Influences of milk components on biofilm formation of Cronobacter spp. (Enterobacter sakazakii)

Aim:  To determine the critical component(s) of skim milk for biofilm formation of Cronobacter species.
Methods and Results:  Biofilm forming ability of 72 Cronobacter strains in skim milk preparation was assayed by crystal violet staining. The results revealed that whey protein and casein are more important determinants of skim milk for biofilm formation than lactose, although there was a wide variation in biofilm forming ability. Biofilm structure and capsular material of six strains exhibiting different biofilm forming ability was investigated via electron microscopes. Scanning electron microscopy showed visually that while the strong biofilm formers (E27B, FSM 30 and 2·82) resulted in almost complete coagulation of skim milk, the weak biofilm formers (55, FSM 290 and 2·84) caused less coagulation. No capsule was clearly delineated in transmission electron micrographs of either strong or weak biofilm formers.
Conclusion:  These results indicate that, for biofilm formation of Cronobacter species in skim milk, nitrogen source is probably a more important determinant than carbohydrate, and that strong biofilm formers are responsible for substantial coagulation of skim milk.
Significance and Impact of the Study:  This study provides information for better understanding of the underlying mechanisms by which Cronobacter species form biofilm in infant formula milk.     

Heat resistance of Cronobacter species (Enterobacter sakazakii) in milk and special feeding formula

Aim:  To determine D - and z -values of Cronobacter species ( Enterobacter sakazakii ) in different reconstituted milk and special feeding formula and the effect of reconstitution of powdered milk and special feeding formula with hot water on the survival of the micro-organism.
Methods and Results:  Five Cronobacter species (four C. sakazakii isolates and C. muytjensii ) were heated in reconstituted milk or feeding formula pre-equilibrated at 52–58°C for various times or mixed with powdered milk or feeding formula prior to reconstitution with water at 60–100°C. The D -values of Cronobacter at 52–58°C were significantly higher in whole milk (22·10–0·68 min) than in low fat (15·87–0·62 min) or skim milk (15·30–0·51 min) and significantly higher in lactose-free formula (19·57–0·66 min) than in soy protein formula (17·22–0·63 min). The z -values of Cronobacter in reconstituted milk or feeding formula ranged from 4·01°C to 4·39°C. Water heated to ≥70°C and added to powdered milk and formula resulted in a > 4 log₁₀ reduction of Cronobacter .
Conclusions:  The heat resistance of Cronobacter should not allow the survival of the pathogen during normal pasteurization treatment. The use of hot water (≥70°C) during reconstitution appears to be an effective means to reduce the risk of Cronobacter in these products.
Significance and Impact of the Study:  This study supports existing data available to regulatory agencies and milk producers that recommended heat treatments are sufficient to substantially reduce risk from Cronobacter which may be present in these products.     

Development of sandwich enzyme-linked immunosorbent assay for the detection of Cronobacter muytjensii (formerly called Enterobacter sakazakii)

This study aimed to produce a polyclonal antibody against Cronobacter muytjensii (C. muytjensii, formerly called Enterobacter sakazakii) and to develop an immunoassay for its detection. The optimum production of rabbit anti-C. muytjensii immunoglobulin G (IgG) and chicken anti-C. muytjensii IgY was reached in weeks 8 and 9, respectively. Purification of rabbit anti-C. muytjensii IgG from immunized rabbit sera was accomplished using the caprylic acid and ammonium sulfate precipitation method. As a result, sodium dodecyl sulfate-polyacrylamide gel electrophoresis produced two bands around 25 and 50 kDa, corresponding to a light and a heavy chain, respectively. The optimized conditions for sandwich enzyme-linked immunosorbent assay were using rabbit anti-C. muytjensii IgG (1 μg/mL) as a detection antibody and chicken anti-C. muytjensii IgY (10 μg/mL) as a capture antibody. In this assay, no cross-reactivity was observed with the other genera of pathogenic bacteria tested, which included Escherichia coli O157:H7, Salmonella typhimurium, Staphylococcus aureus, Bacillus cereus and Listeria monocytogenes. The developed assay did not show cross-reactivity with other tested species of Cronobacter and Enterobacter genera such as C. turicensis, C. sakazakii, E. aerogenes, E. pulveris and E. helveticus. The detection limit of sandwich ELISA for C. muytjensii was found to be 2.0 × 10(4) colony forming units (CFU)/mL. In addition, detection of C. muytjensii in infant formula powder showed a low matrix effect on the detection curve of sandwich ELISA for C. muytjensii, the detection limit being found to be 6.3 × 10(4) CFU/mL. These findings demonstrate that the developed method is able to detect all strains of C. muytjensii. Hence, this ELISA technique has potent application for the rapid and accurate detection of C. muytjensii in dietary foods.     

