Enterobacter Sakazakii Strains Research Articles

Novel duplex real-time Taqman PCR assay for rapid detection of Enterobacter sakazakii in powdered infant formula

To develop a dulplex real-time Taqman polymerase chain reaction(PCR)assay for the detection of Enterobacter sakazakii. Primers and probes were designed based on the sequences of the macromolecular synthesis(MMS)operon and outer member protein A(ompA). Sensitivity and specificity of the assay were evaluated. Detection limit of the assay in pure culture was 4.3×103 CFU/mL and as few as 2 CFU/100 g of Enterobacter sakazakii could be detected in artificially contaminated infant formula through 24 h of enrichment. All of 15 Enterobacter sakazakii strains were successfully identified,no specific amplification were presented when 24 non-Enterobacter sakazakii were tested. The duplex real time Taqman PCR assay developed in study was sensitivity,specific and rapid. It could be used for detection of Enterobacter sakazakii from infant milk powder for food safety.

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.

DETECTION AND DISCRIMINATION OF ENTEROBACTER SAKAZAKII (CRONOBACTER SPP.) BY MID‐INFRARED SPECTROSCOPY AND MULTIVARIATE STATISTICAL ANALYSES

ABSTRACTEnterobacter sakazakii can cause rare but life‐threatening diseases such as meningitis in infants and neonates. Fourier transform infrared (FT‐IR) spectroscopy was used to detect and discriminate between eight E. sakazakii strains, two Enterobacter cloacae strains, three Escherichia coli strains and two Klebsiella pneumoniae strains. FT‐IR vibrational combination bands reflect subtle compositional differences in the cell membranes of E. sakazakii strains, especially in the region between 1,200 and 900 cm−1which contains absorption bands from carbohydrates. Two multivariate statistical analyses including principal component analysis (PCA) and soft independent modeling of class analogy (SIMCA) were used for data analysis. E. sakazakii strains were clearly distinguishable from the other strains by PCA. Based upon SIMCA analysis, 90% of E. sakazakii, 88% of E. cloacae, 91% of E. coli and 91% of K. pneumoniae samples were correctly classified, suggesting that this technique could be used to detect E. sakazakii strains rapidly and accurately.PRACTICAL APPLICATIONSFourier transform infrared (FT‐IR) coupled with multivariate statistical analyses can be used to detect, discriminate and identify Enterobacter sakazakii strains that have been implicated in food safety incidents caused by contaminated infant formula. Compared with traditional microbiological plating methods, this new approach of using FT‐IR could be an alternative means for rapid and accurate detection of bacterial samples that are important in agricultural, food and medical areas.

IDENTIFICATION OF PROTEINS INVOLVED IN INFECTIVITY AND ENTEROTOXIN PRODUCTION IN ENTEROBACTER SAKAZAKII

ABSTRACTEnterobacter sakazakii is a gram‐negative rod bacterium that was formerly known as “yellow‐pigmented Enterobacter cloacae” until 1980. It is an emerging opportunistic pathogen associated with bacterial meningitis in immunocompromised neonates who ingested contaminated powdered infant formula. In several E. sakazakii‐related outbreaks and sporadic cases, powdered infant formula was epidemiologically or microbiologically established as the source of infection. The International Commission on Microbiological Specifications for Foods has ranked E. sakazakii as a “severe hazard for restricted populations.” However, the organism was isolated at very low levels from commercial powdered infant formula and dry environmental samples collected from infant formula factories. In most cases, the contamination did not exceed 1 cfu per 100 g. Progress in understanding the epidemiology of E. sakazakii outbreaks and instituting effective control measures has been poor to date. To clarify the mechanisms involved in infectivity and enterotoxin production, we performed two‐dimensional gel electrophoresis (2‐DE) coupled with matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry and isotope‐coded affinity tags (ICATs)‐LC ‐MS to identify and quantify differences in protein expression between the E. sakazakii strains. We found 23 spots in 2‐DE and 37 proteins in ICAT analysis that were differentially expressed in E. sakazakii. These two methods gave us closely related but different information, suggesting that the two methods are complementary or at least supplementary.PRACTICAL APPLICATIONS Enterobacter sakazakii has been implicated in meningitis or enteritis with 20% to >50% mortality of infants who contract the disease. Our research has been used in verification of Enterobacter sakazakii isolated from infant formula and other milk and milk related foods. We have established standard maps of two Enterobacter sakazakii strains which were isolated from infant formula. Through appearance or absence of specific spots and their isoelectric point or molecular weight comparison, a two dimensional electrophoresis standard map can be developed and used as a powerful tool to differentiate Enterobacter sakazakii from other Enterobacteriaceae. This offers microbiologists powerful tools for Enterobacter sakazakii detection and provides more basic information to investigate the pathogenesis of Enterobacter sakazakii and contamination sources in infant formula production.

