A selective differential medium for Enterobacter sakazakii, a preliminary study

Enterobacter sakazakii can cause fatal invasive infection of neonates associated with the presence of this organism in powdered infant milk formula. A new chromogenic medium (Druggan-Forsythe-Iversen agar, DFI) is described for the selective detection of this emergent pathogen. The medium is based on the alpha-glucosidase reaction which is detected using 5-bromo-4-chloro-3-indolyl-alpha,D-glucopyranoside (XalphaGlc). Ent. sakazakii hydrolyses this substrate to an indigo pigment, producing blue-green colonies on this medium. DFI was compared with the current method of detection on violet red bile glucose agar (VRBGA) followed by pigment production on tryptone soy agar (TSA) after 48-72 h at 25 degrees C and subsequent biochemical profile determination using Biomerieux API20E. Ninety-five clinical and food strains of Ent. sakazakii were detected on the DFI chromogenic medium 2 days sooner than the alternative method. The characteristics of 148 strains representing 17 genera of non-Ent. sakazakii Enterobacteriaceae were compared using the two methods. Only 16/18 Escherichia vulneris strains, 2/3 strains of Pantoea spp. and 1/8 Citrobacter koseri strains gave false positive results on DFI agar. Eight alpha-glucosidase positive strains were identified as Pantoea using their API20E biochemical profile, but had higher percentage identification as Ent. sakazakii using ID32E. Therefore the DFI medium enables the detection of Ent. sakazakii within mixed cultures of Enterobacteriaceae, whereas the organism could be missed when using VRBGA since the latter is a general Enterobacteriaceae selective medium. In addition, the common use of API20E to check yellow pigmented colonies on TSA may lead to false negative results and consequently the acceptance of a batch of infant formula milk (IFM) that contains Ent. sakazakii.     

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.     

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.     

Evaluation of a new enrichment broth for detection of Cronobacter spp. in powdered infant formula

The aim of this study was to investigate the potential of using Al-Holy-Rasco (AR) medium, a novel broth for detection and isolation of Cronobacter spp. in infant formula milk (IFM). The new medium's composition is generic brain heart infusion broth with the addition of 1% NaCl, 15% sucrose, and 0.80 g/liter sodium deoxycholate as selective ingredients. AR broth outperformed Enterobacteriaceae enrichment broth (EE), Enterobacter sakazakii enrichment broth (ESE), modified lauryl sulfate broth, and milk as enrichment media to stimulate the growth of a cocktail of 10 strains of Cronobacter. Additionally, AR broth significantly suppressed the growth of competing non-Cronobacter Enterobacteriaceae as compared with EE, ESE, modified lauryl sulfate broth, and milk. The recovery of desiccated Cronobacter (1 to 5,000 CFU/100 g) from powdered IFM in the presence of competing non-Cronobacter Enterobacteriaceae was determined by EE, ESE, and AR broth with 10 and 15% sucrose. AR broth with 15% sucrose outperformed all other examined broths and recovered Cronobacter from all samples tested at all Cronobacter concentrations. AR broth must be validated before it can be used for rapid detection and isolation of Cronobacter from powdered IFM.     

Short communication: Effects of vacuum freeze-drying on inactivation of Cronobacter sakazakii ATCC29544 in liquid media with different initial inoculum levels

Vacuum freeze-drying is an important food-processing technology for valid retention of nutrients and bioactive compounds. Cronobacter sakazakii has been reported to be associated with severe infections in neonates through consumption of contaminated powdered infant formula. In this study, effects of vacuum freeze-drying treatment for 12, 24, and 36 h on inactivation of C. sakazakii with different initial inoculum levels in sterile water, tryptic soy broth (TSB), skim milk, and whole milk were determined. Results indicated that the lethality rate of C. sakazakii in each sample increased with the extension of vacuum freeze-drying time. With initial inoculum levels of 10² and 10³ cfu/mL, the survival of C. sakazakii in different liquid media was significantly affected by vacuum freeze-drying for 12, 24, and 36 h. In addition, the lethality rates of C. sakazakii in whole milk, skim milk, and TSB was significantly reduced compared with those in sterile water. Furthermore, whole milk showed the strongest protective role for C. sakazakii cells, followed by skim milk and TSB medium. Using the scanning electron microscope, the intracellular damage and obvious distortion of C. sakazakii cells were observed after vacuum freeze-drying for 24 and 36 h compared with the untreated sample, and the injured cells increased with the extension of vacuum-drying time. We concluded that inactivation of vacuum freeze-drying on C. sakazakii cells is related to the food matrix, and a combination with other methods for inactivating C. sakazakii is required for ensuring microbial safety of powdered infant formula.     