Inactivation of Cronobacter spp. (Enterobacter sakazakii) in infant formula using lactic acid,copper sulfate and monolaurin

Aims: To investigate the effect of lactic acid (LA), copper (II), and monolaurin as natural antimicrobials against Cronobacter in infant formula. Methods and Results: The effect of LA (0·1, 0·2 and 0·3% v/v), copper (II) (10, 50 and 100 μg ml^?1) and monolaurin (1000, 2000, and 3000 μg ml^?1) suspended into tween‐80? or dissolved in ethanol against Cronobacter in infant formula was investigated. Reconstituted infant formula and powdered infant formula were inoculated with five strains of Cronobacter spp. at the levels of c. 1 × 10⁶ CFU ml^?1 and 1 × 10³ CFU g^?1, respectively. LA at 0·2% v/v had a bacteriostatic effect on Cronobacter growth, whereas 0·3% v/v LA resulted in c. 3 log₁₀ reduction. Copper (II) at the levels of 50 μg ml^?1 and 100 μg ml^?1 elicited c. 1 and 2 log₁₀ reductions, respectively. The combination of 0·2% LA and 50 μg ml^?1 copper (II) resulted in a complete elimination of the organism. Monolaurin exhibited a slight inhibitory activity against Cronobacter (c. 1·5 log₁₀ difference) compared to the control when ethanol was used to deliver monolaurin. Conclusions: A complete elimination of Cronobacter was obtained when a combination of sublethal concentrations of LA (0·2%) and copper (II) (50 μg ml^?1) was used. Significance and Impact of the Study: The use of the synergistic interactive combination of LA and copper (II) could be beneficial to control Cronobacter in the infant formula industry.     

Resistance of Enterobacter sakazakii (Cronobacter spp.) to environmental stresses

Aim:  To gain a better understanding of the survival and persistence of Enterobacter sakazakii in severe environments.
Methods and Results:  We evaluated the resistance of Ent. sakazakii to various environmental stresses, including heating, drying, water activity ( a _w), and pH. The resistance of Ent. sakazakii to heat varies widely among strains. Most tested strains of Ent. sakazakii exhibited unusual resistance to dry stress, which depends on drying media. Growth of most strains occurred within 24 h at 37°C when the initial a _w of the medium was adjusted to 0·94 with sucrose or sodium chloride. The minimum pH for growth within 24 h at 37°C was 3·9 or 4·1 for most strains tested. Additionally, there did not appear to be any relationship between resistance to stresses and biofilm-forming ability in Ent. sakazakii planktonic cells.
Conclusions:  These results indicate that Ent. sakazakii is much more resistant than other Enterobacteriaceae to environmental stresses. Moreover, it is likely that Ent. sakazakii has cross-resistance to dry and thermal stresses.
Significance and Impact of the Study:  The findings of this study will contribute to an improved understanding of the survival and behaviour of Ent. sakazakii , which will lead to improved strategies for preventing outbreaks of Ent. sakazakii infection.     