Effects of the Reconstitution and Storage Conditions of Powdered Infant Formula (PIF) on the Survival and Growth of Enterobacter sakazakii

The effect of the reconstruction and storage conditions of powdered infant formula (PIF) on the survival and growth of three Enterobacter sakazakii strains, ATCC 29004, HT 022 and HT 028, was investigated. D values of E. sakazakii ATCC 29004 and HT 022 at 60 degrees C were 3.6 and 1.6 min, respectively, and that of HT 028 at 52 degrees C was 1.6 min. The effect of the temperature of the water used for the reconstruction of PIF on the inactivation of the three E. sakazakii strains was also investigated. One to 2 log order inactivation occurred at 70 degrees C, and above 5 log order inactivation at 80 degrees C. Storage tests at 5, 10 and 25 degrees C showed that none of the strains could grow at 5 degrees C, HT 028 grew slightly at 10 degrees C, and at 25 degrees C all three strains started growth after 4 hr incubation and reached up to 8 log CFU/mL after 16 hr incubation. From the above results, it is concluded that a suitable temperature of the hot water for reconstruction of PIF is above 70 degrees C, and the preferred storage temperature of reconstructed PIF, which is recommended to be consumed within 2 hr, is below 5 degrees C.

Enterobacter sakazakii survives spray drying

Four strains of Enterobacter sakazakii were inoculated into 35% reconstituted skim milk at 107 and 102 cfu/g dry wt. After spray‐drying in a Buchi mini spray drier (inlet 160°C and outlet 90°C), the resulting powders were analysed for E. sakazakii by enrichment and enumeration. In all cases, E. sakazakii survived the spray drying process and were detected in the powders with a low inoculum and enumerated in all the powders with the high inoculum for at least 12 weeks. The results emphasize that the controls in place to prevent E. sakazakii from getting to the spray drier are essential.

BIOFILM FORMATION BY ENTEROBACTER SAKAZAKII GROWN IN ARTIFICIAL BROTH AND INFANT MILK FORMULA ON PLASTIC SURFACE

ABSTRACT Biofilm‐forming characteristics of 72 different Enterobacter sakazakii strains on plastic surface were tested. And the influence of the artificial growth medium and infant milk formula (IMF) was also determined. Quantification of biofilm formation was performed with medium including brain heart infusion broth, nutrient broth, tryptic soy broth, 1/20 diluted tryptic soy broth (1/20‐TSB) and differently diluted IMF on plastic microtiter plates. According to the result, reconstituted IMF was the more effective medium for biofilm formation than artificial medium. At normal IMF concentration, 56 strains produced biofilm (77.8%) but most suitable artificial medium and 1/20‐TSB; 12 strains produced biofilm (16.7%). These results indicated that the IMF offers more opportunities of biofilm formation on plastic surface. Therefore, all bottles and equipment should be effectively cleaned for preventing biofilm formation by IMF and E. sakazakii.PRACTICAL APPLICATIONS E. sakazakii could produce biofilm and protect the cells from being affected by disinfectants. It is also more difficult to wash such bacteria off from the surface they attached with biofilm and the chance of cross‐contamination could increase. The ability of biofilm formation could be influenced by different strains and also the growing media. By this study, we observed that IMF is the most effective media for biofilm formation among all the other media tested. Since IMF is the most important vehicle and source of E. sakazakii, it is important to surely clean and disinfect all the processing facilities in the factory and all the utensils, bottles, or teats used for IMF preparation. As biofilm would be formed when contact time increased, it is recommended not to sit the feeding bottles with IMF for a prolonged time to prevent biofilm formation and therefore minimize the opportunity of E. sakazakii infection.