The phenotypic and genotypic characterization of Enterobacter sakazakii strains from infant formula milk

Enterobacter sakazakii is an emerging foodborne pathogen associated with severe diseases in neonates. Infant formula milk (IFM) has been identified as one of the major contaminated sources and a transmission vehicle. To determine the phenotypic and genotypic characterization of this pathogen, 22 E. sakazakii strains isolated from IFM by an FDA-recommended method and PCR on the α-glucosidase gene were subtyped by random amplified polymorphic DNA (RAPD)-PCR, enterobacterial repetitive intergenic consensus (ERIC)-PCR, and antibiotic resistance patterns. At a similarity threshold of 80%, 16 ERIC-PCR fingerprint types were identified with a discriminatory power (D) of 0.933, and 18 RAPD-PCR types were identified with D of 0.973. Resistance to 9 antibiotics tested by disk diffusion assay revealed 6 antibiotic resistance patterns with D of 0.749. The comparison of characterization indicated that RAPD-PCR and ERIC-PCR have high discriminatory power showing genetic diversity of E. sakazakii isolates, and ERIC-PCR patterns showed a closer correlation than RAPD-PCR patterns to phenotypic characterization and the brands of IFM. Overall, the ERIC-PCR typing method could be used for tracing sources of E. sakazakii isolates in the food chain.     

Multiplex PCR coupled with propidium monoazide for the detection of viable Cronobacter sakazakii,Bacillus cereus,and Salmonella spp. in milk and milk products

Cronobacter sakazakii, Bacillus cereus, and Salmonella spp. are common food-borne pathogens. The aim of this study was to develop a sensitive, specific, and rapid method for the simultaneous detection of these 3 pathogens in milk and milk products. Three specific primers were designed based on ompA, invA, and cesB of C. sakazakii, Salmonella spp. and B. cereus, respectively, for use in a multiplex PCR (mPCR). To eliminate false-positive results, cells were pretreated with propidium monoazide (PMA) for the selective elimination of the genomic DNA of dead cells. An internal amplification control was applied as an indicator of false-negative results from the interference of inhibitors in the food matrix. Results showed that, in pure culture, the limits of detection of the assay for C. sakazakii, Salmonella Enteritidis, and B. cereus were 9.5 × 10⁴, 7.4 × 10², and 7.5 × 10² cfu/mL, respectively. Moreover, 8 cfu/mL of viable B. cereus cells were detected after 5 h of enrichment, and 9 cfu/mL of viable C. sakazakii and 7 cfu/mL of Salmonella Enteritidis were detected after 7 h of enrichment in spiked pure milk, walnut peanut milk, and whole-wheat milk. To validate the PMA-mPCR assay, the PMA-mPCR assay and the traditional culture method were performed to detect the 3 bacterial strains in 1,165 milk product samples. The PMA-mPCR assay obtained the same results as the culture-based method. Results demonstrated that the PMA-mPCR assay has excellent sensitivity and specificity for the simultaneous detection of viable C. sakazakii, Salmonella Enteritidis, and B. cereus in milk and milk products.     

