Journal of Applied Botany and Food Quality 88, 97 - 101 (2015), DOI:10.5073/JABFQ.2015.088.013 1 Department of Nutrition and Food Technology, Jordan University of Science and Technology, Irbid, Jordan 2 Department of Nutrition and Food Technology, Al-Balqa Applied University, Salt, Jordan 3 Department of Food Science, University of Manitoba, Winnipeg, Manitoba, Canada 4 North Carolina A&T State University, Food Microbiology and Biotechnology Laboratory, Greensboro, NC, United States Inactivation of Cronobacter sakazakii in reconstituted infant milk formula by plant essential oils Anas A. Al-Nabulsi1*, Saddam S. Awaisheh2, Tareq M. Osaili1, Amin N. Olaimat3, Razan J. Rahahaleh2, Fawzi M. Al-Dabbas2, Lina A. Al-Kharabsheh2, Rabin Gyawali4, Salam A. Ibrahim4 (Received September 24, 2014) * Corresponding author Summary This study aimed to screen the in vitro antimicrobial activity of 10 plant essential oils against 4 Cronobacter sakazakii strains, and use these oils or their combination to control C. sakazakii cocktail at low (3 log10 CFU/ml) and high (6 log10 CFU/ml) contamination levels in reconstituted infant milk formula (RIMF). Cinnamon and fir oils were the most inhibitory to C. sakazakii strains with inhibition zone of 32 to 40 mm at 20 μl/disc (the minimum inhibitory concentrations were 0.16 and 0.625 μl/ml, respectively). The addition of each of cinnamon or fir oil at 1 % (v/v) reduced the C. sakazakii numbers in RIMF by 0.7-0.8 log10 CFU/ml when inoculated with high contami- nation level and by 2.5-3.1 log10 CFU/ml when inoculated with low contamination level. However, the combination of cinnamon and fir oils reduced C. sakazakii numbers at both inoculum levels to unde- tectable levels after 3 h of incubation at 37 °C. The results of the current study indicated that a combination of cinnamon and fir oils has a potent antimicrobial activity which may potentially be used to control C. sakazakii in RIMF. Introduction Cronobacter sakazakii is an opportunistic pathogen that may cause bacteremia, sepsis, meningitis and necrotizing enterocolitis, main- ly in neonates, preterm and low birth weight infants, and immu- nocompromised infants and adults with case fatality rate of 10 to 80 % (NAZAROWEC-WHITE and FARBER, 1997a; YAN et al., 2012). C. sakazakii has the ability to adapt several environmental stresses including chilling, heating, drying and osmotic stresses and natural antibiotics. This characteristic enables the organism to survive in foods and environments to cause foodborne illnesses (LEE and JIN, 2008; OSAILI et al., 2008). Although C. sakazakii has been isolated from several food sources including dairy products, meat products, vegetable origin food, water and other foods (CHAP et al., 2009; FRIEDMANN, 2007; SHAKER et al., 2007), the C. sakazakii infection has been linked epidemiologically to the consumption of contaminated powdered infant milk formula (PIMF). C. sakazakii can be inactivated during milk pasteurization; however, post-processing contamination of PIMF is mainly respon- sible for the presence of C. sakazakii in these products. Furthermore, C. sakazakii has the ability to colonize on the surfaces of rehydrated infant milk formula (RIMF) preparation equipment and utensils such as brushes, bottles, and spoons (NAZAROWEC-WHITE and FARBER 1997a, b). The microbiological safety of PIMF is very critical because infants are known to be particularly more vulnerable to foodborne infec- tions (DRUDY et al., 2006). To reduce the risk of C. sakazakii in- fections, Food and Agriculture Organization and World Health Or- ganization (FAO and WHO, 2004) recommended using hot water (≥70 °C) in reconstitution of PIMF before feeding infants. However, the