Inactivation of Cronobacter sakazakii by ultrasonic waves under pressure in buffer and foods

The objective of this research was to characterize the resistance of Cronobacter sakazakii to ultrasonic waves under pressure (manosonication, MS). The D_MS value (decimal reduction time value) of C. sakazakii in standard conditions (35 °C, 117 μm, 200 kPa, citrate-phosphate buffer pH 7.0) was 0.41 min. This value was higher than that of Yersinia enterocolitica (D_MS = 0.19 min) and lower than those of Salmonella enterica serovar Enteritidis (D_MS = 0.61 min), Listeria monocytogenes (D_MS = 0.86 min), and Enterococcus faecium (D_MS = 1.2 min). Strain studied (ATCC 29544, NCTC 8155, 9238, and 9529), growth temperature (10, 20, 30, and 37 °C), and pH of the treatment media (4.0, 5.0, 6.0, and 7.0) did not significantly change C. sakazakii MS resistance. Conversely, entry into stationary growth phase, decreasing water activity of the treatment media (0.98, 0.96, and 0.94), and treatment in food products (apple and orange juices, chicken and vegetable soups, and rehydrated powdered milk) resulted in up to a 1.6-, 3.9-, and 2.5-fold maximum change in D_MS values, respectively. Whereas an exponential relationship between the amplitude of ultrasonic waves and D_MS values was found, the relationship between static pressure and D_MS values was better described by a quadratic equation. The energy transferred into the medium determined the lethality of the ultrasonic waves regardless of the combination of pressure (0, 50, 100, 200 and 300 kPa) and amplitude (34, 62, 90, 117 and 145 μm) applied. There was an exponential relationship between D_MS values and the power input: an increase of 134 W increased the inactivation rate ten times regardless of the treatment medium. No C. sakazakii cells with sublethally injured cytoplasmic membrane or with sublethal oxidative damage occurred after MS treatments, but the results indicated that damage to the outer membrane preceded microbial death.     

Environmental factors influencing the inactivation of Cronobacter sakazakii by high hydrostatic pressure

The effect of High Hydrostatic Pressure (HHP) on the survival of Cronobacter sakazakii was investigated. Deviations from linearity were found on the survival curves and the Mafart equation accurately described the kinetics of inactivation. Comparisons between strains and treatments were made based on the time needed for a 5-log₁₀ reduction in viable count. The ability of C. sakazakii to tolerate high pressure was strain-dependent with a 26-fold difference in resistance among four strains tested. Pressure resistance was greatest in the stationary growth phase and at the highest growth temperatures tested (30 and 37 °C). Cells treated in neutral pH buffer were 5-fold more resistant than those treated at pH 4.0, and 8-fold more sensitive than those treated in buffer with sucrose added (a_w = 0.98). Pressure resistance data obtained in buffer at the appropriate pH adequately estimated the resistance of C. sakazakii in chicken and vegetables soups. In contrast, a significant protective effect against high pressure was conferred by rehydrated powdered milk. As expected, treatment efficacy improved as pressure increased. z values of 112, 136 and 156 MPa were obtained for pH 4.0, pH 7.0 and a_w = 0.98 buffers, respectively. Cells with sublethal injury to their outer and cytoplasmic membranes were detected after HHP under all the conditions tested. The lower resistance of C. sakazakii cells when treated in media of pH 4.0 seemed to be due to a decreased barostability of the bacterial envelopes. Conversely, the higher resistance displayed in media of reduced water activity may relate to a higher stability of bacterial envelopes.     

Sub-lethal heat treatment affects the tolerance of Cronobacter sakazakii BCRC 13988 to various organic acids, simulated gastric juice and bile solution

Cronobacter spp., formerly Enterobacter sakazakii, are considered emerging opportunistic pathogens and the etiological agent of life-threatening bacterial infections in infants. In the present study, C. sakazakii BCRC 13988 was first subjected to sub-lethal heat treatment at 47 °C for 15 min. Survival rates of the heat-shocked and non-shocked C. sakazakii cells in phosphate buffer solution (PBS, pH 4.0) containing organic acids (e.g. acetic, propionic, citric, lactic or tartaric acid), simulated gastric juice (pH 2.0-4.0), and bile solution (0.5 and 2.0%) were examined. Results revealed that sub-lethal heat treatment enhanced the test organism's tolerance to organic acids, although the extent of increased acid tolerance varied with the organic acid examined. Compared with the control cells, heat-shocked C. sakazakii cells after 120-min of exposure, exhibited the largest increase in tolerance in the lactic acid-containing PBS. Furthermore, although heat shock did not affect the behavior of C. sakazakii in bile solution, it increased the test organism's survival when exposed to simulated gastric juice with a pH of 3.0-4.0.     