Inhibition of Cronobacter sakazakii by Heat Labile Bacteriocins Produced by Probiotic LAB Isolated from Healthy Infants

Cronobacter sakazakii is an opportunistic pathogen that can cause bacteremia, meningitis, and necrotizing enterocolitis, most often in neonates with case‐fatality rates that may reach 80%. The antimicrobial activity of lactic acid bacteria against a wide range of foodborne pathogens is well‐established in different types of food products. The objective of the current study was to investigate the antibacterial activity of Lactobacillus acidophilus and L. casei isolated from feces of healthy infants against different strains of C. sakazakii in agar and a rehydrated infant milk formula (RIMF) model. The inhibition zones of C. sakazakii around L. acidophilus or L. casei ranged from 22 to 32 mm on eMan Rogosa Sharpe (MRS) agar under aerobic conditions, while a slight reduction in antibacterial activity was noted on modified MRS (0.2% glucose) under anaerobic conditions. It was observed that pH‐neutralized cell‐free supernatant (CFS) of L. acidophilus or L. casei was inhibitory against tested C. sakazakii strains. The inhibition zones of neutralized CFS were lower than the antibacterial activities of live cultures. The antibacterial activity of CFS was abolished when CFS from L. acidophilus or L. casei was heated at 60 or 80 °C for either 10 min or 2 h, or treated with trypsin or pepsin. This was considered strong evidence that the inhibition was due to the production of bacteriocins by L. casei and L. acidophilus. Both the CFS and active growing cells of L. casei and L. acidophilus were able to reduce the viability of C. sakazakii in the RIMF model. The results may extend the use of natural antimicrobials instead of conventional preservation methods to improve the safety of RIMF.     

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.     

Prevalence and characterization of Salmonella enterica in dried milk-related infant foods in Shaanxi,China

The aim of this study was to investigate the existence and characteristics of Salmonella enterica in dried milk-related infant foods. Twenty-four (3.4%) of 705 samples, including 5 (2.0%) of 246 powdered infant formula, 18 (4.0%) of 445 infant rice cereal, and 1 (7.1%) of 14 other infant foods, were positive for Salmonella. Fifteen serotypes were identified in 40 Salmonella isolates; Salmonella Duesseldorf (15.0%) and Salmonella Indiana (15.0%) were more frequently detected than other serotypes. Resistance to chloramphenicol (82.5%) was most common, followed by tetracycline (57.5%), ceftiofur (52.5%), kanamycin (52.5%), streptomycin (50.0%), gentamycin (45.0%), nalidixic acid (35.0%), ceftriaxone (32.5%), ciprofloxacin (25.0%), amikacin (20.0%), and cefoxitin (15.0%). Twenty-eight (70.0%) isolates were resistant to ≥8 antimicrobials, with 5 (12.5%) being resistant to 14 antimicrobials. Amino acid substitutions in gyrase A (GyrA) were most frequently detected as Ser83Arg/Asp87Glu and in p53-associated Parkin-like cytoplasmic protein (ParC), they were all Ser80Arg; the quinolone resistance gene qnrS (47.5%) was commonly detected as well as aminoglycoside acetyltransferase [aac(6′)-Ib; 25.0%], qnrA (17.5%), and qnrB (15.0%) genes. Thirty distinct pulsed-field gel electrophoresis patterns were identified among 40 isolates; no identical pulsed-field gel electrophoresis pattern was detected among Salmonella isolates with the same serovar that was recovered in 2010 and 2012. Our results suggest that dried milk-related infant foods could be contaminated with Salmonella and highlight that the dangers to infant health should not be neglected.     

Evaluation of different methods for the detection and identification of Enterobacter sakazakii isolated from South African infant formula milks and the processing environment