Prevalence of Cronobacter species (Enterobacter sakazakii) in follow-on infant formulae and infant drinks

Aims:  To determine the prevalence of Cronobacter spp. ( Enterobacter sakazakii ) in follow-on formula powders commercially available in European countries.
Methods and Results:  A total of 470 samples comprising 31 different products from 18 brand names belonging to seven companies were tested for the presence of Cronobacter species. No milk- or soy-based infant formula powders were found to contain Cronobacter species . However, two cereal-based infant drinks were positive for Cronobacter sakazakii . A review of the published cases spanning the past 48 years did not reveal any fatalities attributable to Cronobacter spp. in children over 3 months.
Conclusions:  The low incidence of Cronobacter in infant powdered drinks, the lack of fatal Cronobacter infections in infants greater than 3 months and the low incidence of Cronobacter -related reported illness in this age group indicated that ingestion of these products presents a low risk for the intended consumers.
Significance and Impact of the Study:  The risk posed to neonates from the consumption of infant formula contaminated with Cronobacter is clear. Risks associated with powdered follow-on formulae intended for consumption by older infants is now under consideration by the World Health Organization. Our data contributes to the body of knowledge available for the assessment of the risk to consumers from these food products.     

Survival and growth of Cronobacter species (Enterobacter sakazakii) in wheat-based infant follow-on formulas

Aims:  To determine the survival and growth characteristics of Cronobacter species ( Enterobacter sakazakii ) in infant wheat-based formulas reconstituted with water, milk, grape juice or apple juice during storage.
Methods and Results:  Infant wheat-based formulas were reconstituted with water, ultra high temperature milk, pasteurized grape or apple juices. The reconstituted formulas were inoculated with Cronobacter sakazakii and Cronobacter muytjensii and stored at 4, 25 or 37°C for up to 24 h. At 25 and 37°C, Cronobacter grew more (>5 log₁₀) in formulas reconstituted with water or milk than those prepared with grape or apple juices ( c. 2–3 log₁₀). The organism persisted, but did not grow in any formulas stored at 4°C. Formulas reconstituted with water and milk decreased from pH 6·0 to 4·8–5·0 after 24 h, whereas the pH of the formulas reconstituted with fruit juices remained at their initial pH values, c. pH 4·8–5·0.
Conclusions:  Cronobacter sakazakii and C. muytjensii can grow in reconstituted wheat-based formulas. If not immediately consumed, these formulas should be stored at refrigeration temperatures to reduce the risk of infant infection.
Significance and Impact of the Study:  The results of this study will be of use to regulatory agencies and infant formula producers to recommend storage conditions that reduce the growth of Cronobacter in infant wheat-based formulas.     

Cronobacter species (formerly known as Enterobacter sakazakii) in powdered infant formula: a review of our current understanding of the biology of this bacterium

Cronobacter species (formerly known as Enterobacter sakazakii) are opportunistic pathogens that can cause necrotizing enterocolitis, bacteraemia and meningitis, predominantly in neonates. Infection in these vulnerable infants has been linked to the consumption of contaminated powdered infant formula (PIF). Considerable research has been undertaken on this organism in the past number of years which has enhanced our understanding of this neonatal pathogen leading to improvements in its control within the PIF production environment. The taxonomy of the organism resulted in the recognition of a new genus, Cronobacter, which consists of seven species. This paper presents an up-to-date review of our current knowledge of Cronobacter species. Taxonomy, genome sequencing, current detection protocols and epidemiology are all discussed. In addition, consideration is given to the control of this organism in the manufacturing environment, as a first step towards reducing the occurrence of this pathogen in PIF.     

Dissemination of Cronobacter spp. (Enterobacter sakazakii) in a powdered milk protein manufacturing facility

The microbial contamination of air filters and possible links to contaminated product in a powdered milk protein-processing facility were investigated. Over a 10-month period, seven air filters, the environment, and powdered product were analyzed for the presence of Cronobacter spp. The effects of air filter installation, maintenance, and subsequent dissemination of Cronobacter were investigated. A total of 30 isolates were characterized by pulsed-field gel electrophoresis (PFGE). PFGE revealed the presence of three clonal populations distributed throughout the manufacturing site. This study highlights the need for proper installation of air filters to limit the dissemination of microorganisms into processing sites.     

