Thermal inactivation of Enterobacter sakazakii in rehydrated infant formula

The presence of low levels of Enterobacter sakazakii in dried infant formula have been linked to outbreaks of meningitis, septicemia, and necrotizing enterocolitis in neonates, particularly those who are premature or immunocompromised. In the current study, the ability of 12 strains of E. sakazakii to survive heating in rehydrated infant formula was determined at 58 degrees C with a submerged coil apparatus. The observed D58-values ranged from 30.5 to 591.9 s, with the strains appearing to fall into two distinct heat resistance phenotypes. The z-value of the most heat-resistant strain was 5.6 degrees C. When dried infant formula containing this strain was rehydrated with water preequilibrated to various temperatures, a more than 4-log reduction in E. sakazakii levels was achieved by preparing the formula with water at 70 degrees C or greater.     

Detergent and sanitizer stresses decrease the thermal resistance of Enterobacter sakazakii in infant milk formula

ABSTRACT:  This study determined the effect of acid, alkaline, chlorine, and ethanol stresses on the thermal inactivation of Enterobacter sakazakii in infant milk formula. Unstressed or stressed cells were mixed with reconstituted powdered infant milk formula (PIMF) at temperatures between 52 and 58 °C for various time periods or mixed with PIMF prior to reconstitution with hot water between 50 and 100 °C. D - and z -values were determined using liner regression analysis. In general, detergent and sanitizer stresses decreased the thermal resistance of E. sakazakii in infant milk formula. The results of this study may be of use to regulatory agencies, manufacturers, and infant caregivers to design heating processes to eliminate E. sakazakii .     

柳江县市售国产婴幼儿配方粉中阪崎肠杆菌污染监测分析

目的 了解市售国产婴幼儿配方粉中阪崎肠杆菌污染状况,为消费预警提供科学依据。方法 分别按2011、2012年版国家食源性致病菌监测工作手册对市场上11家国内生产的32份婴幼儿配方粉进行检测。结果 32份样品检出2株阪崎肠杆菌,检出率为6.25%。其中婴幼儿配方奶粉中阪崎肠杆菌检出率4.35%(1/23);婴幼儿谷物食品中阪崎肠杆菌检出率11.11%(1/9)。结论 柳江县市售部分婴幼儿配方粉中存在阪崎肠杆菌污染,食用安全隐患不容忽视,应加强监测力度,预防和控制阪崎肠杆菌引起的食物中毒事件发生。     

Inactivation of Enterobacter sakazakii in reconstituted infant formula by monocaprylin

Enterobacter sakazakii is an emerging pathogen that causes meningitis, bacteremia, sepsis, and necrotizing enterocolitis in neonates and children, with a mortality rate of 14%. Epidemiological studies have implicated dried infant formula as the principal source of the pathogen. Caprylic acid is a natural eight-carbon fatty acid present in breast milk and bovine milk and is approved as generally recognizable as safe by the U.S. Food and Drug Administration. The objective of this study was to determine the antibacterial effect of monocaprylin (monoglyceride ester of caprylic acid) on E. sakazakii in reconstituted infant formula. A five-strain mixture of E. sakazakii was inoculated into 10-ml samples of reconstituted infant formula (at 6.0 log CFU/ml) followed by 0, 25, or 50 mM (1%) monocaprylin. The samples were incubated at 37 or 23 degrees C for 0, 1, 6, and 24 h and at 8 or 4 degrees C for 0, 6, 24, and 48 h, and the surviving populations of E. sakazakii at each sampling time were counted. The treatments containing monocaprylin significantly reduced the population of E. sakazakii (P < 0.05) compared with the controls. Monocaprylin (50 mM) reduced the pathogen by >5 log CFU/ml by 1 h of incubation at 37 or 23 degrees C and by 24 h of incubation at 8 or 4 degrees C. Results indicate that monocaprylin could potentially be used to inactivate E. sakazakii in reconstituted infant formula; however, sensory studies are warranted before its use can be recommended.     

Survival of Enterobacter sakazakii in a dehydrated powdered infant formula

A quantity of dehydrated powdered infant formula was prepared to contain Enterobacter sakazakii strain 607 at approximately 106 CFU/ml when rehydrated according to the manufacturer's instructions. The survival of the microorganism in the dry formula was followed for 2 years, during which samples periodically were rehydrated and analyzed for viable E. sakazakii. During the initial 5 months of storage at room temperature, viable counts declined approximately 2.4 log cycles. During the subsequent 19 months, the concentration of viable E. sakazakii declined an additional 1.0 log cycle. These results indicate that a small percentage of E. sakazakii cells can survive for extended periods in dehydrated powdered infant formula.     