Enterobactersakazakii is an emerging pathogen associated with life-threatening neonatal infections resulting from the consumption of contaminated powdered infant formula milk (IFM). Recent taxonomic analyses have determined that E. sakazakii comprises a number of genomospecies, and it has been proposed that E. sakazakii be reclassified as a novel genus, "Cronobacter". Accurate methods are required for the rapid detection and identification of this group of micro-organisms, since even low cell numbers have been reported to cause disease. The aim of this study was to evaluate various E. sakazakii detection methods in order to ascertain the most suitable method for detection and identification of these pathogenic agents. Samples from IFM and the environment were evaluated for the presence of E. sakazakii using the isolation steps (pre-enrichment, enrichment and selection) described in the Food and Drug Administration (FDA) method for E. sakazakii detection. Sixty-four isolates (50 from IFM and 14 from the environment) were selected from tryptone soy agar (TSA), regardless of colony appearance, and these isolates were identified by 16S ribosomal DNA (rDNA) sequencing. Thereafter, different culture-dependent and culture-independent methods were evaluated to accurately detect and identify the E. sakazakii isolates. These methods included the assessment of yellow pigment production on TSA, typical colonies on chromogenic Druggan-Forsythe-Iversen (DFI) and Chromocult(R) Enterobacter sakazakii (CES) media and polymerase chain reaction (PCR) using six different species-specific primer pairs described in the literature. Identification of E. sakazakii using yellow pigment production was demonstrated to have a low sensitivity, specificity and accuracy (87%, 71% and 74%, respectively), which lowers the suitability of the FDA method. Chromogenic DFI and CES media were sensitive, specific and accurate (100%, 98% and 98%, respectively) for the detection of E. sakazakii. The specificity of the PCR amplifications ranged from 8% to 92%, emphasising the need for rigorous primer testing against closely related species. Of the primer pairs evaluated, Esakf/Esakr were the most suitable for E. sakazakii detection and identification. The detection limit of Esakf/Esakr was found to be 10(4) CFU/ml. This study demonstrated that no single method was capable of unambiguously confirming the presence and identity of E. sakazakii isolates, that each method had inherent advantages and disadvantages, and that in most cases several methods were required for accurate detection and identification. Further, it was demonstrated that the current FDA method for E. sakazakii detection should be revised in the light of the availability of more sensitive, specific and accurate detection methods.     

Real-time PCR von Enterobacter sakazakii in Säuglingsanfangsnahrung

Enterobacter sakazakii represents a rare opportunistic pathogen that causes a high mortality rate. Especially newborns are infected by contaminated infant formula. To reduce this risk, the EU directive 2073/2005 demands for the absence of the pathogen in 30 ×10 g infant formula. We describe a PCR-/real-time PCR that specifically detected 32 E. sakazakii strains among 21 species of Enterobacteriaceae as well as contaminated specimens among 30 different brands of infant formula. This real-time PCR, performed after a selective enrichment of E. sakazakii, reduces the working time by 1 to 3 days in comparison to the detection methods recommended by the US Food and Drug Administration (FDA) and ISO/DTS 22964 that is still under revision. In contrast to the latter procedure, we detected contaminated specimens of infant formula by the use of the procedure described in this report and the FDA method.

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Zusammenfassung: Enterobacter sakazakii repr?sentiert einen seltenen opportunistischen Erreger, der Infektionen mit hoher Mortalit?tsrate hervorruft. Insbesondere Neugeborene erkranken durch kontaminierte Anfangsnahrung. Zur Risikominimierung fordert die EU-Verordnung 2073/2005 die Abwesenheit des Erregers in 30 ×10 g S?uglingsanfangsnahrung. Ein PCR-/Real-Time PCR-Verfahren wird beschrieben, das zun?chst mit 32 E. sakazakii -St?mmen und weiteren 20 Arten der Enterobacteriaceen geprüft und anschlie?end zur Untersuchung 30 unterschiedlicher Marken von S?uglingsanfangsnahrung verwendet wurde. Nach der Selektivanreicherung führt das Real-Time PCR-Verfahren zu einer Ersparnis von 1 bis 3 Arbeitstagen beim Nachweis von E. sakazakii im Vergleich zum Verfahren der amerikanischen Food and Drug Administration (FDA) und dem noch in Bearbeitung befindlichen Verfahren ISO/DTS 22964. Im Gegensatz zur letztgenannten Untersuchungsmethode erfolgten mehrere Positivnachweise mit dem hier beschriebenen Verfahren und dem der FDA.