improper storage of RIMF may permit a significant growth of C. sakazakii which has a short lag time and generation time in RIMF (NAZAROWEC-WHITE and FARBER, 1997b). Therefore, incorporation of effective antimicrobials may have the potential to reduce the in- fection risk of C. sakazakii in infants by contaminated RIMF (NAIR et al., 2004). Increasing bacterial resistance to drugs has created problems in the treatment of several diseases. Since there is increased concerns re- garding the safety of synthetic compounds and overuse of antibio- tics as preservatives, interest in the development of effective natu- ral antimicrobials as food preservatives has been increased recently (ABEE et al., 1995; NAIR et al., 2004). Several studies investigated the inhibitory effects of natural antimicrobials, such as animal-, plant-, and microorganism-derived antimicrobials against foodborne pathogens (TAJKARIMI et al., 2010; LEE and JIN, 2008). Natural anti- microbial agents derived from plant sources such as essential oils have been recognized and used for centuries in food preservation. Edible, medicinal and herbal oils are potent antimicrobials against foodborne pathogens such as Listeria monocytogenes, Salmonella Typhimurium, Escherichia coli O157:H7, Shigella dysenteriae, Ba- cillus cereus and Staphylococcus aureus (TAJKARIMI et al., 2010). However, few studies have investigated the inhibitory effects of carvacrol, thymol, eugenol and cinnamic acid (LEE and JIN, 2008) or trans-cinnamaldehyde (AMALARADJOU et al., 2009) against C. sakazakii. Other individual or combined essential oils may have a potential to reduce C. sakazakii in RIMF. Therefore the objectives of the current study were to: i) screen the antimicrobial activity of 10 medicinal plant essential oils against 4 strains of C. sakazakii, ii) determine the minimum inhibitory concentration (MIC) of cinnamon and fir oils against C. sakazakii strains, and iii) use these oils or their combination to control C. sakazakii cocktail at low (3 log10 CFU/ml) and high (6 log10 CFU/ml) contamination levels in RIMF. Materials and methods Preparation of C. sakazakii cultures Four strains of C. sakazakii isolated from infant formula in Jordan were obtained from the bacterial culture collection of the Depart- ment of Nutrition and Food Technology at Jordan University of Sci- ence and Technology: CS1; CS14; CS15 and CS19 (SHAKER et al., 2007; CHAP et al., 2009). Each strain of C. sakazakii was kept in Brain Heart Infusion broth (BHI, Oxoid, Basingstoke, UK) contain- ing 20 % glycerol at -40 °C. The strains were individually maintained on BHI agar slants at 4 °C and transferred bi-weekly to maintain viability. To prepare the working cultures, one loopful of each strain was transferred to 10 ml BHI broth and incubated at 37 °C for 24 h. A volume of 100 μl of the overnight strain cultures was transferred to 10 ml BHI broth and incubated for 20-24 h at 37 °C. A cocktail of C. sakazakii strains was prepared by mixing equal proportions of each of the four strains into a sterile container and diluted with a sterile 0.1 % peptone water (Oxoid, Basingstoke, UK) to obtain the desired inoculum level. 