribute to improving the understanding of the behavior of C. sakazakii and C. pulveris isolates in RIMF at various heat stress temperatures and may participate in the effective control of these pathogens in infant food production.

Keywords: Cronobacter sakazakii, Cronobacter pulveris, Thermo-tolerance, D˗value, z˗value, Infant milk formula.

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Introduction

Breast milk is usually the most important source of nutrition for newborns and is essential for survival, immunological development, health, and early growth in life (Baker et al., 2021). Alternatively, non-breast-fed newborns can be fed on commercial powdered infant milk formula (IMF) that provides a nutritious alternative to breast milk (Costa et al., 2020). Infant formula is considered to be a microbiologically high-quality product, however, some microorganisms have been identified in neonatal infections associated with infant formula feeding (Garbaj et al., 2015). Moreover, powdered infant formula (PIF) manufacturing relies on the blending of many soluble substances and additives into dried base powder. This factor may be considered a critical health risk and serve as a main source of foodborne pathogens that transmit to neonates (Yemiş and Delaquis, 2020). Based on some epidemiological studies on the incidence of neonatal infections, Cronobacter spp., particularly C. sakazakii, is the most important foodborne pathogens in PIF (Al-Holy et al., 2010; Yemiş and Delaquis, 2020). Surveillance studies have identified Cronobacter spp. from different environments and foods such as milk powder, cereal formula, various dairy products, dried foods, meat products, spices, vegetables, rice, plants, etc. (Osaili et al., 2009). This ubiquitous microorganism, Gram-negative, non-spore-forming bacilli has been reported to cause septicemia, meningitis, sepsis, and necrotizing enterocolitis in newborns at low infection doses (Chauhan et al., 2020). In addition, C. sakazakii has the ability to withstand and tolerate sever stress conditions for instance high osmotic pressure and extreme dehydration (low water activity) compared to other Cronobacter spp. (Bai et al., 2019; Zhang et al., 2020). Reconstituted infant formula is known to provide an optimal environment for the growth of these pathogens, survive slight heat stress during preparation, and pose a significant potential risk to newborns (Xu et al., 2015). Moreover, some microbiological studies revealed that Cronobacter spp. has the ability to grow rapidly for long periods of time at room temperature in baby bottles (Al-Holy et al., 2009; Azwai et al., 2022). Therefore, World Health Organization (WHO)/FAO suggested that PIF should be reconstituted with water at a temperature of at least 70°C (Zhang et al., 2020). However, this target temperature may be difficult to adjust during home reconstitution of PIF or may not be suitable for direct feeding of neonates. Additionally, the PIF has different reconstituted instructions from different sources which may recommend lukewarm water with an alternative advised temperature (Yang et al., 2015). As a result, serious concerns have been raised about the safety of infant formula during the feeding of neonates worldwide. As C. sakazakii and C. pulveris (recently, Stephan et al. (2014), proposed reclassifying C. pulveris to Franconibacter pulveris) were previously detected in dairy and meat products sold in the Libyan market (Garbaj et al., 2017); therefore, C. sakazakii and pulveris needed to be evaluated for their resistance to heat stress which, is one of the most important factors affecting the behavior of these isolates in reconstituted IMF (RIMF). In contrast, from too many studies on the thermo-tolerance of Cronobacter spp., there is hardly any data available on variability and behavior of single isolate of C. sakazakii in rehydration PIF in countries such as Libya. Therefore, this study was performed to demonstrate the effect of heat stress on Cronobacter spp. (C. sakazakii and C. pulveris isolated from food of animal origin from Libya) in RIMF.

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Materials and Methods

Bacterial strains used in this study

The nine strains of Cronobacter spp. that have been used in this study (Table 1), were obtained from Foodborne Libyan Type Bacterial Collection (FLBC), these strains were isolated from raw milk, powdered (IMF), some dairy products, and meat from different localities in Libya and stored in cryobeads banking system at −80°C (Garbaj et al., 2017). A single bead was streaked with a wire loop under the aseptic condition on Nutrient agar (NA; CM0003, Oxoid, UK) and incubated at 37°C for 24 hours. Cells from single colonies of each strain were streaked on Tryptone Soya agar slants (TSA; Oxoid, UK), incubated at 37°C for 24 hours, and kept refrigerated until they were used.

Inoculum preparation

The bacterial cultures were separately transferred from TSA slants to Brain Heart Infusion broth ((BHI; CM0225, Oxoid, UK). The strains were transferred individually into sterilized test tubes containing 10 ml BHI and incubated at 37°C for 24 hours. Within this stage, the bacteria are considered to be in the early stationary phase, which is known as the most resistant phase to antibiotics.