Evaluation of multiple strains of Enterobacter sakazakii using fatty acid profiles

Fatty acid profiles are useful for identifying Gram-negative Enterobacter sakazakii strains within the family Enterobacteriaceae. The majority of cases of E. sakazakii infection have involved sepsis, meningitis, or enteritis, especially in neonates and infants. Gas chromatography with flame ionization detection (GC-FID) was utilized for the analysis of cellular fatty acid methyl esters (FAMEs). Thirty E. sakazakii strains isolated from food and environmental sources were cultured for 24 h on brain heart infusion (BHI) agar on three different days at 37 °C. Whole cell FAMEs were obtained by saponification, methylation and extraction into hexane:methyl tert-butyl ether. The day to day variations for the 30 E. sakazakii strains for each fatty acid were determined. Gram-negative bacteria commonly contain combinations of straight-chain, unsaturated, hydroxyl, and cyclo fatty acids. Major fatty acids of E. sakazakii strains evaluated in this study were straight chain 12:0, 14:0, 16:0 and unsaturated 16:1, 18:1, and 17:0 ω cyclo 7–8. Analysis of FAMEs from E. sakazakii strains grown on BHI agar by this rapid GC-FID method is highly reproducible and provides a sensitive procedure for identification of this organism. The fatty acid profile assay could be used to rapidly screen infant formula samples for E. sakazakii and reduce the time required for the current assay by up to 5 days.

Dry Stress and Survival Time of Enterobacter sakazakii and Other Enterobacteriaceae in Dehydrated Powdered Infant Formula

Powdered infant formula is not a sterile product, and opportunistic pathogens could multiply in the reconstituted product, resulting in neonatal infections. In this study, the generation of sublethally injured Enterobacteriaceae during desiccation and their persistence in dehydrated powdered infant formula was assessed during a 2.5-year period. The study included 27 strains of Enterobacter sakazakii, Enterobacter cloacae, Salmonella Enteritidis, Citrobacter koseri, Citrobacter freundii, Escherichia coli, Escherichia vulneris, Pantoea spp., Klebsiella oxytoca, and Klebsiella pneumoniae. The number of sublethally injured cells generated during desiccation was lower for K. oxytoca, Pantoea spp., Salmonella Enteritidis, and capsulated strains of E. sakazakii than for the other Enterobacteriaceae. The Enterobacteriaceae could be divided into three groups with respect to their long-term survival in the desiccated state. C. freundii, C. koseri, and E. cloacae were no longer recoverable after 6 months, and Salmonella Enteritidis, K. pneumoniae, and E. coli could not be recovered after 15 months. Pantoea spp., K. oxytoca, and E. vulneris persisted over 2 years, and some capsulated strains of E. sakazakii were still recoverable after 2.5 years.

Differentiation of Enterobacter sakazakii from Closely Related Enterobacter and Citrobacter Species Using Fatty Acid Profiles