98 A.A. Al-Nabulsi, S.S. Awaisheh, T.M. Osaili, A.N. Olaimat, R.J. Rahahaleh, F.M. Al-Dabbas, L.A. Al-Kharabsheh, R. Gyawali, S.A. Ibrahim Medicinal plants and essential oils extraction The essential oils of 10 different medicinal plants (cinnamon, anise, eucalyptol, chamomile, fenugreek, fennel, fir, pooh, qysoom and rosemary) were obtained from a local extraction facility in Amman, Jordan. Essential oils were extracted by using hydro-distillation pro- cedure. The oils were kept in a sealed dark glass vial at 4 ºC until required. Screening of antimicrobial activity of medicinal plant essential oils Disc-diffusion method was used to determine the antimicrobial activity of the 10 essential oils against 4 C. sakazakii strains. Brief- ly, 20 μl of each essential oil was dispensed onto 6 mm-diameter sterile filter paper discs which were dried by air. Each disc was placed on tryptic soy agar (TSA, Oxoid, Basingstoke, UK) plates which were previously inoculated with approximately 5.0 log10 CFU/ ml of the individual C. sakazakii strains. Plates were incubated at 37 °C for 24 h and the inhibitory zones were measured in millimeter using a caliper. The synergistic activity of essential oils mixture that showed the maximum antibacterial activity (cinnamon and fir oils) was prepared by mixing equal volumes of each essential oil, and then 20 μl of the mixture was applied onto 6 mm-diameter sterile filter paper (AWAISHEH, 2013). Determination of the minimum inhibitory concentration of cin- namon and fir oils against C. sakazakii strains MICs of cinnamon or fir oils were determined using 96-wells Micro- Dilution method. A 100 μl of overnight culture containing 6.0 log10 CFU/ml of each C. sakazakii strain and 100 μl of cinnamon and fir oils dilution were added with 200 μl total volume in each well. A two-fold serial dilution was used to yield concentrations of 0.08 to 100 μl/ml essential oils. Then, the sealed plates were incubated for 24 h at 37 °C and C. sakazakii growth in each well was measured by reading the respective absorbance at 600 nm using a microplate reader (ELx 800, Biotek, Highland Park, VT, USA). Absolute di- methyl sulfoxide (DMSO) containing essential oils added to sterile media and DMSO added to the bacterial suspension were used as negative and positive controls, respectively. MIC of the essential oils is the lowest dilution which inhibits the bacterial growth (AWAISHEH, 2013). Relative antimicrobial activity of cinnamon and fir oils combina- tion to selected antibiotics The inhibitory efficacy of cinnamon and fir oils combination at 1 % (v/v) against C. sakazakii cocktail was compared with selected an- tibiotics using disc-diffusion method as described above. Antibiotic discs (6 mm in diameter) of 5 reference antibiotics including: ami- kacin (30 μg), azithromycin (15 μg), ceftriaxone (30 μg), gentamicin (10 μg), and nalidixic acid (30 μg) were obtained from ARCOMEX medical supplies company (Amman, Jordan). The ratio of the inhibi- tion zone (mm) produced by the cinnamon and fir oils combination and the reference antibiotics was used to express the relative anti- microbial activity of cinnamon and fir oils combination. Inactivation of C. sakazakii in reconstituted infant milk formula model by cinnamon and fir oils or their combination Commercial PIMF was reconstituted according to the manufac- turer’s instructions with sterile distilled water. Under aseptic condi- tions, approximately 7.7 g PIMF was reconstituted with 60 ml water and mixed using a magnetic stirrer at 350 rpm for 25 min at 100 °C until completely dissolved. A cocktail culture of 4 strains C. saka- zakii by mixing equal volume from each strain at 4.0 log or 7.0 log CFU/ml was inoculated into 10 ml of RIMF (control) or RIMF con- taining either 1 % (v/v) of cinnamon oil, fir oil, or cinnamon and fir oils mixture. The samples were incubated at 37 °C for 6 h and the C. sakazakii numbers at 0, 3 and 6 h were carried out as described by Osaili et al. (2010) with minor modification. A 100 μl of each sample was spread on plates of Violet Red Bile Agar (VRBA, Ox- oid, Basingstoke, UK) supplemented with 2 % glucose which were incubated at 37 °C for 24 h. Statistical analysis The experiments were replicated 4 times using two samples per