Nonthermal Inactivation of Cronobacter sakazakii in Infant Formula Milk: A Review

Up-to-date, nonthermal technologies and combinations of them, in accordance with the “hurdle technology” concept, are being applied by different research groups in response to calls by the International Food and Human Health Organizations (ESPGHAN, 2004; FAO/WHO, 2006, 2008) for alternatives to thermal control of Cronobacter sakazakii in reconstituted powdered infant formula milk. This review highlights (i) current knowledge on the application of nonthermal technologies to control C. sakazakii in infant formula milk and (ii) the importance of the application of nonthermal technologies for the control of C. sakazakii as part of the development of strategies in the context of improving food safety and quality of this product.     

Impact of environmental stress desiccation, acidity, alkalinity, heat or cold on antibiotic susceptibility of Cronobacter sakazakii

Cronobacter sakazakii is an emerging foodborne pathogen that has been implicated in severe forms of meningitis, septicemia or necrotizing colitis in pre-term neonates. Although illness outbreaks (primarily associated with powdered infant formula, PIF) caused by this pathogen are rare, the case-fatality rate may reach 50%. Successful treatment of C. sakazakii infection is reliant upon clinical use of antibiotics (AB) such as ampicillin. Recent reports showed increased resistance of C. sakazakii to broad-spectrum antibiotics. The objective of this study was to evaluate the effect of extreme pH (3.5 for 30 min or 11.25 for 5 min), cold (4 °C for 24 h), heat (55 °C for 5 min), and desiccation (cells were dried at 40 °C for 2 h and held at 21 °C for 4 d) stresses on susceptibility of five isolated strains of C. sakazakii to streptomycin, gentamicin, kanamycin, neomycin, tetracycline, doxycycline, tilmicosin, florfenicol, ampicillin, amoxicillin, vancomycin, ciprofloxacin and enrofloxacin. All unstressed strains of C. sakazakii were sensitive to streptomycin, gentamycin, kanamycin, ciprofloxacin, enrofloxacin, ampicillin and amoxicillin, but were moderately resistant or resistant to the rest. Exposing cells to alkaline or acidic stress did not change their sensitivity toward streptomycin, gentamycin, kanamycin or ciprofloxacin, but their resistance toward the other AB was increased. Cells stressed by desiccation showed increased sensitivity toward streptomycin, gentamicin, kanamycin, ciprofloxacin, enrofloxacin, ampicillin and doxycycline, but showed resistance toward the others. Cold-stressed cells were more sensitive to streptomycin, gentamicin, kanamycin, and ciprofloxacin compared with heat-stressed cells, but both heat and cold-stressed cells showed increased resistance toward all the other AB. Results obtained will help in understanding the effect of environmental stresses during processing on C. sakazakii susceptibility to AB.     

蓝光对阪崎肠杆菌的杀菌及机制研究

本文以食源性致病菌阪崎肠杆菌为对象,研究了医学领域抗细菌感染蓝光的杀菌作用,并首次对其杀菌机制进行了探究。结果表明,当蓝光剂量大于30 J/cm~2时,对阪崎肠杆菌具有显著杀菌作用,照射剂量达240 J/cm~2时,杀菌率超过8 log 10 CFU。亚致死剂量(0～30 J/cm~2)蓝光照射下,单线态氧(~1O_2)探针SOSG开始出现绿色荧光信号,显示细胞内1O2涌现并造成细胞外壁微小孔洞;活性氧物质(ROS)也迅速产生并逐渐增大至5 a.u.;随后脂质氧化标志物丙二醛(MDA)逐渐增加,显示细胞内产生脂质氧化。上述胞内物质变化导致细胞外膜受损,30 J/cm~2蓝光下细胞外膜渗透性增加了48.96%;此外脂肪酰基谱测定揭示,不饱和脂肪酸C18:2,C16:1和C18:1含量减少并逐渐消失,很可能是细胞外膜损伤的重要原因之一。本研究确证了蓝光下细菌胞内~1O_2、ROS及脂质氧化物的动态变化,更重要是的,揭示了细胞外膜损伤是蓝光杀菌的重要方式之一,因为细菌脂质尤其是不饱和脂肪酸是蓝光杀菌重要靶标,本研究将有助于蓝光杀菌机制的深入探究。     

