Effectiveness of disinfectants in killing Enterobacter sakazakii in suspension, dried on the surface of stainless steel, and in a biofilm

The effectiveness of 13 disinfectants used in hospitals, day-care centers, and food service kitchens in killing Enterobacter sakazakii in suspension, dried on the surface of stainless steel, and in biofilm was determined. E. sakazakii exhibited various levels of resistance to the disinfectants, depending on the composition of the disinfectants, amount and type of organic matrix surrounding cells, and exposure time. Populations of planktonic cells suspended in water (7.22 to 7.40 log CFU/ml) decreased to undetectable levels (<0.30 log CFU/ml) within 1 to 5 min upon treatment with disinfectants, while numbers of cells in reconstituted infant formula were reduced by only 0.02 to 3.69 log CFU/ml after the treatment for 10 min. The presence of infant formula also enhanced the resistance to the disinfectants of cells dried on the surface of stainless steel. The resistance of cells to disinfectants in 6-day-old and 12-day-old biofilms on the surface of stainless steel was not significantly different. The overall order of efficacy of disinfectants in killing E. sakazakii was planktonic cells > cells inoculated and dried on stainless steel > cells in biofilms on stainless steel. Findings show that disinfectants routinely used in hospital, day-care, and food service kitchen settings are ineffective in killing some cells of E. sakazakii embedded in organic matrices.     

Survival and growth of Enterobacter sakazakii in infant rice cereal reconstituted with water, milk, liquid infant formula, or apple juice

AIMS: To determine survival and growth characteristics of Enterobacter sakazakii in infant rice cereal as affected by type of liquid used for reconstitution and storage temperature after reconstitution. METHODS AND RESULTS: A commercially manufactured dry infant rice cereal was reconstituted with water, apple juice, milk, or liquid infant formula, inoculated with a 10-strain mixture of E. sakazakii at populations of 0.27, 0.93, and 9.3 CFU ml(-1), and incubated at 4, 12, 21 or 30 degrees C for up to 72 h. Growth did not occur in cereal reconstituted with apple juice, regardless of storage temperature, or in cereal reconstituted with water, milk, or formula and stored at 4 degrees C. The lag time for growth in cereal reconstituted with water, milk, or formula was decreased as the incubation temperature (12, 21 and 30 degrees C) was increased. Upon reaching maximum populations of 7-8 log10 CFU ml(-1), in some instances populations decreased to nondetectable levels during subsequent storage which was concurrent with decreases in pH. CONCLUSIONS: Enterobacter sakazakii initially at very low populations can rapidly grow in infant rice cereal reconstituted with water, milk, or infant formula. SIGNIFICANCE AND IMPACT OF THE STUDY: Reconstituted infant rice cereal can support luxuriant growth of E. sakazakii. Reconstituted cereal that is not immediately consumed should be discarded or stored at a temperature at which E. sakazakii and other food-borne pathogens cannot grow.     

Effects of preculturing conditions on lag time and specific growth rate of Enterobacter sakazakii in reconstituted powdered infant formula

Enterobacter sakazakii can be present, although in low levels, in dry powdered infant formulae, and it has been linked to cases of meningitis in neonates, especially those born prematurely. In order to prevent illness, product contamination at manufacture and during preparation, as well as growth after reconstitution, must be minimized by appropriate control measures. In this publication, several determinants of the growth of E. sakazakii in reconstituted infant formula are reported. The following key growth parameters were determined: lag time, specific growth rate, and maximum population density. Cells were harvested at different phases of growth and spiked into powdered infant formula. After reconstitution in sterile water, E. sakazakii was able to grow at temperatures between 8 and 47 degrees C. The estimated optimal growth temperature was 39.4 degrees C, whereas the optimal specific growth rate was 2.31 h(-1). The effect of temperature on the specific growth rate was described with two secondary growth models. The resulting minimum and maximum temperatures estimated with the secondary Rosso equation were 3.6 degrees C and 47.6 degrees C, respectively. The estimated lag time varied from 83.3 +/- 18.7 h at 10 degrees C to 1.73 +/- 0.43 h at 37 degrees C and could be described with the hyperbolic model and reciprocal square root relation. Cells harvested at different phases of growth did not exhibit significant differences in either specific growth rate or lag time. Strains did not have different lag times, and lag times were short given that the cells had spent several (3 to 10) days in dry powdered infant formula. The growth rates and lag times at various temperatures obtained in this study may help in calculations of the period for which reconstituted infant formula can be stored at a specific temperature without detrimental impact on health.     