ex- periment (N=8). All data represents the mean value ± standard devia- tion (SD). One-way ANOVA and multiple range tests were used to evaluate differences among means using the least significance differ- ence (LSD) test at a significance level of P < 0.05. Results and discussion Antimicrobial activity of 10 medicinal plant essential oils The antimicrobial activities of essential oils obtained from 10 dif- ferent plant species against 4 C. sakazakii strains were determined (Tab. 1). These plants showed to have some antimicrobial proper- ties against wide range of foodborne pathogens (AWAISHEH, 2013). The average zone of inhibition with 20 μl of essential oils ranged from 0.0 to 40.0 mm. Cinnamon and fir oils were the strongest anti- microbials against tested C. sakazakii strains with a range of inhibi- tion zone of 32 to 40 mm. However, chamomile and rosemary oils showed weak antimicrobial activities against C. sakazakii strains with inhibition zone range of 4.5 to 6.0 and 3.0 to 8.0, respectively. While other plant (anise, eucalyptol, fennel, fenugreek, pooh, and qysoom) essential oils did not exhibit antimicrobial activity against tested C. sakazakii strains. AL-NABULSI et al. (2009) also found that herbal infant teas such as chamomile and fennel supported growth of C. sakazakii at 37 and 21 °C. The antimicrobial activity tested essential oils (except cinnamon oil) in the present study against C. sakazakii strains Tab. 1: Antimicrobial activity of 10 medicinal plant essential oils (20 μl/disc) against 4 C. sakazakii strains using disc diffusion method at 37 °C. Inhibition zone diameter (mm) 1, 2 Anise Cinnamon Chamomile Eucalyptol Fennel Fenugreek Fir Pooh Rosemary Qysoom C. sakazakii CS1 0.0±0.0c 32.0±3.1a 5.0±0.2b 0.0±0.0c 0.2±0.1c 0.2±0.1c 34.0±2.1a 2.0±0.1c 3.0±0.2bc 2.0±0.1c C. sakazakii CS14 1.0±0.1c 35.0±2.9a 5.0±0.1b 0.2 ± 0.1c 0.5±0.1c 1.0±0.1c 32.0±2.1a 1.5±0.1c 3.0±0.2bc 1.5±0.1c C. sakazakii CS15 0.5±0.1c 40.0±3.4a 4.5±0.2b 0.5 ± 0.1c 1.0±0.1c 0.0±0.0c 40.0±1.8a 1.0±0.1c 4.5±1.0bc 1.0±0.1c C. sakazakii CS19 1.0 ±0.1c 38.0±2.1a 6.0±0.2b 0.5 ± 0.1c 1.0±0.1c 1.0±0.1c 36.0±2.4a 1.0±0.1c 8.0±0.4b 1.0±0.1c 1 Values are mean of 4 replicates ± standard deviation 2 Values with different letters in the same row are significantly different (P < 0.05). Control of C. sakazakii in infant milk formula 99 C. sakazakii have not been investigated as they did not show inhibi- tory effect against C. sakazakii strains. However, several studies reported their inhibitory activity against other foodborne pathogens (BAĞCI and DIĞRAK, 1996; KON and RAI, 2012). The findings of the current study are consistent with those of PRABUSEENIVASAN et al. (2006) and KON and RAI (2012) who found that cinnamon oil had the strongest inhibitory activity among 21 to 35 plant essential oils against Gram-negative and Gram-positive bacteria. BAĞCI and DIĞRAK (1996) also found that essential oil from different species of fir had antimicrobial activity against Bacillus spp., Pseudomo- nas aeruginosa, L. monocytogenes, Klebsiella pneumoniae, Enter- obacter aerogenes and S. aureus. Minimum inhibitory concentration of cinnamon and fir oils against C. sakazakii strains Based on the results of disc diffusion method for screening of anti- microbial activity of 10 plant essential oils against C. sakazakii strains, cinnamon and fir oils showed the highest zone of inhibition and these were selected to determine their MIC values against C. sakazakii strains (Tab. 2). The MICs of cinnamon and fir oils were 0.16 and 0.62 μl/ml, respectively, against all tested C. sakazakii strains. The MICs of essential oils such as carvacrol, thymol, eugenol and cinnamic acid was found to be 1.25, 1.25, 5.0 and >5.0 