The phenotypic and genotypic characterization of Korean isolates of Cronobacter spp. (Enterobacter sakazakii)

This study was conducted to investigate the phenotypic and genotypic characteristics of Korean isolates of Cronobacter spp. (Enterobacter sakazakii). A total of 43 Cronobacter spp., including 5 clinical isolates, 34 food isolates, 2 environmental isolates, and 2 reference strains (C. sakazakii ATCC 29004 and C. muytjensii ATCC51329) were used in this study. Korean isolates of Cronobacter spp. were divided into 11 biogroups according to their biochemical profiles and 3 genomic groups based on the analysis of their 16S rRNA gene sequences. Biogroups 1 and 2 contained the majority of isolates (n=26), most of which were contained in 16S rRNA cluster 1 (n=34). Korean isolates of Cronobacter spp. showed diverse biochemical profiles. Biogroup 1 contained C. sakazakii GIHE (Gyeonggido Research Institute of Health and Environment) 1 and 2, which were isolated from babies that exhibited symptoms of Cronobacter spp. infection such as gastroenteritis, sepsis, and meningitis. Our finding revealed that Biogroup 1, C. sakazakii, is more prevalent and may be a more pathogenic biogroup than other biogroups, but the pathogenic biogroup was not represented clearly among the 11 biogroups tested in this study. Thus, all biogroups of Cronobacter spp. were recognized as pathogenic bacteria, and the absence of Cronobacter spp. in infant foods should be constantly regulated to prevent food poisoning and infection caused by Cronobacter spp.     

Comprehensive approaches to molecular biomarker discovery for detection and identification of Cronobacter spp. (Enterobacter sakazakii) and Salmonella spp

Cronobacter spp. (formerly Enterobacter sakazakii) and Salmonella spp. are increasingly implicated internationally as important microbiological contaminants in low-moisture food products, including powdered infant formula. Estimates indicate that 40 to 80% of infants infected with Cronobacter sakazakii and/or Salmonella in the United States may not survive the illness. A systematic approach, combining literature-based data mining, comparative genome analysis, and the direct sequencing of PCR products of specific biomarker genes, was used to construct an initial collection of genes to be targeted. These targeted genes, particularly genes encoding virulence factors and genes responsible for unique phenotypes, have the potential to function as biomarker genes for the identification and differentiation of Cronobacter spp. and Salmonella from other food-borne pathogens in low-moisture food products. In this paper, a total of 58 unique Salmonella gene clusters and 126 unique potential Cronobacter biomarkers and putative virulence factors were identified. A chitinase gene, a well-studied virulence factor in fungi, plants, and bacteria, was used to confirm this approach. We found that the chitinase gene has very low sequence variability and/or polymorphism among Cronobacter, Citrobacter, and Salmonella, while differing significantly in other food-borne pathogens, either by sequence blasting or experimental testing, including PCR amplification and direct sequencing. This computational analysis for Cronobacter and Salmonella biomarker identification and the preliminary laboratory studies are only a starting point; thus, PCR and array-based biomarker verification studies of these and other food-borne pathogens are currently being conducted.     

