1. Enterobacter Sakazakii Preaperd by: Sahar Salem Abu Naseer Presented to: Dr. Abedlraouf Elmanama

2. Genus Enterobacter  Enterobacter is one of the coliform groups of bacteria in the Enterobacteriaceae family.  They are frequent colonizer of hospitalized patients, and can be responsible for a variety of nosocomial infections.  Enterobacter species rarely cause disease in an otherwise healthy individual.

3. Genus Enterobacter  Enterobacter species are widely distributed in nature, occuring in :  fresh water  soil  sewage  plants  vegetables  animal and human feces.

4. Genus Enterobacter  The most frequently isolated species from enterobacter are E. cloacae and E. aerogenes, followed by E. sakazakii.

5. Enterobacter species  Other species rarely encountered in the clinical specimen include:  Enterobacter asburiae  Enterobacter gergoviae  Enterobacter taylorae  Enterobacter hormaechei  and Enterobacter cancerogenus.  Enterobacter agglomerans has been removed from the genus Enterobacter and renamed Pantoea agglomerans

6. Enterobacter species  There are four additional species are found in the inviroment or as plant pathogens and have not been found in human clinical specimens:  E.intermedius  E.dissolvens.  E.nimipressuralis.  E.pyrinus.

7. Enterobacter sakazakii  Gram negative rod shap.  Named in honor of Japanes microbiologist Riichi Sakazaki.

8. Enterobacter sakazakii  The organism was called "yellow-pigmented Enterobacter cloacae".  until 1980 when It was introduced as a new species based on: Differences in DNA-DNA hybridization. Biochemical reactions. Antibiotic susceptibility

10. Common source of Enterobacter sakazakii:  The bacterium can be detected in the gut of healthy humans.  It can also be found in the gut of animals as well as in the environment.  Enterobacter sakazakii can be isolated from a wide variety of foods.  The only food that has been identified with illness is reconstituted milk-based powdered infant formula.

11. Common source of Enterobacter sakazakii:  A recent study has isolated E. sakazakii from the guts of a laboratory colony of Mexican fruit flies, Anastrepha ludens.  A technical report from a pest exterminating company in the United States records the presence of Enterobacter sakazakii in the house fly (Musca domestica), although the exact location of the bacterium on or in the fly is unclear.

12. E.Sakazakii infection  Enterobacter sakazakii is a foodborne pathogen that has been associated with sporadic cases and outbreaks causing :  Meningitis.  Necrotizing enterocolitis.  And sepsis especially in neonates.

13. E.sakazakii infection  It has caused disease in all age groups.  children less than 1 year old are at particular risk. especially:  Neonates (first 28 days) Particularly pre-term infants.  Low birth weight infants.  Immunocompromised infants.

14. E.sakazakii infection  Infants of HIV-positive mothers are also at risk both because they may specifically require infant formula and may be more susceptible to infection.  In addition, asymptomatic infants have been identified with E. sakazakii in their stools or urine.

15. E.sakazakii infection  It is rarely causes a primary infection in healthy individuals, but has been implicated in burn, wound, and urinary tract infections and occasionally septicemia and meningitis.  Most reported cases are nosocomial and involve infants.  In most cases the infection has been linked to milk-based powdered infant formula products

16. E.sakazakii infection  The fatality rate has ranged from 30% – 80%, but has declined to under 20% in recent years. Where infection does not result in death.  the affected infant may have permanent neurological or developmental deficiencies.

17. E.sakazakii infection  Fewer cases of E sakazakii infection have been reported in adults, and these have mainly been in elderly patients with serious underlying medical conditions.

18. E.sakazakii infection  Infant is more susceptible than adult for many reasons :  The stomach of newborns, especially of premature babies, is less acidic than that of adults: a possible important factor contributing to the survival of an infection with E. sakazakii in infants.  Underdeveloped immune system of infant.  Lack of competitive intestinal microflora.  High iron levels in IFM Contribute too.

19. Infant formula contamination:  There are two main routes by which E. sakazakii can enter reconstituted infant formula: A) Through intrinsic contamination: -through the raw material used for producing the formula -through contamination of the formula or other dry ingredients after pasteurisation b) Through external contamination: -During reconstitution and handling.

21. Virulence factor of E.sakazakii.  There is very little known about virulence factors and pathogenicity of E. sakazakii. o Enterotoxin-like compounds were produced by some strains. o Using tissue cultures, some strains produced a cytotoxic effect.

