Desiccation and persistence of Cronobacter spp. (E. sakazakii) in infant formula. 1 st Intl Conference on Cronobacter UCD January 2009

Desiccation and persistence of Cronobacter spp. (E. sakazakii) in infant formula 1 st Intl Conference on Cronobacter UCD January 2009

Desiccation and persistence of Cronobacter spp. (E. sakazakii) in infant formula 1 st Intl Conference on Cronobacter UCD January 2009

Desiccation and persistence of Cronobacter spp. (E. sakazakii) in infant formula 1 st Intl Conference on Cronobacter UCD January 2009

Structure! " # $!! %

Characteristics of Cronobacter spp. &'()* + $ "

Low impact journal! Only 5 pages,--).'/!0 Six strains of Cr. sakazakii 3 from Franco and Jeff Enteral feeding tubes and baby bottles FAO/WHO 2004 One PAGE! JEMRA Risk Model FAO/WHO 2004 Key method reference: Druggan, Forsythe & Silley (1993)

Sources of Cronobacter spp.

Being right for the wrong reason!

Sources of Cronobacter spp. Plants Mixed salad vegetables Rice seed Herbs & spices Grain Cereal Rhizosphere Ingredients Dried flour or meal (corn, soy, wheat and rice) Dried sodium caseinate Starches Potato flour Products Powdered infant formula Follow-on formula Weaning foods Milk powder Dried foods Dried vegetables and spices Infant cereals Adult cereals Tofu Iced tea Other Household dust Hospital air Preparation equipment For more details see Friedemann 2007

Sources of Cronobacter spp. Yellow pigmentation Oxygen radical protection Capsule Attachment and desiccation resistance Efflux pumps Essential oil resistance

Desiccation persistence

Desiccation persistence,*.- 1 Caubilla-Barron & Forsythe 2007

Desiccation persistence Cronobacter spp. Strain Capsulation Source TSA VRBGA Difference Cr. sakazakii 1 No Clinical 8.6 4.9 3.7 Cr. sakazakii 4 Clinical 8.2 7.1 1.1 Cr. sakazakii 23 PIF 7.9 3.2 4.7 Cr. muytjensii 3 Clinical 7.9 6.7 1.2 Cr. sakazakii 2 Yes Clinical 8.5 7.6 0.9 Cr. sakazakii 7 PIF 8.3 7.7 0.6 Cr. sakazakii 10 Nuts 9.0 8.5 0.5 Cr. sakazakii 13 Herb 8.6 8.1 0.5 Cr. sakazakii 25 PIF 8.7 8.0 0.7 Cr. turicensis 9 Baby food 8.1 7.6 0.5 Caubilla-Barron & Forsythe 2007

Desiccation persistence 8 6 Viable count (log cfu/ml) 4 2 P antoea spp. C. freundii S. Enteritidis 0 0 5 10 15 20 25 30 Time (months) Pantoea spp. C. freundii Salm. Enteritidis Caubilla-Barron & Forsythe 2007

Desiccation persistence 8 6 Viable count (log cfu/ml) 4 2 0 0 6 12 18 24 30 Time (months) Strain number C. sakazakii 1 C. sakazakii 2 C. sakazakii 25 C. turicensis C. muytjensii Caubilla-Barron & Forsythe 2007

Desiccation persistence 8 Viable count (log cfu/ml) 6 4 2 Biphasic decline Subpopulation more resistant? 0 0 6 12 18 24 30 Time (months) Caubilla-Barron & Forsythe 2007

Capsule and biofilm formation Intrinsic (<1 cfu/g) Equipment (Reported) People (unknown) Reconstitution Ambient temperature storage Baby stomach Probability of ingestion and Probability of infection Biofilm formation (recontamination) Forsythe 2005

Capsule and biofilm formation Organism Source Capsule production a Polyvinylchloride (log 10 cfu/tube) Av Max Min Cr. sakazakii NCTC 11467 T Infant clinical - 5.7 6.3 4.0 Cr. sakazakii ATCC 12868 Unknown +++ 4.6 4.8 4.3 Cr. sakazakii 1 PIF +++ 6.3 6.4 5.8 Cr. sakazakii 2 PIF +++ 5.5 5.7 4.9 Cr. sakazakii 3 PIF +++ 5.3 5.6 5.0 Cr. sakazakii 4 PIF ++ 5.6 5.8 5.3 Cr. sakazakii 5 PIF ++ 4.6 4.9 4.0 Cr. sakazakii 6 PIF + 5.9 6.2 5.2 Cr. sakazakii 7 Unknown + 4.6 4.9 4.5 Cr. sakazakii 8 Unknown - 5.3 5.5 4.4 Cr. sakazakii 9 Environmental - 6.2 6.6 5.5 Poster P14

Thermal resistance Organism D 55 min Cr. sakazakii 3-49 Cr. malonaticus 6 Cr. malonaticus 3 Cr. turicensis 3 Cr. muytjensii 3 Cr. dublinensis 3 Salm. Senftenberg 36 E. hormaechei 2-54 Poster P25

D-values of unstressed and stressed Cronobacter spp. in reconstituted PIF Temperature Unstressed Stress treatment ( C) Desiccation Starvation Heat Cold 52 15.3 8.7 17.5 7.4 14.4 54 4.5 2.1 6.7 2.1 3.9 56 2 0.8 2.1 0.8 1.5 58 0.5 0.3 0.7 0.3 0.5 2,--3

Mechanisms of resistance Compatible solutes Trehalose and maltose are both disaccharides of glucose Trehalose has two (11)-glycosidic linkages and no reducing sugar residue Maltose (14)-glycosidic linkage, plus reducing end sugar 456 677!!27! 7,78' Breeuwer et al. 2003

Mechanisms of resistance Compatible solutes Proteins peptides amino acids ecotine ecotine peptides proline betaine betaine trehalose sugars choline taurine carnitine choline taurine carnitine Breeuwer et al. 2003

Mechanisms of resistance "..

Mechanisms of resistance Proteins associated with desiccation and osmotic stress adaptation DNA protecting protein under starved conditions Dps Cold shock-like protein CspC OmpC OmpA Thermoregulated motility protein Super oxide dismutase Chaperonoin GroES ABC-type proline/glycine betaine transport systems Hypothetical protein Mfla_1164 Hypothetical protein Mfla_1165 Hypothetical protein Psyc_0523 Trehalose uptake Capsule production Putative gene in C. sakazakii BAA-894 Yes Yes Yes Yes Yes Yes Yes Yes NF NF Yes Yes* Yes* / :;<!!!! 7 9/ For more detail see Reidel & Lehner 2007

Summary Thank you Origin of Cronobacter Physiological resistance traits Cross-protection : heat and desiccation stress Desiccation during processing Induction of thermotolerance reconstitution temperature efficacy Capsulation - lower proportion of sublethally injured cells which revive and multiply and form biofilms!