# Presentation: 418147

## Enterobacter sakazakii Case Reports and Outbreaks Involving Infants as Reported in the Peer-Reviewed English Medical LiteratureKarl C. KlontzEpidemiology TeamCenter for Food Safety and Applied NutritionFood and Drug Administration

## Case Definition- E. sakazakii infection

- An infant meeting at least one of the following criteria:
- •	*E. sakazakii* recovered from _>_ 1 of the following normally sterile sites:
- 		▫ Blood
- 		▫ Cerebrospinal fluid
- 		▫ Brain tissue
-                ▫ Urine
- •	Outbreak of necrotizing enterocolitis and *E. sakazakii* recovered from blood, stool, or stomach aspirate of > 1 infant
- •	Bloody diarrhea, *E. sakazakii* recovered from stool in pure culture

## Number Reports E. sakazakii Infection – English-Language Peer-Reviewed Literature

- _Age group_		    _No. references_        _No. cases (%)_
- Infants				20                       48 (83)
- Children (>1 yr), adults                      7                       10 (17)
- _Age group_                     _No. cases_              _Percent_
- 0 – 1 month		37                         64
- >1 – 12 months		10		17
- >1 – 4 years		  4 		  7
- > 4 years                               6                         10  (median age = 74 yr)
- > Not stated                          _1 _                          _2_   (infant, but ? age)
- > Total	                              58                       100

## Initial Reports of E. sakazakii infection: Chronology

- Urmenyi (1961)
- Jöker (1965)
- Monroe (1979)
- Kleiman (1981)
- Adamson (1981)
- Muytjens (1982)
- Jiménez (1982)*
- Muytjens (1983)      Case series (1st study to propose possible link
- 		                   to powdered formula)

- Case reports

**Notes:**
First seven reports involved single cases or, in the instance of Urmenyi (1961) and Muytjens (1982), two cases.

Jimenez report (1982) was first report of infection (urosepsis) in an adult (76-yr-old with hx rectal adenocarcinoma)

## E. sakazakii Infections in Infants: Selected Clinical Features – Peer-Reviewed Literature (N=48)

- Gender: 52% female
- Syndromes: Meningitis (58%), Sepsis/bacteremia (17%); Necrotizing enterocolitis (29%)
- Overall case-fatality: 33%  (15/46 for whom info available)
- _Birth weight (g)_	_Number_	_No. Died (%)_
- 	< 1000 		     10                           2  (20)
- 	1000-1499                            9                           2  (22)
- 	1500-2499		      11                           7  (64)
- 	> 2500                                10                            3  (30)

## Muytjens et al. (1983): Analysis of 8 cases of neonatal meningitis and sepsis due to E. sakazakii

- Reanalyzed 20 *Enterobacter* strains isolated from CSF (going back 6 yrs) and 25 strains from blood (2 yrs) in the Netherlands
- Hypothesis: *E. sakazakii* may not have been identified originally because organism produces minimal pigment at 36° C
- Using various biochemical and growth parameter systems (25 ° C), 8 strains identified as *E. sakazakii*
- All 8 strains confirmed by CDC as *E. sakazakii*
- Clinical data for *E. sakazakii* cases obtained retrospectively

**Notes:**
Hypothesis: Because pigment production, a distinguishing characteristic of E. sakazakii, is greatly diminished at the usual incubation temperature of 36 degrees C, it seemed likely that a number of E. sakazakii isolates were not recognized as atypical E. cloacae in the past.  The investigators therefore decided to reanalyze a number of Enterobacter strains isolated from CSF and blood to investigate more cases of E. sakazakii infection.

## Muytjens et al. (1983): Clinical features of 8 cases of neonatal meningitis and sepsis due to E. sakazakii

- 3/8 cases delivered by C-section
- Progress in first few days of life was normal
- First signs of illness occurred between days 4-8 of life
- 2/8 cases had necrotizing enterocolitis + meningitis
- 75% case-fatality rate; 2 survivors “retarded”
- Death occurred within hours – several days
- “Hemorrhagic encephalitis”

## Muytjens et al. (1983): Epidemiologic features of 8 cases of neonatal meningitis and sepsis due to E. sakazakii

- 6/8 cases birth weights < 2,500 grams (lowest: 850 g)
- 5/8 cases premature (gestation _<_ 36 wk)
- 5/8 cases occurred at same general hospital
- Other 3 cases born in 3 other hospitals
- Temporal clustering: 3/5 cases in general hospital became ill within 3 months; 2/3 cases in other hospitals within 2 months

## Muytjens et al. (1983): Environmental Sampling Results

- Environmental sampling at pediatric ward of general hospital
*E. sakazakii* recovered from prepared formula, dish brush, and stirring spoon, but not from powdered formula and water
- Plasmid profiles: “3 or 4 of 5 isolates from patients at the general hospital were probably the same strain”
- Plasmid profiles differed between cases vs. prepared formula
- (1990 note: “No cases observed at general hospital since powder formula replaced by liquid formula 8 years ago.”)

**Notes:**
Sampling conducted at general hospital where 5/8 cases occurred

E. sakazakii recovered “several times” from prepared formula

The plasmid profiles of strains from patients 2 through 5 are interesting: patients 3 through 5 were infected within 3 months of one another and thus are closely related in both time and location.  Each of the three strains from these patients contained two plasmids, 70 and 37 megadaltons.  The strain from patient 2 had a similar but not identical profile.  It contained a 70-megadalton plasmid, but its second plasmid was 39-megadaltons, rather than 37.  This strain was isolated 2 years before the strains from patients 3 through 5 were.