Distribution,prevalence and persistence of Cronobacter (Enterobacter sakazakii) in the nonprocessing and processing environments of five milk powder factories

Aims: To characterise the occurrence of Cronobacter in milk powder factories. Methods and Results: Cronobacter was isolated from 32% of 298 environmental samples from five factories. More isolations occurred in nonprocessing (49%) than processing areas (29%), although the greatest occurrence was in a single milk powder area during shutdown maintenance (81%) and the lowest after reinstatement of production hygiene practices (6%). Clonal analysis using PFGE placed 129 isolates into 49 groups. Most clones (45) were unique to each factory and seven were isolated in both milk powder and other areas of the same factory including tanker bays, evaporator rooms, an employee’s shoes and external roofs. Cronobacter was not isolated from raw milk processing areas. Within powder areas, 17 clones occurred at more than one and up to eight locations and six occurred more than once at the same location. Between four and seven clones were in the powder areas at each factory. The most prevalent and persistent clones were isolated from external roofs above spray driers, in air treatment areas and where high foot traffic occurs. Conclusions: Cronobacter is dispersed widely at milk powder factories. This study suggests that distribution is assisted by movement of air, milk powder and personnel and that new hygiene strategies will be needed to reduce prevalence. Significance and Impact of the Study: Knowledge of occurrence is essential for the development of strategies to control dissemination of Cronobacter within factories and reduce risk of entry into powdered milk products.     

Rapid Detection and Simultaneous Genotyping of Cronobacter spp. (formerly Enterobacter sakazakii) in Powdered Infant Formula Using Real-time PCR and High Resolution Melting (HRM) Analysis

Cronobacter spp. is an emerging pathogen that causes meningitis, sepsis, bacteremia, and necrotizing enterocolitis in neonates and children. The present study developed an assay integrating real-time PCR and high resolution melting (HRM) analysis targeting the OmpA gene for the specific detection and rapid identification of Cronobacter spp. (formerly Enterobacter sakazakii) in powdered infant formula. Eleven Cronobacter field isolates and 25 reference strains were examined using one pair of primers, having the accuracy of 100% in reference to conventional methods. The assay was proved to be highly sensitive with a detection limit of 10² CFU/ml without pre-enrichment, and highly concordant (100%) when compared with ISO-IDF 22964 in 89 actual samples. The method performed for Cronobacter spp. detection was less than 24 h, drastically shortened, compared to several days using standard culturing method, it is probe-free and reduces a risk of PCR carryover. Moreover, all Cronobacter strains examined in this study were genotyped into two species according to their HRM profiles. The established method should provide a molecular tool for direct detection and simultaneous genotyping of Cronobacter spp. in powdered infant formula.     

Development of a novel screening method for the isolation of "Cronobacter" spp. (Enterobacter sakazakii)

A differential medium, "Cronobacter" screening broth, has been designed to complement agars based on hydrolysis of chromogenic alpha-glucopyranoside substrates. The broth was evaluated using 329 Enterobacteriaceae strains (229 target isolates), spiked/naturally contaminated samples, and a parallel comparison with current methods for raw materials, line/end products, and factory environment samples.     

Occurrence of Cronobacter spp. in retail foods

Aims: To study the occurrence of Cronobacter spp. in foods and to investigate the phenotypic properties of the strains isolated. Methods and Results: A total of 53 strains of Cronobacter spp. isolated from 399 food samples were identified using conventional biochemical methods and MALDI‐TOF mass spectrometry. Foods of plant origin were the most frequently contaminated samples. No Cronobacter spp. were found in infant milk formula, wheat‐based infant food, pasteurized and raw cow milk, mincemeat, chicken, chickpea and potato dumpling powder. The individual species were identified as Cronobacter sakazakii (54·7%), Cronobacter malonaticus (28·4%), Cronobacter dublinensis (7·5%), Cronobacter muytjensii (7·5%) and Cronobacter turicensis (1·9%). Cronobacter sakazakii and C. malonaticus belong to biotype 1, 2, 2a, 3, 4 and 5, 5a, respectively. Cronobacter dublinensis strains were subdivided into biotypes 6 and 12. All strains were resistant to erythromycin and two of them were resistant to both erythromycin and tetracycline. Conclusions: Cronobacter spp. were isolated from various food samples pre‐eminently of plant origin and dried food ingredients. Significance and Impact of the Study: These findings will increase and detail our knowledge of the presence and diversity of Cronobacter spp. in foods.     