Individual cell lag time distributions of Cronobacter (Enterobacter sakazakii) and impact of pooling samples on its detection in powdered infant formula

Cells of six strains of Cronobacter were subjected to dry stress and stored for 2.5 months at ambient temperature. The individual cell lag time distributions of recovered cells were characterized at 25 °C and 37 °C in non-selective broth. The individual cell lag times were deduced from the times taken by cultures from individual cells to reach an optical density threshold. In parallel, growth curves for each strain at high contamination levels were determined in the same growth conditions. In general, the extreme value type II distribution with a shape parameter fixed to 5 (EVIIb) was the most effective at describing the 12 observed distributions of individual cell lag times. Recently, a model for characterizing individual cell lag time distribution from population growth parameters was developed for other food-borne pathogenic bacteria such as Listeria monocytogenes. We confirmed this model’s applicability to Cronobacter by comparing the mean and the standard deviation of individual cell lag times to populational lag times observed with high initial concentration experiments. We also validated the model in realistic conditions by studying growth in powdered infant formula decimally diluted in Buffered Peptone Water, which represents the first enrichment step of the standard detection method for Cronobacter. Individual lag times and the pooling of samples significantly affect detection performances.     

奶粉中阪崎克罗诺杆菌生长与失活建模的研究进展

奶粉中含有丰富的营养物质,极易被微生物污染,而阪崎克罗诺杆菌是污染奶粉的主要病原菌之一。本文对国内外阪崎克罗诺杆菌生长、失活建模的研究进行综述,生长模型的建立为病原微生物的监测提供有效信息,失活模型的建立能反映传统、新型微生物控制技术和历史应激对阪崎克罗诺杆菌的失活作用效果。最后,针对目前阪崎克罗诺杆菌在预测建模研究中存在的问题进行探讨,并对阪崎克罗诺杆菌的研究方向提出建议,以期为今后的研究提供参考。     

The presence of endotoxin in powdered infant formula milk and the influence of endotoxin and Enterobacter sakazakii on bacterial translocation in the infant rat

Lipopolysaccharide (LPS) is a heat stable endotoxin that persists during the processing of powdered infant formula milk (IFM). Upon ingestion it may increase the permeability of the neonatal intestinal epithelium and consequently bacterial translocation from the gut. To determine the level of endotoxin present in IFM, 75 samples were collected from seven countries (representing 31 brands) and analysed for endotoxin using the kinetic colorimetric Limulus amoebocyte lysate (LAL) assay. The endotoxin levels ranged from 40 to 5.5 x 10(4) endotoxin units (EU) per gram and did not correlate with the number of viable bacteria. The neonate rat model was used to address the risk of endotoxin-induced bacterial translocation from the gut. Purified Escherichia coli LPS was administered to rat pups followed by inoculation with Enterobacter sakazakii ATCC 12868. Bacteria were isolated from the mesentery, spleen, blood and cerebral spinal fluid (CSF) of endotoxin-treated rats due to enhanced gut and blood brain barrier penetration. Histological analysis of the colon showed marked distension of the mucosal and muscular layers. It is plausible that the risk of neonatal bacteraemia and endotoxemia, especially in neonates with immature innate immune systems, may be raised due to ingestion of IFM with high endotoxin levels.     

Validation of radio-frequency dielectric heating system for destruction of Cronobacter sakazakii and Salmonella species in nonfat dry milk

Cronobacter sakazakii and Salmonella species have been associated with human illnesses from consumption of contaminated nonfat dry milk (NDM), a key ingredient in powdered infant formula and many other foods. Cronobacter sakazakii and Salmonella spp. can survive the spray-drying process if milk is contaminated after pasteurization, and the dried product can be contaminated from environmental sources. Compared with conventional heating, radio-frequency dielectric heating (RFDH) is a faster and more uniform process for heating low-moisture foods. The objective of this study was to design an RFDH process to achieve target destruction (log reductions) of C. sakazakii and Salmonella spp. The thermal destruction (decimal reduction time; D-value) of C. sakazakii and Salmonella spp. in NDM (high-heat, HH; and low-heat, LH) was determined at 75, 80, 85, or 90°C using a thermal-death-time (TDT) disk method, and the z-values (the temperature increase required to obtain a decimal reduction of the D-value) were calculated. Time and temperature requirements to achieve specific destruction of the pathogens were calculated from the thermal destruction parameters, and the efficacy of the RFDH process was validated by heating NDM using RFDH to achieve the target temperatures and holding the product in a convection oven for the required period. Linear regression was used to determine the D-values and z-values. The D-values of C. sakazakii in HH- and LH-NDM were 24.86 and 23.0 min at 75°C, 13.75 and 7.52 min at 80°C, 8.0 and 6.03 min at 85°C, and 5.57 and 5.37 min at 90°C, respectively. The D-values of Salmonella spp. in HH- and LH-NDM were 23.02 and 24.94 min at 75°C, 10.45 and 12.54 min at 80°C, 8.63 and 8.68 min at 85°C, and 5.82 and 4.55 min at 90°C, respectively. The predicted and observed destruction of C. sakazakii and Salmonella spp. were in agreement, indicating that the behavior of the organisms was similar regardless of the heating system (conventional vs. RFDH). Radio-frequency dielectric heating can be used as a faster and more uniform heating method for NDM to achieve target temperatures for a postprocess lethality treatment of NDM before packaging.     