Capillary gas chromatography with flame ionization detection (GC-FID) was used to determine the cellular fatty acid (CFA) profiles of 134 Enterobacter sakazakii strains, and these were compared to the CFA profiles of other closely related Enterobacter and Citrobacter species. For GC-FID analysis, whole cell fatty acid methyl esters (FAMEs) from cells cultured on brain heart infusion (BHI) agar at 37 degrees C for 24 h were obtained by saponification, methylation, and extraction into hexane/methyl tert-butyl ether. A database for E. sakazakii was prepared using fatty acid profiles from the 134 strains. Major fatty acids of E. sakazakii strains evaluated in this study were straight-chain 12:0, 14:0, and 16:0, unsaturated 18:1 omega7c, and 17:0 omegacyclo 7-8. Principal component analysis (PCA) based on CFA profiles for E. sakazakii strains shows separation of E. sakazakii subgroups A and B. The CFA profiles for E. sakazakii and Enterobacter cloacae show that there are several fatty acids, 14:0, 17:0 omegacyclo 7-8, 18:1 omega7c, and summed 16:1 omega6c/16:1 omega7c, that differ significantly between these two species. A PCA model based on CFA profiles for E. sakazakii strains clearly shows separation of E. sakazakii from closely related Enterobacter and Citrobacter species. Analysis of FAMEs from E. sakazakii strains grown on BHI agar by a rapid GC-FID method can provide a sensitive procedure for the identification of this organism, and this analytical method provides a confirmatory procedure for the differentiation of E. sakazakii strains from closely related Enterobacter and Citrobacter species.

Effect of High-Pressure Processing on Strains of Enterobacter sakazakii

Four strains of Enterobacter sakazakii were inoculated into tryptic soy broth and reconstituted powdered infant formula and exposed to high-pressure processing. Pressures of 200, 400, and 600 MPa were used for each medium for 1 min. E. sakazakii was reduced by more than 6 log (strains A and B) in both media at 600 MPa. Strain B was significantly (P ≤ 0.05) more pressure resistant than the other strains, with just more than a 3-log reduction at 600 MPa in both media. The reconstituted infant formula has a significant (P ≤ 0.05) protective effect for certain strains and pressures (strain B at 400 MPa and strain D at 400 and 600 MPa). Differences in log reductions between media (milk and broth) were also observed for certain strains and specific pressures (strain B at 400 MPa and strain D at 400 and 600 MPa; P ≤ 0.05). This research showed that E. sakazakii, when present in reconstituted powdered infant formula, can be submitted to high-pressure processing (600 MPa for 1 min) and achieve log reductions ranging from 3 to 6.84, depending on the strain.

Biofilm Formation, Extracellular Polysaccharide Production, and Cell-to-Cell Signaling in Various Enterobacter sakazakii Strains: Aspects Promoting Environmental Persistence

Enterobacter sakazakii is considered an opportunistic pathogen and has been implicated in food-associated cases of meningitis or enteritis, especially in neonates and infants. The organism has been detected in various types of food and in food production units, but so far only powdered infant formula has been linked to outbreaks of disease. Survival and persistence in such environments requires the ability to adapt to high osmotic potentials and/or dry conditions. Fifty-six E. sakazakii strains were evaluated for several features important for persistence and survival: (i) biofilm formation and the putative production of cellulose as one of the components of the extracellular matrix, (ii) adherence to hydrophilic and hydrophobic surfaces, (iii) the production of extracellular polysaccharides, and (iv) the ability of E. sakazakii to produce cell-to-cell signaling molecules. Pellicle and flock formation was observed in 21 of the strains grown in Luria-Bertani broth and in 44 of the strains grown in brain heart infusion broth. Calcofluor-stained fibrils, observed microscopically in every (fragile or rigid) pellicle, suggested the presence of cellulose as an extracellular compound in this type of biofilm. Twelve isolates did not form any pellicle or flocks under either condition. Twenty-three of the isolates exhibited the potential to adhere to glass surfaces in shaken cultures, and 33 strains showed biofilm formation at the air-solid interface of polyvinyl chloride microtiter wells. Sixteen isolates adhered to both surfaces. Twenty-four of the isolates tested produced a milky, viscous mass, considered as extracellular polysaccharide. High-performance liquid chromatography analysis of the polysaccharide revealed the presence of glucose, galactose, fucose, and glucuronic acid. Thin-layer chromatography analyses performed on ethyl acetate extracts of cell-free supernatants of the 56 strains indicated the presence of two different types of acylated homoserine lactones (3-oxo-C6-HSL and 3-oxo-C8-HSL). These findings illustrate the ability of E. sakazakii to produce cell-to-cell signaling molecules.