Survival characteristics of environmental and clinically derived strains of Cronobacter sakazakii in infant milk formula (IMF) and ingredients

Aims: The study aimed to compare survival of Cronobacter sakazakii strains in plant‐derived infant milk formula (IMF) ingredients and their thermotolerance in reconstituted IMF. Methods and Results: Inulin and lecithin were inoculated with isolates of C. sakazakii including the typed clinical strains, NCTC 11467^T and BAA 894; a mutant strain in which the wcaD gene had been disrupted; and two environmental strains isolated from IMF processing facilities. Samples were stored and examined for C. sakazakii. All strains were still detectable in both matrices after 338 days storage, except for the mutant strain that was no longer detectable at that time. Higher numbers of the environmental strains were recoverable after 338 days than the clinical strains. The thermotolerance of the five strains was investigated in reconstituted IMF at 55, 60 and 65°C. The clinically derived type strain, NCTC 11467^T, and the mutant strain were shown to be significantly more thermotolerant than other strains tested. Conclusions: Environmental strains were more persistent than the clinical strains in inulin and lecithin, indicating that patho‐adaptation may have contributed to a reduction in the desiccation tolerance phenotype. However, the thermotolerance results could indicate that the ability to produce extracellular polysaccharide decreases thermotolerance. Significance and Impact of the Study: These results indicate that desiccation resistance may play a role in survival of C. sakazakii in dry IMF ingredients and processing plants; however, this trait may be of less importance in clinical environs.     

Inactivation effect of X-ray treatments on Cronobacter species (Enterobacter sakazakii) in tryptic soy broth, skim milk, low-fat milk and whole-fat milk*

Aims:  To determine the inactivation effect of X-ray treatments on Cronobacter ( E. sakazakii ) in tryptic soy broth (TSB), skim milk (0% fat), low-fat milk (1% and 2%) and whole-fat milk (3·5%).
Methods and Results:  X-rays were produced using the RS 2400 generator system (Rad Source Technologies Inc.). Cronobacter (in TSB), inoculated skim milk (0% fat), low-fat milk (1% and 2% fat) and whole-fat milk (3·5% fat) were treated with 0·0, 0·1, 0·5, 0·75, 1·0, 2·0, 3·0, 4·0, 5·0 and 6·0 kGy X-ray doses. Surviving bacteria in the TSB and inoculated milk, before and after treatment, were enumerated using plating method onto trypticase soy agar. Greater than 7·0-log CFU reduction in Cronobacter population was observed with 4·0, 5·0, 6·0, 6·0 and 6·0 kGy X-ray in the TSB, skim milk, 1% fat milk, 2% fat milk and 3·5% fat milk, respectively.
Conclusions:  Treatment with X-rays significantly ( P  <   0·05) reduced Cronobacter to less than detectable limits (<1 log CFU ml⁻¹) in skim milk at 5·0 kGy and milk with 1% fat content and greater at 6·0 kGy dose levels. The D-value for Cronobacter in TSB was significantly ( P  <   0·05) lower than those in milk samples.
Significance and Impact of the Study:  Treatment with X-rays could be an effective and safe alternative technology to control pathogenic bacteria ( Cronobacter ) in the dairy industry.     

New method to assess mitophagy flux by flow cytometry

Mitochondrial autophagy, also known as mitophagy, is an autophagosome-based mitochondrial degradation process that eliminates unwanted or damaged mitochondria after cell stress. Most studies dealing with mitophagy rely on the analysis by fluorescence microscopy of mitochondrial-autophagosome colocalization. However, given the fundamental role of mitophagy in the physiology and pathology of organisms, there is an urgent need for novel quantitative methods with which to study this process. Here, we describe a flow cytometry-based approach to determine mitophagy by using MitoTracker Deep Red, a widely used mitochondria-selective probe. Used in combination with selective inhibitors it may allow for the determination of mitophagy flux. Here, we test the validity of the use of this method in cell lines and in primary cell and tissue cultures.