mmol/L, respectively against 2 C. sakazakii strains (LEE and JIN, 2008). The results of current study are similar to those reported by GHOSH et al. (2013) who found that MIC of cinnamaldehyde was 0.263 mg/ml against C. sakazakii. Similarly, KON and RAI (2012) found that MIC of cinnamon oil was 0.2 mg/ml against S. aureus. The MIC of fir oil in the current study was slightly lower than those reported by OH et al. (2007) who found that the MICs of fir oil against E. coli and Staphylococcus epidermidis were 2.05 and 3.81 mg/ml, respectively. Relative antimicrobial activity of cinnamon and fir oils combina- tion to selected antibiotics The effectiveness of cinnamon and fir oils combination on inhibition of C. sakazakii cocktail was compared to the standard antibiotics (Tab. 3). The cinnamon and fir oils combination showed higher anti- microbial activity against C. sakazakii than the reference antibio- tics. The combination of cinnamon and fir oils significantly inhibited the growth of C. sakazakii cocktail by 38.5 mm on plates. However, ceftriaxone had the strongest inhibitory effect (20.5 mm) among antibiotics tested against C. sakazakii cocktail, while gentamicin showed the weakest activity (8.0 mm). The relative antimicrobial ac- tivity of cinnamon and fir oils combination to gentamicin, nalidixic acid, amikacin, azithromycin and ceftriaxone were 482 %, 412 %, 405 %, 385 % and 188 %, respectively. Although the inhibitory effect of cinnamon and fir oil combinations against C. sakazakii was ob- vious, it should be noted that the concentration of the oil mixtures (20 μl, approximately 20 mg) was greater than the concentrations of the standard reference antibiotics (10 to 30 μg). Previous studies have demonstrated the antimicrobial resistance of C. sakazakii to the antibiotics (AL-NABULSI et al., 2011; FARAJNIA et al., 2009; SAAED and MUSSALAM, 2011). Therefore, using natural antimicrobial agents such as cinnamon and fir oils may be used as alternative to overcome emerging of antibiotics resistance thus reducing the risk associated with C. sakazakii infection. Inactivation of C. sakazakii in RIMF by cinnamon and fir oils or their combination The inhibitory effects of cinnamon and fir oils or their combination against a 4 strains C. sakazakii cocktail at high (6.0 log10 CFU/ml) or low (3.0 log10 CFU/ml) inocula in RIMF during 6 h of incubation at 37 °C was investigated (Tab. 4). In the control samples (without essential oils), C. sakazakii gradually increased from 3.1 and 6.1 log10 CFU/ml to reach 6.3 and 8.4 log10 CFU/ml, respectively after 6 h at 37 °C. The addition of 1 % cinnamon or fir oils alone reduce the viability of C. sakazakii with low inoculum level by 3.1 and 2.5 log10 reduction of C. sakazakii, respectively, compared to the control. Similarly, with higher initial inoculum level (6.0 log10 CFU/ mL), cinnamon or fir oils reduced the numbers of C. sakazakii by 0.7 and 0.8 log10 CFU/ml, respectively, after 6 h. However, the C. sakazakii cells were not detected when RIMF incorporated with 1 % of cinnamon and fir oils mixture after 3 h of incubation at both inoculum levels. This demonstrates that the combination of cinna- mon and fir oils could be used as a practical approach to reduce the risk of C. sakazakii in RIMF and to improve the safety of these con- sumable products. The antimicrobial effect of cinnamon oil has been well studied against E. coli O157:H7, Salmonella, and L. monocyto- genes (OUSSALAH et al., 2007). But there are still limited studies on C. sakazakii strain Tab. 2: Minimum inhibitory concentration (MIC) values of cinnamon and fir oils against 4 C. sakazakii strains at 37 °C. MIC (μl/ml)1 Cinnamon Fir C. sakazakii CS1 0.160 0.625 