Enterobacter sakazakii in food and beverages (other than infant formula and milk powder)

The ubiqitous microorganism Enterobacter sakazakii is a rare contaminant of infant formula and may cause severe systemic infection in neonates. So far, other food is not known to cause E. sakazakii-infections. The scarce information about the ecology of E. sakazakii and the uncertainty concerning the source of infection in children and adults warrant a summary of the current knowledge about the presence of this opportunistic microorganism in food other than infant formula. This review systematizes publications on the presence of E. sakazakii in food and beverages until June 2006. Food other than infant formula has been rarely investigated for the presence of E. sakazakii. Nevertheless, this microorganism could be isolated from a wide spectrum of food and food ingredients. E. sakazakii was isolated from plant food and food ingredients like cereal, fruit and vegetables, legume products, herbs and spices as well as from animal food sources like milk, meat and fish and products made from these foods. The spectrum of E. sakazakii-contaminated food covers both raw and processed food. The kind of processing of E. sakazakii-contaminated food was not restricted to dry products. Fresh, frozen, ready-to-eat, fermented and cooked food products as well as beverages and water suitable for the preparation of food, were found to be contaminated by E. sakazakii. Although E. sakazakii-contaminated food do not have general public health significance, measures for prevention should consider the presence of E. sakazakii in food, food ingredients, their processing and preparation as possible source of contamination, colonization or infection.     

Gamma radiation sensitivity of Enterobacter sakazakii in dehydrated powdered infant formula

The observed Enterobacter sakazakii D10-values for tryptic soy broth and dehydrated powdered infant formula were 0.27 +/- 0.05 and 0.76 +/- 0.08 kGy, respectively. A decrease of approximately 3 log in the dehydrated powdered infant formula was obtained by irradiation with 3.0 kGy or rehydration with hot water at 80 degrees C. No recoverable bacteria were found in the powdered infant formula irradiated at 5.0 kGy and stored, either before or after rehydration. A radiation dose of up to 5.0 kGy had no marked effect on the sensory properties of the dehydrated powdered infant formula after rehydration and heating. Gamma radiation could potentially be used to inactivate E. sakazakii in dehydrated powdered infant formula; however, nutritional studies need to be conducted before the use of radiation can be recommended.     

Inactivation of Enterobacter sakazakii in infant milk formula by gamma irradiation: determination of D10-value

ABSTRACT:  Enterobacter sakazakii is an emerging foodborne pathogen that has caused several cases of meningitis and necrotizing entercolitis in infants and has been associated with infant formulas. Five strains of E. sakazakii were inoculated individually into brain heart infusion broth and rehydrated or dehydrated infant milk formula and exposed to ionizing radiation. E. sakazakii strains in brain heart infusion broth and rehydrated infant milk formula (RIMF) were exposed to irradiation dose of up to 1 kGy while strains in dehydrated infant milk formula (DIMF) were exposed to irradiation dose of up to 9 kGy. The D₁₀-values were determined by using a linear regression model. Average calculated D₁₀-values ranged from 0.21 to 0.29 kGy, 0.24 to 0.37 kGy, and 1.06 to 1.71 kGy in brain heart infusion broth, RIMF, and DIMF, respectively. The results obtained from this study will be useful for powdered infant milk formula industries to reduce the risk associated with E. sakazakii .     

婴幼儿配方食品中阪崎肠杆菌的计数结果不确定度评定

[目的] 了解专项抽查的婴儿配方食品中的阪崎肠杆菌指标状况,并探讨阪崎肠杆菌的计数结果的不确定度评定方法。[方法] 按照GB 4789.1-2010 《食品微生物学检验 总则》和GB 4789.40-2016 《食品微生物学检验 阪崎肠杆菌检验》进行了抽样和检测, 依据JJF1059.1-2012 《测量不确定度评定与表示》及贝塞尔统计学方法对计数结果进行不确定度评定。[结果]22批次婴儿配方食品中有1批次检出阪崎肠杆菌,检出率为4.55%, 该批次样品中阪崎肠杆菌平均计数为25.5MPN/100g,计数结果扩展标准不确定度为2.72MPN/100g。[结论] 本次评估依据阪崎肠杆菌检计数检验国家标准,对4次保温培养节点进行了分析,结果表明固液混合过程,酵液取样体积,阪崎肠杆菌显色平板上可疑菌落选择和MPN法重复计数的四个不确定度较大,是记数检验过程中的重要环节。     

Enterobacter sakazakii: biological properties and significance in dried infant milk formula (IMF) powder†

Enterobacter sakazakii is an emerging opportunistic pathogen associated with rare but life‐threatening cases of meningitis, necrotizing enterocolitis and sepsis in premature infants. The bacterium has been isolated at low levels from dried infant milk formulas and is a cause of concern to all manufacturers. The threat posed by E. sakazakii is further compounded by a paucity of knowledge in key areas, specifically its ecology and physiology. This organism now represents a serious challenge to all those involved in the manufacture of infant formula products.     