反式肉桂醛与温和加热结合对复原婴幼儿牛乳中阪崎克罗诺肠杆菌的抑杀作用

研究反式肉桂醛与温和加热结合对复原婴幼儿牛乳中阪崎克罗诺肠杆菌的抑杀作用。将3 种阪崎克罗诺 肠杆菌的混合菌株（浓度约6.6（lg（CFU/mL）））接种于含有不同质量分数反式肉桂醛（0、0.1%、0.2%、0.3% 和0.4%）的复原婴幼儿牛乳样品中,将样品置于25、45、50、55 ℃培养,并在不同的时间点对样品中存活的阪崎 克罗诺肠杆菌涂布计数。为探究反式肉桂醛与温和加热结合的抑杀机制,实验利用LIVE/DEAD?细菌活性检测试 剂盒和场发射扫描电子显微镜探究细胞膜完整性及细胞形态。结果表明：0.4%的反式肉桂醛在25 ℃处理90 min、 45 ℃处理20 min、50 ℃处理10 min及55 ℃处理10 min使复原婴幼儿牛乳中阪崎克罗诺肠杆菌总数降低至检出限 以下。与反式肉桂醛及温和加热单独作用相比,反式肉桂醛结合温和加热对阪崎克罗诺肠杆菌有显著的抑杀效果 （P＜0.05）,并且随温和加热温度的升高及反式肉桂醛质量分数的增加,效果更加明显。温和加热与反式肉桂醛 结合会影响细胞膜的通透性并使细胞破碎瓦解。以上结果表明：反式肉桂醛与温和加热结合有潜力在冲调乳粉过程 中应用,从而降低阪崎克罗诺肠杆菌的感染风险。     

Inactivation of Saccharomyces cerevisiae in pineapple, grape and cranberry juices under pulsed and continuous thermo-sonication treatments

Pineapple, grape and cranberry juice were thermo-sonicated (24 kHz, 400 W, 120 μm) at 40 °C, 50 °C and 60 °C during 10 min at continuous and pulsed mode. Inactivation of Saccharomyces cerevisiae was tested from 0 to 10 min; color and pH were measured. Survivor’s curves were fitted with Weibull distribution, four parameter model and modified Gompertz equation. The acoustic energy (AE) was also calculated. S. cerevisiae was inactivated in the treatments at 60 °C, with the continuous mode being more effective. Grape juice showed total inactivation (7-log) after 10 min. Results showed that pH and color changed significantly (p < 0.05); ultrasound may promote chemical reactions and extract some components. The modified Gompertz equation showed the best fit. Energy analysis showed that pineapple juice (4287.02 mW/ml) required a higher amount of energy; grape juice showed the lowest value (3112.13 mW/ml). Ultrasound represents a viable option for juice pasteurization.     

Reservoir and routes of transmission of Enterobacter sakazakii (Cronobacter spp.) in a milk powder-producing plant