Comparison of media for the isolation of Enterobacter sakazakii

Enterobacter sakazakii is associated with neonatal infections and is occasionally present at low levels (<1 CFU/g) in powdered infant formula milk (IFM). It has been previously reported that some E. sakazakii strains do not grow in standard media for Enterobacteriaceae and coliform bacteria; therefore, a reliable method is needed for recovery of the organism. Three E. sakazakii enrichment broths-Enterobacteriaceae enrichment broth (EE), E. sakazakii selective broth (ESSB), and modified lauryl sulfate broth (mLST)-were compared with a novel broth designed for maximum recovery of E. sakazakii, E. sakazakii enrichment broth (ESE). One hundred seventy-seven strains (100%) grew in ESE, whereas between 2 and 6% of strains did not grow in EE, mLST, or ESSB. E. sakazakii possesses alpha-glucosidase activity, and a number of selective, chromogenic agars for E. sakazakii isolation based on this enzyme have been developed. E. sakazakii isolation agar produced fewer false-positive colonies than did Druggan-Forsythe-Iversen agar. However, the latter supported the growth of more E. sakazakii strains. It was also determined that 2% of E. sakazakii strains did not produce yellow pigmentation on tryptone soya agar at 25 degrees C, a characteristic frequently cited in the identification of E. sakazakii. The recovery of desiccated E. sakazakii (0.2 to 2000 CFU/25 g) from powdered IFM in the presence of a competing flora was determined with various enrichment broths and differential selective media. Current media designed for the isolation and presumptive identification of E. sakazakii do not support the growth of all currently known E. sakazakii phenotypes; therefore, improvements in the proposed methods are desirable.     

Performance of media for recovering stressed cells of Enterobacter sakazakii as determined using spiral plating and ecometric techniques

A study was done to determine the performance of differential, selective media for supporting resuscitation and colony development by stressed cells of Enterobacter sakazakii. Cells of four strains of E. sakazakii isolated from powdered infant formula were exposed to five stress conditions: heat (55 degrees C for 5 min), freezing (-20 degrees C for 24 h, thawed, frozen again at -20 degrees C for 2 h, thawed), acidic pH (3.54), alkaline pH (11.25), and desiccation in powdered infant formula (water activity, 0.25; 21 degrees C for 31 days). Control and stressed cells were spiral plated on tryptic soy agar supplemented with 0.1% pyruvate (TSAP, a nonselective control medium); Leuschner, Baird, Donald, and Cox (LBDC) agar (a differential, nonselective medium); Oh and Kang agar (OK); fecal coliform agar (FCA); Druggan-Forsythe-Iversen (DFI) medium; violet red bile glucose (VRBG) agar; and Enterobacteriaceae enrichment (EE) agar. With the exception of desiccation-stressed cells, suspensions of stressed cells were also plated on these media and on R&F Enterobacter sakazakii chromogenic plating (RF) medium using the ecometric technique. The order of performance of media for recovering control and heat-, freeze-, acid-, and alkaline-stressed cells by spiral plating was TSAP > LBDC > FCA > OK, VRBG > DFI > EE; the general order for recovering desiccated cells was TSAP, LBDC, FCA, OK > DFI, VRBG, EE. Using the ecometric technique, the general order of growth indices of stressed cells was TSAP, LBDC > FCA > RF, VRBG, OK > DFI, EE. The results indicate that differential, selective media vary greatly in their abilities to support resuscitation and colony formation by stressed cells of E. sakazakii. The orders of performance of media for recovering stressed cells were similar using spiral plating and ecometric techniques, but results from spiral plating should be considered more conclusive.     