C. sakazakii CS14 0.160 0.625 C. sakazakii CS15 0.160 0.625 C. sakazakii CS19 0.160 0.625 1 Values are mean of 4 replicates Tab. 3: Relative antimicrobial activity for combination of cinnamon and fir oils (20 μl/disc) to selected antibiotics against a 4 strains C. sakazakii cocktail using disc diffusion method at 37 °C. C. sakazakii Cocktail Reference antibiotics (concentration (μg/disc))1 Amikacin Azithromycin Ceftriaxone Gentamicin Nalidixic acid (30 μg) (15 μg) (30 μg) (10 μg) (30 μg) Inhibition zone diameter 38.5±3.5a 9.5±1.5c 10.0±2.1c 20.5±2.2b 8.0±1.5c 9.0±1.4c (mm) 2, 3 Relative antimicrobial 100 % 405 % 385 % 188 % 482 % 412 % Activity 4 1 Concentration of the oil mixtures is greater than concentrations of the standard reference antibiotics. 2 Results are means of 4 replicates ± standard deviation. 3 Values with different letters in the same row are significantly different (P < 0.05). 4 Relative antimicrobial activity of cinnamon and fir oils mixture = (inhibition zones of cinnamon and fir oils mixture ÷ inhibition zones of antibiotic) x 100 % Cinnamon and fir oils mixture (20 μl/disc)1 100 A.A. Al-Nabulsi, S.S. Awaisheh, T.M. Osaili, A.N. Olaimat, R.J. Rahahaleh, F.M. Al-Dabbas, L.A. Al-Kharabsheh, R. Gyawali, S.A. Ibrahim essential oils against C. sakazakii in RIMF. The results of current study are similar to those reported by AMALARADJOU et al. (2009) who found that 0.5 % trans-cinnamaldehyde reduced the C. saka- zakii numbers to undetectable levels (> 6 log10 CFU/ml reduction) by 4 h at 37 or 23 °C and by 10 h at 8 or 4 °C). It has been reported that mechanisms of cinnamaldehyde (the major component of cinna- mon oil) action is related to inhibition of different enzymes involved in cytokinesis, action as an ATPase inhibitor and/or disturb the cell membrane (HYLDGAARD et al., 2012). KIM et al. (2009) also reported that muscadine seed extracts reduced C. sakazakii numbers (6 log10 CFU/ml) to undetectable levels by 1 h at 37 °C. OSAILI et al. (2009) found that C. sakazakii had lower growth in wheat-based infant follow-on formulas reconstituted with grape or apple juices com- pared to those reconstituted with water or milk 25 and 37 °C. The potent antimicrobial activity of these plant compounds is attributed due to the presence of several phenolic compounds, organic acids, and variety of active components in essential oils (KIM et al., 2009; AMALARADJOU et al., 2009). The phenolic compounds may cause bacterial membrane damage and disrupt the cell wall peptidoglycan. Also, the hydroxyl group in phenolic compounds may bind the ac- tive sites of enzymes and change their substrate affinity. Moreover, the lipid solubility of phenolic compounds and their degree of steric hindrance may also contribute to their overall antimicrobial activity (CEYLAN and FUNG, 2004). Conclusions Among 10 plant essential oils, cinnamon and fir oils showed the strongest antimicrobial activity against C. sakazakii strains with a range of inhibition zone of 32 to 40 mm. The relative antimicrobial activity (inhibition zone ratio) of 1 % cinnamon and fir oils mix- ture compared to amikacin, azithromycin, ceftriaxone, gentamicin and nalidixic acid ranged from 188 % to 482 % against C. sakazakii cocktail. Cinnamon and fir oils combination at 1 % (v/v) eliminated the C. sakazakii numbers (> 6 log10 CFU/ml reduction) by 3 h at 37 °C. FAO/WHO (2004) recommended that RIMF should be stored at room temperature for less than 2 h. It is evident from the results of this study that combination of cinnamon and fir oils caused a strong and rapid antimicrobial effect against C. sakazakii, therefore cinnamon and fir oils combination may have a potential to control C. sakazakii in RIMF. However, effect of cinnamon and fir oils on the sensory attributes of RIMF is required. References ABEE, T., KROCKEL, L., HILL, C., 1995: Bacteriocins: modes of action and potentials in food preservation and control of food poisoning. Int. J. Food Microbiol. 