Several outbreaks of Cronobacter spp. (Enterobacter sakazakii) have been described as food-borne illness in neonates and infants. Powdered infant formula has been identified as a source of infection, especially in hospital nurseries, where a bulk of formula nutrient is prepared for the whole day and instructions for preparation are not always followed correctly. Neonates who are underweight or immunosuppressed are especially at risk for an E. sakazakii infection. Considering that milk powder is the main ingredient of powdered infant formula, we analyzed the incidence and distribution of E. sakazakii in a milk powder-producing plant. We looked specifically at the spray-drying towers and roller dryers. Selected isolates from samples taken from the environment and final product were typed by pulsed-field gel electrophoresis to investigate the epidemiology of the organism within the production area of the plant. Seven pulsed-field gel electrophoresis types were detected in the spray-drying area, which presumably entered the plant through an aperture for process air and an improperly controlled roller shutter. Furthermore, textile filters for exhaust air of both the spray-drying towers were identified as internal reservoirs of the pathogen. For economic reasons, powder from the textile filters is reintroduced into the product flow; this can contaminate the final product. For the production of milk powder to be used as an ingredient of powdered infant formula, it was suggested to terminate the process of reintroducing the filtered powder into the product flow. A second transmission route was identified in the roller dryer section of the factory. It could be shown that contaminated milk concentrate could pass the process unheated, thus leading to a contamination of the product with E. sakazakii.     

Heat shock effects on the viability of Cronobacter sakazakii during the dehydration,fermentation, and storage of lactic cultured milk products

In the present study, the viability of heat-shocked and non-shocked Cronobacter sakazakii, a foodborne pathogen, after drying and during the fermentation as well as storage of lactic cultured milk was evaluated. It was found that heat shock increased the viability of C. sakazakii. The pure culture of C. sakazakii, regardless of heat shock, grew rapidly in skim milk with a viable population of ca. 8.59–8.70 log cfu/ml after ca. 48 h of cultivation. Thereafter, the viable population of C. sakazakii remained stable. While in the mix culture with Streptococcus thermophilus or Lactobacillus bulgaricus, a marked reduction in the viable population of C. sakazakii was noted after 24 h of cultivation in skim milk. Nevertheless, at the end of fermentation, the heat-shocked C. sakazakii had a viable population of 5.93–6.01 log cfu/ml, which is significantly higher (P < 0.05) than that of non-shocked cells of 4.96–4.99 log cfu/ml. While the presence of C. sakazakii did not affect the growth of lactic acid bacteria in skim milk. Additionally, heat shock was found to enhance the survival of C. sakazakii after freeze-drying or spray-drying and during the storage of the lactic fermented milk products (pH 4.3) at 5 °C for 48 h.     

婴幼儿奶粉中阪崎克罗诺杆菌隐性污染状况分析及针对性检测技术研究进展

婴幼儿奶粉中存在的阪崎克罗诺杆菌对婴幼儿危害性极强,卫生标准中对该微生物有严格限制,但检出现象仍然时有发生。存在许多因素导致阪崎克罗诺杆菌在婴幼儿奶粉生产线中的潜伏,本文阐述了其在生产空间中的暴露特征,重点论述逆境胁迫下该菌耐受表型和活的非可培养表型形成的隐性污染对监测和清除污染残留的不利影响。讨论了各类检测技术的优缺点,强调检测灵敏度、回收率和区分活的非可培养状态菌对于控制隐性污染的重要性。以期为阪崎克罗诺杆菌的监测和防控提供新思路,为保障婴幼儿奶粉生产安全提供参考和建议。     

Development of an immobilization and detection method of Enterobacter sakazakii from powdered infant formula

Enterobacter sakazakii is an emerging opportunistic pathogen that is associated with rare but life-threatening cases of meningitis, necrotizing enterocolitis, and sepsis in premature and full-term infants. In the present study, a procedure was developed for immobilization of E. sakazakii with zirconium hydroxide coupled with detection by a species-specific duplex PCR, based on 16s-23s rDNA internal transcribed spacer (ITS) and ompA gene. Specificity of duplex PCR was tested against two-type strains, six isolates of E. sakazakii and other eight non-E. sakazakii species. When pure culture of E. sakazakii was used for immobilization, total recovery rate ranged from 79.4% to 99.6% of input bacteria, and the detection limit of duplex PCR was 3x10(5)CFU/ml. Different levels of E. sakazakii were inoculated into 90ml reconstituted powdered infant formula (PIF), and detection limit of duplex PCR was 3x10(0)CFU/ml with 24-30h enrichment after immobilization. When the experiment was performed in the presence of 10(2)CFU/ml Salmonella typhimurium, the detection limit of duplex PCR was not affected after enrichment. Seven out of 13 commercial PIF were detected positive by duplex PCR after immobilization, while only three were positive by biological methods. This study demonstrates that the combination of immobilization method with duplex PCR is easy, rapid, and efficient, and may have applications for the detection of E. sakazakii in more PIF samples.     