Detection of Enterobacter sakazakii in dried infant milk formula by cationic-magnetic-bead capture

Enterobacter sakazakii has been associated with life-threatening infections in premature low-birth-weight infants. Contaminated infant milk formula (IMF) has been implicated in cases of E. sakazakii meningitis. Quick and sensitive methods to detect low-level contamination sporadically present in IMF preparations would positively contribute towards risk reduction across the infant formula food chain. Here we report on the development of a simple method, combining charged separation and growth on selective agar, to detect E. sakazakii in IMF. This protocol can reliably detect 1 to 5 CFU of E. sakazakii in 500 g of IMF in less than 24 h.     

Effectiveness of Disinfectants in Killing Enterobacter sakazakii in Suspension, Dried on the Surface of Stainless Steel, and in a Biofilm

The effectiveness of 13 disinfectants used in hospitals, day-care centers, and food service kitchens in killing Enterobacter sakazakii in suspension, dried on the surface of stainless steel, and in biofilm was determined. E. sakazakii exhibited various levels of resistance to the disinfectants, depending on the composition of the disinfectants, amount and type of organic matrix surrounding cells, and exposure time. Populations of planktonic cells suspended in water (7.22 to 7.40 log CFU/ml) decreased to undetectable levels (<0.30 log CFU/ml) within 1 to 5 min upon treatment with disinfectants, while numbers of cells in reconstituted infant formula were reduced by only 0.02 to 3.69 log CFU/ml after the treatment for 10 min. The presence of infant formula also enhanced the resistance to the disinfectants of cells dried on the surface of stainless steel. The resistance of cells to disinfectants in 6-day-old and 12-day-old biofilms on the surface of stainless steel was not significantly different. The overall order of efficacy of disinfectants in killing E. sakazakii was planktonic cells > cells inoculated and dried on stainless steel > cells in biofilms on stainless steel. Findings show that disinfectants routinely used in hospital, day-care, and food service kitchen settings are ineffective in killing some cells of E. sakazakii embedded in organic matrices.     

Heat resistance of Desulfotomaculum nigrificans spores in soy protein infant formula preparations.

The heat resistance of Desulfotomaculum nigrificans spores was determined in soy protein infant formula preparations. Methods of sporulation were developed and evaluated. D. nigrificans spores of highest heat resistance were produced in a 40% infusion of spent mushroom compost. Fraction-negative D121 degrees C-values obtained in modified soy formula were 25.8 min for spores of ATCC 7946 produced at 55 degrees C and 54.4 min for an isolate designated RGI 1, which was sporulated at 66 degrees C. From the fraction-negative D-values, z-values were obtained of 6.7 degrees C for ATCC 7946 and 9.5 degrees C for RGI 1. Survivor-curve D121 degrees C-values were 5.6 min for ATCC 7946 and 2.7 min for RGI 1 sporulated at 55 degrees C and heated in modified soy formula. Corresponding D121 degrees C-values in Butterfield phosphate buffer (pH 7.2) were 3.3 min (ATCC 7946) and 1.1 min (RGI 1). The z-values generated from survivor-curve D-values were similar to those obtained by using fraction-negative procedures. In all instances the inactivation kinetics appeared to be linear. The isolate designated RGI 1, when sporulated at 66 degrees C and heated in a modified infant soy formula, exhibited an extraordinary heat resistance far in excess of previous reports.     

Slow rehydration improves the recovery of dried bacterial populations.

Slow rehydration of bacteria from dried inoculant formulations provided higher viable counts than did rapid rehydration. Estimates were higher when clay and peat powder formulations of Rhizobium meliloti, Rhizobium leguminosarum biovar trifolii, and Pseudomonas putida, with water activities between 0.280 and 0.650, were slowly rehydrated to water activities of approximately 0.992 before continuing the dilution plating sequence. Rhizobium meliloti populations averaged 6.8 x 10(8) cfu/g and 1328 cfu/alfalfa seed greater when slowly rehydrated from bulk powder and preinoculated seeds, respectively. Bulk powder samples were slowly rehydrated to 0.992 water activity by the gradual addition of diluent, followed by a 10-min period for moisture equilibration. Preinoculated seed samples were placed in an environmental chamber at 24 degrees C with relative humidity greater than 80% for 1 h to allow moisture absorption. "Upshock," osmotic cellular stresses that occur during rehydration, was reduced when dried microbial formulations were slowly rehydrated and equilibrated before becoming fully hydrated in the dilution plating sequence. These procedures may also be applicable when estimating total viable bacterial populations from dried soil or other dry formulations.     