Predictive modelling of Enterobacter sakazakii inactivation in bovine milk during high-temperature short-time pasteurization

A linear model was derived to describe the thermal inactivation of Enterobacter sakazakii in bovine whole milk in a high-temperature short-time pilot scale pasteurizer. Integrated lethal effect, or pasteurization effect (PE), was obtained by converting times at different temperatures in the various sections of the pasteurizer to the equivalent time at the reference temperature (72°C). PE was then related, by a simple linear function, to the log₁₀ of the % viable counts with a power transformation of the PE values to improve linear fit. R² values for the three E. sakazakii trials varied from 0.941 to 0.959. Inter-trial variation was incorporated into the model using @RISK™ simulation software, and a comparison between models for E. sakazakii and Listeria monocytogenes revealed that L. monocytogenes was more heat-resistant. Output from simulations confirmed that treatment at 68°C for 16 s can ensure (at the 1st percentile) a 5-log reduction of E. sakazakii.     

Real-time monitoring of luciferase-tagged Cronobacter sakazakii in reconstituted infant milk formula

The aim of this study was to examine the potential of using a lux-tagged Cronobacter sakazakii strain to monitor growth of the bacterium in various liquids. C. sakazakii was transformed with plasmid p16S lux, and integration of the plasmid at the desired site on the chromosome was confirmed by PCR. The growth of the lux-tagged strain was similar to that of the non-lux-tagged strain, and the integrated plasmid was stable when cells were cultured in the absence of antibiotic. Growth of the lux-tagged strain was monitored in real time in Luria-Bertani broth, skim milk, and infant milk formula by using both the Luminoskan luminometer and the Xenogen IVIS imager. Bioluminescence could be detected when the lux-tagged strain was cocultured with other bacteria. The effect of monocaprylin and nisin on the growth of C. sakazakii in milk was monitored by measuring bioluminescence. In conclusion, growth of a lux-tagged C. sakazakii can be monitored in real time in both clear and opaque liquids by measuring bioluminescence. lux-tagged C. sakazakii strains could be potentially used in high-throughput assays to monitor the effects of various infant milk formula compositions on growth of the bacterium.     

Inhibition of growth of Enterobacter sakazakii in reconstituted infant formula by the lactoperoxidase system

Neonatal bacteremia and meningitis caused by the opportunistic pathogen Enterobacter sakazakii have been associated with the consumption of reconstituted powdered infant formula. Lactoperoxidase (LPO), present in mammalian milk, is known to inhibit the growth of enteric pathogens. We undertook a study to determine if the lactoperoxidase system (LPOS) will inhibit the growth of E. sakazakii in a milk-based powdered infant formula reconstituted with water. Initially at 0.04 CFU/ml, E. sakazakii grew to 2.40 to 2.74 log CFU/ml in reconstituted infant formula held at 30 or 37 degrees C for 8 h and to 0.6 log CFU/ ml in formula held for 12 h at 21 degrees C. The pathogen was not detected (less than 1 CFU/227 ml) by enrichment of formula treated with 10 to 30 microg/ml LPO and stored for 24 h at 37 degrees C or 30 microg/ml LPO and stored for 24 h at 30 degrees C. Populations of E. sakazakii, initially at 4.40 log CFU/ml of reconstituted infant formula containing 5 microg/ml LPO, did not increase significantly (P > 0.05) for up to 12 h at 21 and 30 degrees C. Populations either decreased significantly or were unchanged in formula supplemented with 10 microg/ml LPO and stored at 21, 30, or 37 degrees C for up to 24, 8, and 8 h, respectively. Results indicate that LPOS can be used to control the growth of E. sakazakii in reconstituted infant formula, thereby potentially reducing the risk of neonatal infections resulting from consumption of formula that may be contaminated with the pathogen.     