28, 169-185. AL-NABULSI, A.A., OSAILI, T.M., ZAIN ELABEDEEN, N.A., JARADAT, Z.W., SHAKER, R.R., KHEIRALLAH, K.A., TARAZI, Y.H., HOLLEY, R.A., 2011: Impact of environmental stress desiccation, acidity, alkalinity, heat or cold on antibiotic susceptibility of Cronobacter sakazakii. Int. J. Food Microbiol. 146, 137-143. AL-NABULSI, A.A., OSAILI, T.M., SHAKER, R.R., OLAIMAT, A.N., AYYASH, M.M., HOLLEY, R.A., 2009: Survival of Cronobacter species in recon- stituted herbal infant teas and their sensitivity to bovine lactoferrin. J. Food Sci. 74, 479-484. AMALARADJOU, M.A., HOAGLAND, T.A., VENKITANARAYANAN, K., 2009: Inactivation of Enterobacter sakazakii in reconstituted infant formula by trans-cinnamaldehyde. Int. J. Food Microbiol. 129, 146-149. AWAISHEH, S.S., 2013: Efficacy of Fir and Qysoom essential oils, alone and in combination, in controlling Listeria monocytogenes in vitro and in RTE meat products model. Food Control 34, 657-661. BAĞCI, E., DIĞRAK, M., 1996: Antimicrobial activity of essential oils of some Abies (Fir) species from Turkey. Flav. Fragr. J. 11, 251-256. CEYLAN, E., FUNG, D.Y.C., 2004: Antimicrobial activity of spices. J. Rapid Meth. Autom. Microbiol. 12, 1-55. CHAP, J., JACKSON, P., SIQUEIRA, R., GASPAR, N., QUINTAS, C., PARK, J., OSAILI, T., SHAKER, R., JARADAT, Z., HARTANTYO, S.H.P., ABDULLAH SANI, N., ESTUNINGSIH, S., FORSYTHE, S.J., 2009: International survey of Cronobacter sakazakii and other Cronobacter spp. in follow up for- mulas and infant foods. Int. J. Food Microbiol. 136, 185-188. DRUDY, D., MULLANE, N.R., QUINN, T., WALL, P.G., FANNING, S., 2006: Enterobacter sakazakii: an emerging pathogen in powdered infant for- mula. Clin. Infect. Dis. 42, 996-1002. FARAJNIA, S., ALIKHANI, M.Y., GHOTASLOU, R., NAGHILI, B., NAKHLBAND, A., 2009: Causative agents and antimicrobial susceptibilities of urinary tract infections in the northwest of Iran. Int. J. Infect. Dis. 13, 140-144. FOOD AND AGRICULTURE ORGANIZATION/WORLD HEALTHORGANIZATION, 2004: Joint FAO/WHO workshop on Enterbacter sakazakii and other microorganisms in powdered infant formula, Geneva, 2.-5. Feb. 2004. FRIEDEMANN, M., 2007: Enterobacter sakazakii in food and beverages (other than infant formula and milk powder). Int. J. Food Microbiol. 116, 1-10. GHOSH, I.N., PATIL, S.D., SHARMA, T.K., SRIVASTAVA, S.K., PATHANIA, R., NAVANI, N.K., 2013: Synergistic action of cinnamaldehyde with silver nanoparticles against spore-forming bacteria: a case for judicious use of silver nanoparticles for antibacterial applications. Int. J. Nanomedicine 8, 4721-4731. HYLDGAARD, M., MYGIND, T., MEYER, R.L., 2012: Essential oils in food preservation: mode of action, synergies and interactions with food matrix components. Front. Microbiol. 3(12), 1-24. KIM, T.J., SILVA, J.L., WENG, W.L., CHEN, W.W., CORBITT, M., JUNG, Y.S., 2009: Inactivation of Enterobacter sakazakii by water-soluble musca- dine seed extracts. Int. J. Food Microbiol. 129, 295-299. KON, K., RAI, M., 2012: Antibacterial activity of Thymus vulgaris essential oil alone and in combination with other essential oils. Nus. Biosci. 4, Tab. 4: C. sakazakii numbers (log10 CFU/ml) in RIMF in the presence of 1% (v/v) of cinnamon oil, fir oil, or their combination at 37 °C for 6 h.1, 2 Treatments C. sakazakii numbers (log10 CFU/ml) at low level C. sakazakii numbers at (log10 CFU/ml) at high level 0 h 3 h 6 h 0 h 3 h 6 h Control 3.08±0.22a 4.70±0.50a 6.26±0.40a 6.08±0.34a 7.11±0.61a 8.37±0.43a Cinnamon oil 3.04±0.25a 3.00±0.19b 3.18±0.25b 6.04±0.45a 6.07±0.55b 7.67±0.51b Fir oil 3.08±0.15a 3.18±0.23b 3.78±0.21b 6.04±0.50a 6.20±0.45a 7.56±0.66b Cinnamon and fir oils Mixture 3.05±0.12a ND ND 6.08±0.52a ND ND 1 Values are means of 4 replicates ± standard deviation. 