改良环介导等温扩增技术快速检测婴儿配方奶粉中的阪崎肠杆菌

[目的]采用改良环介导等温扩增(LAMP)技术,快速检测婴儿配方奶粉中的阪崎肠杆菌.[方法]以阪崎肠杆菌(ATCC29544)的16S-23S rRNA间区序列作为靶序列,设计内、外引物和环引物,通过肉眼观察白色沉淀,判断检测结果.[结果]LAMP检测阪崎肠杆菌的灵敏度为0.101 CFU/mL,人工污染阪崎肠杆菌的婴儿配方奶粉的检出限为1.1 CFU/g.采用试剂盒提取DNA,从样品处理到报告结果,耗时1 h.而对照,PCR检测阪崎肠杆菌的灵敏度为101 CFU/mL,人工污染阪崎肠杆菌的婴儿配方奶粉的检出限为1100 CFU/g.采用同样方法提取DNA,从样品处理到报告结果,耗时3 h.[结论]因此,LAMP检测婴儿配方奶粉中的阪崎肠杆菌灵敏度高,耗时短,方法简便.     

Cloning and sequencing of the ompA gene of Enterobacter sakazakii and development of an ompA-targeted PCR for rapid detection of Enterobacter sakazakii in infant formula

Enterobacter sakazakii is an emerging, infant formula-borne pathogen that causes severe meningitis, meningoencephalitis, sepsis, and necrotizing enterocolitis in neonates and infants, with a high fatality rate. Traditional detection methods take up to 7 days to identify E. sakazakii. The outer membrane protein A gene (ompA), along with its flanking sequences from E. sakazakii (ATCC 51329), was cloned in the pGEM-T Easy vector and sequenced. Comparison of the nucleotide and deduced amino acid sequences of the ompA gene with other sequences available in the GenBank database revealed a high degree of homology with ompA genes of other gram-negative bacteria belonging to the Enterobacteriaceae. Based on regions of the ompA gene unique to E. sakazakii, two primers were synthesized to develop and optimize an E. sakazakii-specific PCR. The PCR amplified a 469-bp DNA product from all E. sakazakii strains tested but not from other bacteria. Experiments to determine the sensitivity of the PCR indicated that it could detect as few as 10(3) CFU/ml of E. sakazakii bacteria in infant formula directly and 10(-1) CFU/ml after an 8-h enrichment step. We conclude that this PCR, combined with enrichment culturing, has the potential to be used as a rapid tool for detecting the presence of E. sakazakii in infant formula.     

Rapid inactivation of <Emphasis Type="Italic">Cronobacter sakazakii</Emphasis> on copper alloys following periods of desiccation stress

Cronobacter spp. have been identified as the causative agent in meningitis and necrotizing enterocolitis in premature infants which can be linked to the bacterium's desiccation resistance and persistence in powdered infant formula. In this study we examined the efficacy of copper cast alloys in contact killing of Cronobacter sakazakii following periods of desiccation stress. Cronobacter sakazakii cells suspended in Tryptic Soy Broth (TSB) were killed within 10 min while kept moist on 99.9% copper alloys and within 1 min of drying on 99.9% copper alloys. Survival times were unchanged after cells suspended in TSB were desiccated for 33 days. Cronobacter sakazakii cells suspended in infant formula were killed within 30 min under moist conditions and within 3 min of drying on 99.9% copper alloys. However, when desiccated in infant formula for 45 days, survival times decreased to 10 and 1 min in moist and dry conditions, respectively. In contrast, no decrease in viable cells was noted on stainless steel surfaces under the experimental conditions employed in this study. Cronobacter sakazakii was rapidly killed on copper alloys under all testing conditions of this study indicating that desiccation and copper ion resistance do not prolong survival. These results could have important implications for the utilization of copper in the production and storage of powdered infant formula.     