Growth of Enterobacter sakazakii in reconstituted infant formula as affected by composition and temperature

The ability of Enterobacter sakazakii to cause infections in infants, coupled with its documented presence in some lots of commercially manufactured powdered infant formula, raises a concern about the potential for its growth in reconstituted formula, with consequent increased safety risk. A study was done to determine these characteristics in four commercial milk-based powdered infant formulas and two soy-based formulas reconstituted with water and inoculated with a 10-strain mixture of E. sakazakii at populations of 0.02 and 0.53 CFU/ml (ca. 13 CFU/100 g and ca. 409 CFU/100 g of powdered formula, respectively). Reconstituted formulas were stored at 4, 12, 21, and 30 degrees C, and populations were monitored up to 72 h. E. sakazakii did not grow in formulas stored at 4 degrees C, although it was detected by enrichment of all formulas 72 h after reconstitution. Initially at a population of 0.02 CFU/ml, E. sakazakii grew to populations > or = 1 log CFU/ml of reconstituted formulas held at 12, 21, and 30 degrees C for 48, 12, and 8 h, respectively. At an initial population of 0.53 CFU/ml, the pathogen grew to populations > or = 1 log CFU/ml in reconstituted infant formula held at 12 and 21 degrees C for 24 and 8 h, respectively, and to populations 2.55 to 3.14 log CFU/ml when held at 30 degrees C for 8 h. Populations initially at 0.02 and 0.53 CFU/ml of reconstituted formula increased to < or = 0.25 and 0.4 log CFU/ml, respectively, when formulas were held at 30 degrees C for 4 h. Growth was not greatly influenced by the composition of formulas. Results show that the hang time for reconstituted infant formula held at temperatures in neonatal intensive care units should be no longer than 4 h. Portions of reconstituted infant formula not fed to infants should be stored at < or = 4 degrees C, a temperature at which E. sakazakii will not grow.     

Growth and survival of Enterobacter sakazakii in human breast milk with and without fortifiers as compared to powdered infant formula

Enterobacter sakazakii infections often involve debilitated neonates consuming contaminated reconstituted powdered infant formula. There is the possibility that expressed human breast milk can become contaminated with E. sakazakii in the hospital or home setting and through the use of contaminated breast milk fortifiers. In addition, although breast milk has been shown to have some antimicrobial effects, this has not been extensively researched in regards to E. sakazakii. Thus, we examined the survival and growth of 9 strains of E. sakazakii in breast milk, human breast milk with fortifiers and powdered infant formula at 10, 23 and 37 degrees C. The average generation times for clinical, food and environmental isolates in breast milk were 0.94+/-0.04, 0.75+/-0.04 and 0.84+/-0.04 h at 23 degrees C; and 0.51+/-0.03, 0.33+/-0.03 and 0.42+/-0.03 h at 37 degrees C, respectively. E. sakazakii was able to survive up to 12 days in breast milk with fortifiers at 10 degrees C. However, its average generation times among replicates and isolate sources ranged from 11.97+/-3.82 to 27.08+/-4.54 h in breast milk at 10 degrees C. Interestingly, average generation times in breast milk with fortifiers at 23 degrees C (0.83+/-0.05, 0.93+/-0.06 and 0.96+/-0.06 h) and 37 degrees C (0.41+/-0.04, 0.51+/-0.05 and 0.54+/-0.05 h) were longer than in powdered infant formula and breast milk at the same temperatures, indicating that human breast milk fortifiers may have an inhibitory effect on the growth of E. sakazakii. However, the intrinsically ascribed antimicrobial properties of breast milk do not appear to inhibit the growth of this foodborne pathogen in-vitro.     

乳粉中坂崎肠杆菌的检测

阐述了坂崎肠杆菌（Enterobacter sakazakii)的由来、致病性及其检测史，重点介绍FDA的婴儿配方奶粉中坂崎肠杆菌的分离与计数检测程序，同时提供定量结果表示方式，并探讨了坂崎肠杆菌鉴别要点及检测发展趋势。     

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.     

Rapid, specific detection of Enterobacter sakazakii in infant formula using a real-time PCR assay

Enterobacter sakazakii is a rare cause of invasive infection with high mortality rates in neonates. Powdered milk-based infant formulas have been associated with the E. sakazakii-related outbreaks in premature or other immunocompromised infants. In this study, an assay was developed for the specific detection of E. sakazakii in infant formula using an application of the fluorogenic 5' nuclease assay (TaqMan). A set of primers and probe was designed using the E. sakazakii partial macromolecular synthesis operon: the rpsU gene 3' end and the primase (dnaG) gene 5' end. The specificity of the assay was evaluated using 68 Enterobacter and 55 non-Enterobacter strains. The newly developed assay enables us to detect 100 CFU/ ml in pure culture and in reconstituted infant formula in 50 cycles of PCR without enrichment. The assay was specific enough to discriminate E. sakazakii from all other Enterobacter and non-Enterobacter strains tested. The developed real-time PCR assay could save up to 5 days and eliminate the need for plating samples on selective or diagnostic agars and for biochemical confirmation steps. The real-time PCR assay could be used to rapidly screen infant formula samples for E. sakazakii and would be a boon to food industries and regulatory agencies.