22. Virulence factor of E.sakazakii  There appear to be differences in virulence among E. sakazakii strains, and some strains may be non-pathogenic.  Brain abscesses due to E. sakazakii and the related bacterium (Citrobacter koseri) are morphologically similar and may be due to similar virulence mechanisms.

23. Survival factor of Enterobacter sakazakii:  The survival of Enterobacter sakazakii in powdered infant formula may be partially due to the ability of the microorganism to survive desiccation (drying) during manufacture and afterwards.  E.sakazakii formed a biofilm on surfaces including latex, silicon and to a lesser extent, stainless steel. These materials are commonly used for infant feeding equipment and in preparation areas.

24. Survival factor of Enterobacter sakazakii:  The thermotolerance of the organism was similar to other Enterobacteriaceae such as Salmonella in rehydrated milk powder. Therefore the standard high-temperature short time pasteurisation process of 15 seconds at 72 °C will inactivate the organism.

25. Laboratory Dignosis  Biochemical characteristics:  Oxidase negative.  IMVIC reaction --++.  Urease: negative.  Lysine decarboxylase: negative.  Arginine dihydrolase: positive.  Ornithine decarboxylase: positive.

26.  Fermentation of (lactose, sucrose, raffinose, rhamnose and melibiose).  Non fermentation of D-sorbitol and adonitol.  Delayed production of DNAase.  Production of α-glucosidase.  Absence of phosphoamidas

27. ReactionTest E. aerogenesE. cloacaeE.sakazakii +--Lysine decarboxylase -++Arginine dihydrolase +++Ornithine decarboxylase +++KCN, growth in +++ sucroseFer me ntat ion of +--adonitol +++raffinose ++-D-sorbitol +-_D-arabitol --+Yellow pigment

28. Culture identification of E.sakazakii : In 2002 the FDA published a recommended method for isolating and identifying E. sakazakii from infant formula: 1. pre-enrichment in sterile water. 2. enrichment in EE Broth. E.E. Broth uses glucose instead of lactose to make the medium a test for all enterobacteriaceae including non lactose fermenting organisms.

29. Culture identification of E.sakazakii : 3. Followed by plating into VRBG Agar (Violet Red Bile Glucose Agar). Used for detecting and enumerating Enterobacteriaceae in food and dairy products. It is not possible to select for E. sakazakii colonies from VRBGA plates on the basis of colony morphology as they will appear the same as other Enterobacteriacea

30. Culture identification of E.sakazakii :

31. 4. Then sub culture into Tryptone Soya Agar:  Requires a long (48- to 72-h) incubation time to produce characteristic yellow-pigmented colonies.  yellow-pigmented Enterobacteriaceae can be isolated from clinical specimens and fresh water such as: E.hermanii and E.vulneris.

32. Culture identification of E.sakazakii :  Yellow-pigmented colonies are confirmed as E. sakazakii by oxidase and other biochemical tests.

33. differential media of E.sakazakii. 1- Oxoid Chromogenic Enterobacter sakazakii Agar. - It was developed for faster detection of E.sakazakii in infant formula and other food samples to allows recovery and detection of E. sakazakii in just 2days – 3 days faster than by conventional methods.

34. differential media of E.sakazakii.  Description of the media:  Chromogenic Enterobacter sakazakii Agar is based on the a-glucosidase reaction.  detected by incorporating the chromogenic substrate 5- bromo-4-chloro-3-indolyl-a,D-glucopyranoside in the medium.  The enzyme α-glucosidase, present in E. sakazakii, hydrolyses the substrate producing blue-green colonies on this pale yellow medium.

35. differential media of E.sakazakii.  Not: -Proteus vulgaris is also weakly a-glucosidase positive and could grow to give colonies of a similar colour to E. sakazakii. On this medium Proteus spp. grow as grey colonies. -They produce hydrogen sulphide in the presence of ferric ions forming ferrous sulphide.

36. differential media of E.sakazakii.

37. 2- Ok media: - the fluorogenic substrate of α-glucosidase is used as a selective marker (Methylumbelliferyl-α-d- glucoside). - Media contain also bile salt to select against gram positive media.

38. differential media of E.sakazakii.  This bacterium showed strong fluorogenic characteristics clearly distinguishable from other microorganisms.

39. Treatment:  E sakazakii is generally more susceptible to antimicrobials than are other Enterobacter species.  Ampicillin and gentamicin were referred to as the "gold standard" for treatment of E sakazakii meningitis in neonates.

40. What do we do now??