Detection of Enterobacter sakazakii Strains by Real-Time PCR

A precise 5′ nuclease (TaqMan) real-time PCR was developed and validated in house for the specific detection of Enterobacter sakazakii isolates. Specifically designed nonpatented primers and a hydrolysis (TaqMan) probe were used to target the 16S rRNA gene. All 27 E. sakazakii and 141 non–E. sakazakii strains tested with the real-time PCR were identified correctly. To monitor false-negative results, an internal amplification control was coamplified with the same primers used for the E. sakazakii DNA. The detection probability of the assay was 56% when an E. sakazakii cell suspension containing 102 CFU/ml was used as template in the PCR (0.5 CFU per reaction) and 100% with a 103 CFU/ml suspension. This PCR assay should be very useful for the diagnostic detection of E. sakazakii in foods, especially powdered infant formula, after cultural enrichment.

16S rRNA gene based analysis of Enterobacter sakazakii strains from different sources and development of a PCR assay for identification

BackgroundE. sakazakii is considered to be an opportunistic pathogen, implicated in food borne diseases causing meningitis or enteritis especially in neonates and infants. Cultural standard identification procedures for E. sakazakii include the observation of yellow pigmentation of colonies and a positive glucosidase activity. Up to now, only one PCR system based on a single available 16S rRNA gene sequence has been published for E. sakazakii identification. However, in our hands a preliminary evaluation of this system to a number of target and non-target strains showed significant specificity problems of this system. In this study full-length 16S rRNA genes of thirteen E. sakazakii strains from food, environment and human origin as well as the type strain ATCC 51329 were sequenced. Based on this sequence data a new specific PCR system for E. sakazakii was developed and evaluated.ResultsBy phylogenetic analysis of the new full-length 16S rRNA gene sequence data obtained we could show the presence of a second phylogenetic distinct lineage within the E. sakazakii species. The newly developed 16S rRNA gene targeting PCR system allows identification of E. sakazakii strains from both lineages. The assay's ability to correctly identify different E. sakazakii isolates as well as to differentiate E. sakazakii from other closely related Enterobacteriaceae species and other microorganisms was shown on 75 target and non-target strains.ConclusionBy this study we are presenting a specific and reliable PCR identification system, which is able to correctly identify E. sakazakii isolates from both phylogenetic distinct lines within the E. sakazakii species. The impact of this second newly described phylogenetic line within the E. sakazakii species in view of clinical and food safety aspects need further investigation.

DNA relatedness between Enterobacter sakazakii and other members of the genus Enterobacter

A DNA-DNA hybridization study (nitrocellulose filter method) was carried out with 13 strains of Enterobacter sakazakii and 38 strains belonging to other Enterobacter species ( E. cloacae, E. amnigenus, E. intermedium and E. gergoviae). E. sakazakii strains were highly related (mean relative binding ratio ± standard deviation: 89 % ± 10) to the strain R 16–76 (labelled DNA). No close relationship was found with the other Enterobacter species. The low relative binding ratios were not due to a difference in genome size (as shown by reciprocal binding experiments).

DNA relatedness between Enterobacter Sakazakii and other members of the genus Enterobacter

A DNA-DNA hybridization study (nitrocellulose filter method) was carried out with 13 strains of Enterobacter sakazakii and 38 strains belonging to other Enterobacter species (E. cloacae, E. amnigenus, E. intermedium and E. gergoviae). E. sakazakii strains were highly related (mean relative binding ratio +/- standard deviation: 89% +/- 10) to the strain R 16-76 (labelled DNA). No close relationship was found with the other Enterobacter species. The low relative binding ratios were not due to a difference in genome size (as shown by reciprocal binding experiments).