阪崎克罗诺肠杆菌致病性机理研究进展

阪崎克罗诺肠杆菌是一种革兰氏阴性无芽孢肠道杆菌,可导致新生儿和免疫功能不全的婴幼儿严重脑膜炎、菌血症和坏死性结肠炎,是一种非常重要的食源性条件致病菌。目前,国内外对阪崎克罗诺肠杆菌的致病性及其致病机理的研究报道非常有限。本文通过阪崎克罗诺肠杆菌外膜蛋白A、脂多糖、生物膜、宿主细胞骨架、铁获得机制、海藻糖的存在、超广谱β-内酰胺酶的产生和细胞间相互作用等方面对阪崎克罗诺肠杆菌的致病性机理进行综述。     

阪崎克罗诺杆菌ATCC 29544免疫原性蛋白的鉴定

克罗诺杆菌是一种新型的食源性致病菌。迄今为止,对阪崎克罗诺杆菌的免疫原和潜在毒力因子了解很少,而免疫原性蛋白在细菌的致病性方面发挥着重要的作用。本研究利用免疫蛋白组学的方法,鉴定了阪崎克罗诺杆菌ATCC 29544的免疫原性蛋白。利用固定后的阪崎克罗诺杆菌ATCC 29544菌体免疫新西兰兔,制备多克隆抗体;采用蔗糖密度梯度离心法,提取蛋白质,并利用双向电泳对蛋白质进行分离;以制备的多克隆抗体作为一抗进行免疫印迹分析,对具有免疫反应的蛋白点进行质谱鉴定,结果共鉴定出7种具有明显免疫反应的蛋白质。     

phoP调节子对阪崎克罗诺肠杆菌环境耐受力的影响

为研究克阪崎罗诺肠杆菌中phoP基因与环境耐受力的关系,采用Red同源重组方法构建阪崎克罗诺肠杆菌的phoP基因缺失菌株,并从生长曲线、酸碱耐受、温度耐受等方面,研究phoP基因缺失前后阪崎克罗诺肠杆菌环境耐受力的变化。结果表明：phoP基因缺失菌株的生长速度明显延缓,该基因的缺失减弱了菌对外界酸碱和高温的耐受性,并缩短了热致死时间;phoP基因缺失菌株抵御胆盐环境的能力亦有所下降,与野生型菌株相比,它在4%、8%、12%胆盐溶液环境中的存活率分别降低了19.93%（P＜0.05）、40.73%（P＜0.01）和45.84%（P＜0.01）。此外phoP基因缺失菌株对渗透压的耐受力与野生株相比也显著降低（P＜0.05）。综合以上结果表明,phoP基因有助于阪崎克罗诺肠杆菌应对外界环境刺激。     

A comparative study between overlay method and selective-differential media for recovery of stressed Enterobacter sakazakii cells from infant formula

This study compares the performance of different selective-differential media with the overlay method for recovery of stressed cells of Enterobacter sakazakii from infant formula milk (IFM). Five different selective-differential media were used in this study: OK medium, violet red bile agar (VRBA), Druggan-Forsythe-Iversen agar (DFI), Enterobacteriaceae enrichment (EE) agar, and fecal coliform agar (FCA). Tryptic soy agar supplemented with 0.1% sodium pyruvate (TSAP) was used as a control. The overlay method involved applying a thin layer (8ml) of each of the selective media onto TSAP after spreading a sample onto TSAP. Reconstituted IFM was inoculated by ca 1x10(7)CFU/ml of a mixture of four strains of E. sakazakii and subjected to different stress conditions: heat (55 degrees C for 10min), a freeze-thaw cycle (-20 degrees C for 24h, thawed at room temperature, frozen again at -20 degrees C, and thawed), acidic pH (pH 3.56 for 15min), alkaline pH (pH 11.04 for 15min), and desiccation (E. sakazakii was inoculated onto powdered IFM at a level of ca 1x10(6)CFU/g, held at 21 degrees C, water activity of the inoculated product was 0.29 and examined at 0, 15, and 30d). No major differences were noticed between the control (TSAP) and the overlay methods. However, the overlay method recovered significantly higher numbers of stressed E. sakazakii cells compared to selective-differential media. Also, the selective-differential media exhibited some variability in terms of their capabilities to recover stressed cells of E. sakazakii. Among all the examined selective-differential media, DFI performed better for recovering stressed E. sakazakii cells. This study suggests that the overlay method may serve as a potential alternative to direct selective plating for best recovery of E. sakazakii from IFM.