Preparation of RIMF

IMF was purchased from local supermarkets and pharmacies in Tripoli, Libya. The recommended amount of powder was added and reconstitution of IMF in sterile distilled water was carried out according to the manufacturer’s guideline (two scoops equal 8.6 g per 60 ml of warm water). Ten milliliters (ml) of RIMF were added into sterile test tubes and sterilized at 121°C for 15 minutes to get rid of the background microorganisms.

Thermo-tolerance of Cronobacter spp.

Thermo-tolerance of Cronobacter spp. was tested according to Al-Holy et al. (2009). The reconstituted milk was prepared and inoculated with Cronobacter spp., as follows: IMF was reconstituted according to the manufacturer’s instructions. About 10 ml samples of RIMF were sterilized at 121°C for 15 minutes. The isolate was transferred from TSA slants into (BHI) broth and incubated at 37°C for 24 hours. Ten-fold serial dilution of the BHI culture was performed in buffered peptone water solutions (RM 001˗500G HI media, India) by adding 1 ml from cultured BHI onto 99 ml of buffered peptone water dilution. A portion of 100 µl from each dilution was then aseptically plated (0.1 ml in duplicate) onto (TSA) (Oxoid CM 131) and incubated at 37°C for 24 hours (stationary phase).

Table 1. Cronobacter strains used in this study.

Finally, the dilution that had a microbial load of 10⁸ CFU/ml was used for the inoculation of the prepared RIMF.

Heat treatment was conducted in a water bath at 48°C, 52°C, 56°C, 60°C, 64°C, and 66°C. The tubes were immersed completely in the heated water bath where the temperature was controlled and adjusted at the target temperature. Following each heat treatment, each tube was then removed from the water bath and immersed immediately into ice slush. At each heat treatment, 100 µl from the inoculated IMF was taken in duplicate at different time points: 0, 3, 6, 9, 12, 15, and 18 minutes. Quantification of the colonies was then performed using the overlay method (Al-Holy et al., 2008). The samples were spread-plated on TSA supplemented with 0.1% (w/v) of sodium pyruvate. The plates were then incubated for 2 hours at 37°C and a thin layer (8 ml) of violet red bile agar (HIMIDIA M 581, India) was overlaid onto TSA and the plates were re-incubated for an additional 22 hours at 37°C. Standard regression analysis was performed by log-linear models in Excel (Microsoft 2010) and the D-value was determined by taking the negative reciprocal of the slope. The z-value was calculated using linear regression of the log D-values at six temperatures: 48°C, 52°C, 56°C, 60°C, 64°C, and 66°C.

Statistical analysis

Data were presented as mean ± SD. The decimal reduction time (D-value), the time required at a certain temperature to reduce a specific microbial population, and the z-value (the number of degrees of temperature change necessary to change the D-value) were determined by using Microsoft Excel (log-linear models).

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Results

The microbial quantity of each strain showed relatively similar values, it ranged from 10⁷ to 10⁹ CFU/ ml. Table 2 shows decimal reduction times (D˗value and z˗value) of C. sakazakii and C. pulveris strains inoculated in RIMF. In this study, D˗values were calculated at six different temperatures. Results in Table 2 revealed that the D˗values of all strains of C. sakazakii (CS1, CS3, CS4, CS5, and CS6) at 48°C, 52°C, 56°C, 60°C, 64°C, and 66°C were in the ranges 7.29–23.47, 2.77–15.50, 0.62–1.04, 0.62–1.02, 0.62–1.00, 0.62–1.00 minutes, respectively, while, the z˗ values ranged from 2.50°C to 4.28°C. Results represented in Table 2 also revealed the D˗values of all strains of C. pulveris (CP1, CP2, CP3, CP4) were in the ranges 7.60–22.32, 1.42–8.45, 0.62–1.08, 0.62–0.78, 0.62–0.78, 0.62–0.79 minutes at 48°C, 52°C, 56°C, 60°C, 64°C, and 66°C, respectively, whereas, the z˗ values were ranged from 3.33°C to 4.89°C. Figures 1 and 2 illustrated the decimal reduction time curve (D˗values) of all C. sakazakii and C. pulveris isolates in RIMF at 48°C–66°C. While atypical thermal death time curve of the same isolates of C. sakazakii and C. pulveris in RIMF at 48°C –66°C was shown in Figure 3.