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Eingegangen: 18. Januar 2007     

Comparative Study of a DNA Hybridization Method and the Conventional Culture Procedure for Detection of Salmonella in Foods

A commercial DNA hybridization assay (DNAH) for rapid detection of Salmonella in foods was compared to the conventional culture procedure. The DNAH method employed preenrichment, selective, and post-enrichment steps (44°hr) prior to performing the assay (4hr). Confirmation of positive DNAH assays was accomplished using standard culture methods for isolation of Salmonella. More than 1,600 samples were tested, representing 23 food types and including naturally contaminated, artificially inoculated, and uninoculated foods. Based on the data generated, the DNAH method was as productive as the standard culture method for detection of Salmonella in all foods and was significantly better than the culture method for certain foods.     

A simple and sensitive aptasensor with rolling circle amplification for viable Cronobacter sakazakii detection in powdered infant formula

Cronobacter sakazakii is a foodborne, emerging opportunistic pathogen that causes severe bacteremia, necrotizing enterocolitis, and sepsis with a mortality rate of up to 80%. In this study, we developed a simple and sensitive fluorescent turn-off aptasensor with rolling circle amplification assay for viable C. sakazakii detection in powdered infant formula. The results showed that the proposed aptasensor has good performance and specificity for detecting viable C. sakazakii in pure culture and powdered infant formula samples within 3 h. Under the optimal reaction conditions, there is a linear relationship between fluorescent intensity at 490 nm and logarithmic concentration of C. sakazakii in the range of 2.7 × 10⁵ to 2.7 × 10² cfu/mL, with a limit of detection of 2.7 × 10² cfu/mL in pure culture. The proposed aptasensor achieved a recovery of 104 to 111% in pure culture, and 96 to 107% in spiked powdered infant formula samples. The proposed aptasensor does not require complicated DNA extraction steps or antibodies, and can be performed at 37°C, making it a convenient and sensitive strategy for C. sakazakii detection.     

Screening of specific nucleic acid targets for Cronobacter sakazakii and visual detection by loop-mediated isothermal amplification and lateral flow dipstick method in powdered infant formula

Due to the lack of specific genes for rapid detection methods of Cronobacter sakazakii in food samples, whole genome sequence analysis was performed in this investigation using the basic local alignment search tool. Forty-two DNA fragments unique to C. sakazakii were mined, then primers were designed and screened by PCR and loop-mediated isothermal amplification (LAMP). Two primer sets, CS1 and CS31, were found as specific and stable primers, with their corresponding nucleic acid targets the CSK29544_00235 gene and CSK29544_03484 gene, respectively. Furthermore, compared with 3 genes reported previously, these 2 genes were verified as more specific to C. sakazakii among Cronobacter species, by sequence similarity alignment using Cronobacter MLST databases (http://pubmlst.org/cronobacter). The specificity of the LAMP reaction approached 100% by using 48 bacterial strains, which included 22 C. sakazakii strains. Subsequently, LAMP was combined with visual lateral flow dipstick (LFD) based on the above 2 nucleic acid targets, and was demonstrated as a rapid, efficient method with high specificity. Finally, the detection sensitivity of this assay system for pure cultures and artificially contaminated milk was measured as 4.5 × 10⁰ cfu/mL and 5.7 × 10¹ cfu/g, respectively. Total time to detection for this assay was within 2 h. Thus, the establishment of this LAMP-LFD method shows great significance and potential for rapid detection of C. sakazakii in powdered infant formula.     

Rapid detection of genus Cronobacter in powdered infant formula milk

Rapid and specific detection of Cronobacter spp. in powdered infant formula milk (IFM) is of great importance for health and safety reasons. In the present study, two rapid and specific methods, the immunochromatographic strip (ICT) and the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), were tested for the detection of Cronobacter spp. in IFM. IFM samples spiked by Cronobacter spp. were correctly detected as positive by both methods. These results were verified by the classical cultivation microbiological method (ISO/TS 22964:2006). All three methods were used for the analyses of 13 IMF samples from a local market with identical results. Only one IFM sample was found to be positive. Both tested methods considerably reduced the total detection time, to 24?h (ICT) and 46?h (MALDI-TOF MS), whereas the reference ISO/TS 22964:2006 method needs 140?h.     