2 Values with different letters in the same column are significantly different (P < 0.05). ND = Not detected (Detection level was < 1 log10 CFU/ml) Control of C. sakazakii in infant milk formula 101 50-56. LEE, S.Y., JIN, H.H., 2008: Inhibitory activity of natural antimicrobial com- pounds alone or in combination with nisin against Enterobacter saka- zakii. Lett. Appl. Microbiol. 47, 315-321. NAIR, M.K.M., JOY, J., VENKITANARAYANAN, K., 2004: Inactivation of Enterobacter sakazakii in reconstituted infant formula by monocaprylin. J. Food Prot. 67, 2815-2819. NAZAROWEC-WHITE, M., FARBER, J.M., 1997a: Enterobacter sakazakii: a review. Int. J. Food Microbiol. 34, 103-113. NAZAROWEC-WHITE, M., FARBER, J.M., 1997b: Incidence, survival, and growth of Enterobacter sakazakii in infant formula. J. Food Prot. 60, 226-230. OH, H.J., AHN, H.M., SO, K.H., KIM, S.S., YUN, P.Y., JEON, G.L., RIU, K.Z., 2007: Chemical and antimicrobial properties of essential oils from three coniferous trees Abies koreana, Cryptomeria japonica, and Torreya nu- cifera. J. Appl. Biol. Chem. 50, 164-169. OSAILI, T.M., SHAKER, R.R., AYYASH, M.M., AL-NABULSI, A.A., FORSYTHE, S.J., 2009: Survival and growth of Cronobacter species (Enterobacter sakazakii) in wheat-based infant follow-on formulas. Lett. Appl. Micro- biol. 48, 408-412. OSAILI, T.M., AL-NABULSI, A.A., SHAKER, R.R., AYYASH, M.M., OLAIMAT, A.N., ABU AL-HASAN, A.S., QADORA, K.M., HOLLEY, R.A., 2008: Ef- fects of extended dry storage in powdered infant milk formula on suscep- tibility of Enterobacter sakazakii to hot water or ionizing irradiation. J. Food Prot. 71, 934-939. OSAILI, T.M., AL-NABULSI, A.A., SHAKER, R.R., AL-HOLY, M.M., AL- HADDAQ, M.S., OLAIMAT, A.N., AYYASH, M.M., AL TA’ANI, M.K., FORSYTHE, S.J., 2010: Efficacy of the thin agar layer method for the recovery of stressed Cronobacter spp. (Enterobacter sakazakii). J. Food Prot. 73, 1913-1918. OUSSALAH, M., CAILLET, S., SAUCIER, L., LACROIX, M., 2007: Inhibitory effects of selected plant essential oils on the growth of four pathogenic bacteria: E. coli O157:H7, Salmonella typhimurium, Staphylococcus au- reus and Listeria monocytogenes. Food Control 18, 414-420. PRABUSEENIVASAN, S., JAYAKUMAR, M., IGNACIMUTHU, S., 2006: In vitro antibacterial activity of some plant essential oils. BMC Complem. Al- tern. Med. 6, 39. SAEED, H.A., MUSALLAM, R.M., 2011: First report on Enterobacter saka- zakii from Sudanese patients. Afr. J. Microbiol. Res. 5, 2374-2379. SHAKER, R., OSAILI, T., AL-OMARY, W., JARADAT, Z., AL-ZUBY, M., 2007: Isolation of Enterobacter sakazakii and other Enterobacter sp. from food and food production environments. Food Control 18, 1241-1245. TAJKARIMI, M.M., IBRAHIM, S.A., CLIVER, D.O., 2010: Antimicrobial herb and spice compounds in food. Food Control 21, 1199-1218. YAN, Q.Q., CONDELL, O., POWER, K., BUTLER, F., TALL, B.D., FANNING, S., 2012: Cronobacter species (formerly known as Enterobacter sakazakii) in powdered infant formula: a review of our current understanding of the biology of this bacterium. J. Appl. Microbiol. 113, 1-15. Address of the corresponding author: Anas A. Al-Nabulsi, Department of Nutrition and Food Technology, Jordan University of Science and Technology, Irbid, Jordan E-mail: anas_nabulsi@just.edu.jo © The Author(s) 2015. This is an Open Access article distributed under the terms of the Creative Commons Attribution Share-Alike License (http://creative- commons.org/licenses/by-sa/4.0/).