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Discussion

IMF product has been the focus of studies on the inactivation and inhibition of Cronobacter as the primary vehicle of contamination (Muytjens et al., 1983). The thermal and non-thermal electromagnetic radiation effects were thought to be involved in the mechanism of microbial killing (Najdovski et al., 1991). The thermal resistance of Cronobacter spp. is considered a vital parameter likely assisting infection of infant formula. Thus, the extent of thermo-tolerance between different Cronobacter strains was evaluated by determining the thermal resistance rate by calculating the D-value and z-value of each strain. According to our findings, significant differences in thermal resistance were observed between the isolates (Table 2). It has been reported that the D₄₈ value ranged from 7.29 minutes for CS5 to 23.47 minutes for CS1, and the D₅₂ value ranged from 2.77 minutes for CS4 to 15.50 minutes for CS3. This is in agreement with some studies that reported this pathogen had a different and wide range of heat resistance (Edelson-Mammel and Buchanan, 2004; Al-Holy et al., 2009). Moreover, D₆₀ ranged from 0.62 minutes for CS5 to 1.02 minutes for CS4 in this current study, which is in agreement with (Walsh et al., 2011) who found that D₆₀ ranged from 0.10 to 0.73 minutes. However, D₆₀ of Cronobacter isolates was 5.74 minutes which indicates a high heat resistance (Awadallah et al., 2018). This may be due to the examined Cronobacter strains being isolated from animal feces. These results revealed that it is relatively difficult to compare D-values among researchers. This could be attributed to the fact that the bacterial thermo-tolerance can generally be influenced by several factors including physiological conditions, the growing temperature of the inoculum, and heating menstruum (fat, solids, and sugar concentration) besides bacterial recovery method (Nazarowec-White and Farber, 1997). Moreover, these variations in D˗values may be due to differences in the environmental condition of the inspected isolates (Chauhan et al., 2020). The z˗value was also calculated in this study, it ranged from 2.50°C to 4.28°C, which was lower than previously reported for C. sakazakii. Both studies by Edelson-Mammel and Buchanan (2004) and Iversen et al. (2004) found that the z˗values were ranging from 5.6°C to 5.8°C. Nevertheless, Al-Holy et al. (2009) reported much higher z-values, 3.76°C and 10.11°C. Furthermore, the highest z˗ value (17.42°C) was reported for C. sakazakii by (Awadallah et al., 2018). With regard to C. pulveris, the D˗value and z˗value have not been reported to date. As shown in Table 2, the z˗value ranged from 3.33°Cto 4.89°C which was relatively similar to C. sakazakii results. In comparison, D₄₈ ranged from 7.60 to 22.32 minutes while D₅₂ ranged from 1.42 to 8.45 minutes which were lower than that reported for the same D-values of C. sakazakii. On the other hand, the findings of D_56, D_60, D₆₄, and D₆₆ values for C. pulveris strains were correspondingly equivalent to that reported for C. sakazakii. All of these results were in agreement with the WHO which recommends using hot water at more than 70°C to prepare infant formula to reduce the risk of viable Cronobacter cells (Al-Nabulsi et al., 2009).

Table 2. D- and z-values of tested Cronobacter strains.

Fig. 1. Thermal inactivation of C. sakazakii strains (CS1, CS3, CS4, CS5 and CS6) at 48°C, 52°C, 56°C, 60°C, 64°C, and 66°C in RIMF.

Fig. 2. Thermal inactivation of C. pulveris strains (CP1, CP2, CP3 and CP4) at 48°C, 52°C, 56°C, 60°C, 64°C, and 66°C in RIMF.

Fig. 3. Thermal death time curve of strains of C. sakazakii (left) and C. pulveris (right) in RIMF

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Conclusion

In conclusion, IMF has been recognized as the major vehicle of transmission of C. sakazakii especially when prepared under unhygienic conditions. C. sakazakii was previously isolated from infant products sold in the Libyan market. The thermal resistance of Cronobacter spp. is considered a vital parameter assisting infection of infant formula. In this work, the extent of thermos-tolerance amongst dissimilar Cronobacter isolates was evaluated by calculating D-value and z-value for each isolate. The results of this study indicate that a significant disparity in thermal resistance between C. sakazakii isolates is noted. Some C. sakazakii isolates can grow and proliferate at cooling temperatures with time. With regard to C. pulveris, the z˗ value was relatively similar to C. sakazakii results. It is likely that the usage of hot water at more than 70°C is essential to prepare infant formula as WHO recommends. To date, only a few studies considered the growth variation in heat resistance amongst C. sakazakii strains in infant products. Moreover, this is the first study in Libya investigating the thermos-tolerance of Cronobacter species in RIMF. This study may contribute to reducing the risk of C. sakazakii survival during the preparation of infant food.

Authors’ contributions

A.M.G, S.M.A, and S.A.F. planned and designed the study. S.M.A, S.A.F, J.A.S, A.F.L, and F.T.G carried out the laboratory work. All authors A.M.G, S.A.F, S.M.A, J.A.S, A.F.L, H.L.E, F.T.G, H.T.N, A.A.E.S. and I.M.D contributed equally to the analysis and presentation of data in addition to the preparation and revision of the manuscript. All authors have read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

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