Effect of environmental stresses on the sensitivity of Enterobacter sakazakii in powdered infant milk formula to gamma radiation

Aim:  To evaluate the effect of starvation, heat, cold, acid, alkaline, chlorine and ethanol stresses on the resistance of Enterobacter sakazakii in powdered infant milk formula (PIMF) towards gamma radiation.
Methods and Results:  Stressed cells of E. sakazakii ATCC 51329 and four other food isolate strains were mixed individually with PIMF, kept overnight at room temperature, and then exposed to gamma radiation up to 7·5 kGy. The D ₁₀-values were determined using linear regression and for the stressed E. sakazakii strains these values ranged from 0·82 to 1·95 kGy.
Conclusions:  Environmental stresses did not significantly change the sensitivity of most E. sakazakii strains to ionizing radiation.
Significance and Impact of the Study:  Data obtained established that most forms of environmental stress are unlikely to significantly enhance the resistance of E. sakazakii strains to lethal, low dose irradiation treatment.     

Survival of Aerobic and Anaerobic Bacteria in Chicken Meat During Freeze-Dehydration, Rehydration, and Storage

Total and anaerobic counts were ascertained on boneless, cooked, cubed, frozen chicken meat. We determined survival of aerobes and anaerobes in the natural flora after the meat was freeze-dehydrated and rehydrated at room temperature for 30 min and at 50, 85, and 100 C for 10 min. Total and anaerobic counts of bacteria in the rehydrated meat were established during storage of samples at 4, 22, and 37 C-until a spoilage odor was detected. Samples were also inoculated with Clostridium sporogenes and were dried and rehydrated at 100 C and stored at 37 C. Approximately 21% of the aerobes and 37% of the anaerobes survived drying and rehydration at room temperature. Many genera of aerobes, anaerobes, and facultative anaerobes survived drying and rehydration at 50 C; only sporeformers survived rehydration at 85 or 100 C. Low-temperature (4 C) storage of rehydrated meat produced ample shelf life (over 20 days), whereas storage at the higher temperature resulted in a shelf life of less than 30 hr. Approximately 81% of the C. sporogenes cells survived rehydration at 100 C and grew to over 10(7) cells within 40 hr. Our study presents additional data for adequate microbiological control in processing of freeze-dehydrated meat. Also, it points out the natural selection for sporeformers at high temperature of rehydration, stressing the need for consumer education in product handling for safety purposes.     

Genetic characterization of thermal tolerance in Enterobacter sakazakii

Enterobacter sakazakii is an opportunistic pathogen that causes meningitis and necrotizing enterocolitis in neonates. Here we characterized the thermal tolerance of E. sakazakii isolates obtained from powdered infant formula and other food products in Japan. Isolates were categorized into three classes according to their thermal tolerance, and differential gene expression analysis showed that the heat-resistant clones expressed a higher level of infB (which encodes a translation initiation factor), than did the heat-sensitive isolates. Gene expression and DNA polymorphism analyses suggested that this gene target might be useful to unequivocally detect and identify heat-resistant clones, permitting epidemiological surveillance for this pathogen.     

Desiccation and heat tolerance of Enterobacter sakazakii

AIMS: Enterobacter sakazakii is an opportunistic pathogen which has been isolated at low levels from powdered infant formulas. This study was performed to demonstrate that Ent. sakazakii is not particularly thermotolerant, but can adapt to osmotic and dry stress. METHODS AND RESULTS: We determined the heat, osmotic and dry stress resistance of Ent. sakazakii. The D-value at 58 degrees C ranged from 0.39 to 0.60 min, which is comparable with that of other Enterobacteriaceae, but much lower than reported previously (Nazarowec-White and Farber 1997, Letters in Applied Microbiology 24: 9-13). However, stationary phase Ent. sakazakii cells were found to be more resistant to osmotic and dry stress than Escherichia coli, Salmonella and other strains of Enterobacteriaceae tested. Further analysis indicated that the dry resistance is most likely linked to accumulation of trehalose in the cells. CONCLUSIONS: The high tolerance to desiccation provides a competitive advantage for Ent. sakazakii in dry environments, as found in milk powder factories, and thereby increases the risk of postpasteurization contamination of the finished product. SIGNIFICANCE AND IMPACT OF THE STUDY: Understanding of the physiology and survival strategies of Ent. sakazakii is an important step in the efforts to eliminate this bacterium from the critical food production environments.     