奶粉中坂崎肠杆菌检测方法研究进展

坂崎肠杆菌为肠杆菌科肠杆菌属，奶粉（尤其是婴幼儿配方奶粉）中坂崎肠杆菌的污染可引发严重的公共卫生问题，其主要检测方法包括传统的细菌学培养方法及各种基于聚合酶链式反应（PcR）的分子生物学检测方法，改进的培养方法和快速特异的分子生物学方法已成为坂崎肠杆菌检测的重要发展方向。     

Increased thermal tolerance in Cronobacter sakazakii strains in reconstituted milk powder due to cross protection by physiological stresses

Cronobacter sakazakii (C. sakazakii) is an opportunistic, neonatal, and food borne pathogen primarily associated with the contamination of powdered infant formula (PIF). The pathogen is reported to overcome the food safety barriers such as increased acidity, heat treatment, and so on. This study evaluates the thermal tolerance of C. sakazakii strains independently at 52, 55, and 58°C in reconstituted PIF after exposure to physiological stresses: refrigeration (4°C for 24 hr), starvation (37°C for 48 hr), and desiccation (25°C for 4 days). The Log₁₀ CFU/ml and D-values indicated that survival rate of all the strains decreased significantly (p < .05) after desiccation as compared to those of the control condition (without stress exposure). However, cold stress increased the thermal tolerance of all strains at all temperatures (52, 55, and 58°C) as indicated by increased D-values. Among the tested strains, C. sakazakii strain N15 was found to be the most resistant to thermal treatment after each stress exposure as depicted by principal component analysis (PCA). No apparent correlation between thermal tolerance and starvation stress was observed. The findings indicate that prior exposure to stress conditions may induce cross protection to thermal treatment in C. sakazakii.     

Evaluation of reference samples for the detection of Salmonella

Reference samples consisting of spray-dried milk artificially contaminated with Salmonella typhimurium II 505 contained in a gelatin capsule have been developed to evaluate the standard method for the detection of Salmonellae in foods and feeding stuffs. The present study was carried out to evaluate these reference samples. Salmonella isolations from reference samples in the absence of food were satisfactory in 4 out of 6 laboratories when Müller-Kauffmann tetrathionate broth prepared according to ISO-6579 (TBB-ISO) was used and in all laboratories when the secondary enrichment medium was the magnesium chloride-malachite green broth of Rappaport-Vassiliadis (RV). The proportion of Salmonella isolations from reference samples decreased from 80% to 47% with TBB-ISO and from 95% to 63% with RV in the presence of food. Most negative results were obtained with foods such as minced pork and minced beef, which had high aerobic and Enterobacteriaceae plate counts. It is concluded that the reference samples can be used to test the performance of media and the reproducibility of methods when it is used without the addition of food. The results obtained for Salmonella isolations in the presence of food questioned the role of numbers and types of competitive micro-organisms. In particular these latter results indicated that more research is needed to gain a better understanding of the Salmonella isolation procedure.     

A New Validated Real-Time PCR-Based Method for the Specific and Fast Detection of Cronobacter spp. in Infant Formula

In this study, a real-time polymerase chain reaction (PCR)-based method was designed for the fast detection of Cronobacter spp. (a newly proposed genus formerly known as Enterobacter sakazakii) in infant formula. The real-time PCR was positively tested with 70 Cronobacter strains, including members of all the species of this genus, and 88 non-Cronobacter strains. This new PCR-based system was validated against the reference standard ISO/TS 22964: 2006 (ISO International Organization for Standardization 2006) using 70 food matrices including powdered infant formula, follow-up formula, and hydrolyzed cereals for infants. The detection limit of the technique was found to be of 1 cfu in 10 g of food, fulfilling the requirements of the European Commission. The time of analysis, which comprises around 3–6 days using the reference method, is considerably reduced to less than 24 h using the real-time PCR-based system hereby described, allowing food industry a faster release of the stocks for commercialization. Moreover, this method includes an internal amplification control, co-amplified during each PCR run to verify the results.