Rapid detection of Enterobacter sakazakii using TaqMan real-time PCR assay

Enterobacter sakazakii is an emerging food pathogen, which induces severe meningitis and sepsis in neonates and infants, with a high fatality rate. The disease is generally associated with the ingestion of contaminated infant formula. In this study, we describe the development of a real-time PCR protocol to identify E. sakazakii using a TaqMan probe, predicated on the nucleotide sequence data of the 16S rRNA gene obtained from a variety of pathogens. To detect E. sakazakii, four primer sets and one probe were designed. Five strains of E. sakazakii and 28 non-E. sakazakii bacterial strains were used in order to ensure the accuracy of detection. The PCR protocol successfully identified all of the E. sakazakii strains, whereas the 28 non-E. sakazakii strains were not detected by this method. The detection limits of this method for E. sakazakii cells and purified genomic DNA were 2.3 CFU/assay and 100 fg/assay, respectively. These findings suggest that our newly developed TaqMan real-time PCR method should prove to be a rapid, sensitive, and quantitative method for the detection of E. sakazakii.     

The growth profile, thermotolerance and biofilm formation of Enterobacter sakazakii grown in infant formula milk

AIMS: To study the growth, thermotolerance and biofilm formation of the emergent pathogen Enterobacter sakazakii in infant formula milk (IFM). METHODS AND RESULTS: The temperature range, death kinetics and biofilm formation of E. sakazakii were determined using impedance microbiology and conventional methods. In IFM the organism grew as low as 6 degrees C and optimally at 37-43 degrees C. In faecal coliform tests, 23% of strains (n = 70) produced gas from lauryl sulphate broth (LSB) at 44 degrees C after 48 h incubation. Three strains failed to grow in LSB at any of the temperatures. The D-value of cells suspended in IFM was determined between 54 and 62 degrees C. The resultant z-value was 5.7 degrees C. The organism was able to adhere and grow on latex, polycarbonate, silicon and to a lesser extent stainless steel. CONCLUSIONS: Enterobacter sakazakii was able to grow at refrigeration temperatures and on infant-feeding equipment. The thermotolerance of the organism was similar to other Enterobacteriaceae and should be killed during standard pasteurization treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: Enterobacter sakazakii has been associated with infant meningitis through consumption of contaminated IFM. Enterobacter sakazakii is able to grow in IFM during storage at refrigeration temperatures and attach to infant-feeding equipment, which may become reservoirs of infection.     

Cronobacter ('Enterobacter sakazakii'): current status and future prospects

The genus Cronobacter accommodates the 16 biogroups of the emerging opportunistic pathogen known formerly as Enterobacter sakazakii . Cronobacter spp. are occasional contaminants of milk powder and, consequently, powdered infant formula and represent a significant health risk to neonates. This review presents current knowledge of the food safety aspects of C ronobacter , particularly in infant formula milk powder. Sources of contamination, ecology, disease characteristics and risk management strategies are discussed. Future directions for research are indicated, with a particular focus on the management of this increasingly important bacterium in the production environment.     

Fate of Bacteria in Chicken Meat During Freeze-Dehydration, Rehydration, and Storage

Total plate counts were determined on boneless cooked, cubed chicken meat obtained from a commercial processor. Survival of the natural flora was determined after the meat was freeze-dehydrated and rehydrated at room temperature for 30 min and 50, 85, and 100 C for 10 min. Total counts of bacteria in the rehydrated samples were determined during storage of the meat at 4, 22, and 37 C until spoilage odor was detectable. Meat samples were inoculated with Staphylococcus aureus, then dried, rehydrated, and stored at the same temperatures. Numbers of surviving organisms in the inoculated samples were determined with use of both selective and nonselective media. Representative genera surviving the various rehydration treatments were determined. Approximately 32% of the bacteria in the meat survived during dehydration and rehydration at room temperature. Many numbers and types of vegetative bacteria also survived rehydration at 50 C. When meat was rehydrated at 85 or 100 C, the initial count was less than one per gram. The only organisms isolated from samples rehydrated at 85 or 100 C were of the genus Bacillus. S. aureus in inoculated samples survived dehydration and rehydration at 60 C. Storage of all rehydrated samples at 4 C gave a good shelf life (18 or more days). The study indicates that freeze-dehydrated meat should be produced with adequate microbiological control and that such meat should be rehydrated in very hot water.