Quote from Muytjens and Kollee (Letter, Pediatric Infectious Disease Journal 1990;9:372-3.  Goes on to say: “It may be concluded that formula feeding is a mode of transmission of E. sakazakii in the newborn.”

## E. sakazakii and Powdered Infant Formula: Developments From 1961-1988

- 1980 (Farmer): 1/57 isolates came from “can of dried milk”
- 1983 (Muytjens)
- 		▫ *E. sakazakii* recovered from prepared formula
- 		▫ Recommended sampling of “milk” as part of investigations
- 1988 (Muytjens)
- 		▫ 141 powdered infant formula products obtained from 35 	      	countries tested for presence of *Enterobacteriaceae*
*	**        *▫ 52% samples +*	*
- 		 ▫ 20 powders (14%) from 13 countries yielded *E. sakazakii*
*	**	** **▫ *Concentration of *Enterobacteriaceae* uniformly  _<_ 1 CFU/g*  *

**Notes:**
Farmer (198)) paper: proposes the name Enterobacter sakazakii for the organism that had, until that time, been called “yellow-pigmented Enterobacter cloacae”.  

Characterization conducted on 57 strains sent to CDC between 1970-1977 that were eventually identified as E. sakazakii  The vast majority of isolates were from clinical specimens but 2 were not: one from a “can of dried milk” and the other from a “physician/’s stethescope.”

Interesting to note that members of the family Enterobacteriaceae were cultured from 53% fo the 141 powdered formula products.  The species isolated more often than E. sakazakii were Enterobacter agglomerans and Enterobacter cloacae.

All powders met the requirements of the U.N. Food and Agricultural Organization (fewer than 3 colony-forming units of Enterobacteriaceae/g of powder).

## Three Cases of Neonatal Meningitis Caused by E. sakazakii in Powdered Formula (Biering [1989-Iceland])

- Illnesses in 1986, 1987 (2 within one month)
- 2 infants normal at birth (36- and 38-wk gestation); 1 Down’s
- All 3 did well until day 5 when they became ill
- 1 died, 2 recovered with severe neurologic sequelae
- All 3 fed powdered infant formula
- 1st study to show, in illness setting,* E. sakazakii* recovery (low numbers) from freshly prepared formula from previously unopened cans
- No recovery from environment (milk kitchen, utensils, ward)

**Notes:**
Down’s baby died.

Other two recovered with neurologic sequelae: one severely retarded and quadriplegic at 2 years; the other moderately delayed in all developmental areas and with seizure disorder.

“The organism seemed to be present in the milk powder in low numbers, since it could only be isolated from freshly prepared formula by subculture after 4 h of incubation, and not by direct plating.

As authors’ title states: strong case for causality established

## Three Cases of Neonatal Meningitis Caused by E. sakazakii in Powdered Formula (Biering [1989-Iceland])

- In addition to 3 ill cases, 1 colonization
- 4 *E. sakazakii* strains isolated from neonates had same plasmid profile as 22 formula strains
- Anecdotal evidence that formula bottles occasionally kept at 35-37 C for extended periods in bottle heaters
- But in 1 instance, recovery (direct plating) from bottle that had been refrigerated
- “...milk powder can be the mode of transmission for *E. sakazakii* meningitis or sepsis in neonates...”

**Notes:**
4 E. sakazakii strains isolated from neonates had same plasmid profile as 22 formula strains (also had same multilocus enzyme electrophoresis pattern and antibiotic sensitivities).

No further cases occurred after bottle heaters were no longer used.

## Outbreaks of E. sakazakii infections linked to powdered infant formula: Historical cohort studies

- Simmons et al. (1989):  *Enterobacter sakazakii* Infections in Neonates Associated with Intrinsic Contamination of a Powdered Infant Formula (*Infect Control Hosp Epidemiol*)
- Van Acker et al. (2001): Outbreak of Necrotizing Enterocolitis Associated with *Enterobacter sakazakii* in Powdered Milk Formula (*J Clin Microbiol*)
- CDC.  *Enterobacter sakazakii* Infections Associated with the Use of Powdered Infant Formula-Tennessee, 2001.  *MMWR* 2002;51:297-300

## E. sakazakii Infections in Neonates Associated with Intrinsic Contamination of Powdered Infant Formula (Simmons-1989)

- Feb-Mar 1988: bacteremia (2 infants), UTI (1), bloody diarrhea (1)
*E. sakazakii* recovered from stool of all 4 infants
- Investigation revealed all 4 had been fed same powdered formula
- Formula prepared in blender; rinsed with tap water between uses
- Blender yielded heavy growth of *E. sakazakii*
- No further clinical isolates once blender sterilized
- Historical cohort study in NICU assessed risk factors for infection and colonization (2/7-3/13)

**Notes:**
Blender occasionally cleaned with handwashing agents as well: chlorhexidene and hexachlorophene

Historical cohort study (2/7-3/13) began 4 days before first isolate recovered and ended the day of the last isolate.  All infants in the NICU during this period were included in the historical cohort study.  Any baby culture positive for E. sakazakii was considered a case.

Culturing of infants was not done systematically during this period but only as ordered by the attending physician for evaluation of clinical illness.

## E. sakazakii Infections in Neonates Associated with Intrinsic Contamination of Powdered Infant Formula (Simmons-1989)

-                           Infected/Colonized
-                              Yes         No
-                Yes          4              1           5
- Formula
-                  No            0             40        40
-             Relative risk = undefined; p = 0.0006

## E. sakazakii Infections in Neonates Associated with Intrinsic Contamination of Powdered Infant Formula (Simmons-1989)

- Implicated elemental formula given 1° to most premature infants
- Samples obtained from opened, yet unmixed, can powdered formula
- Powder cultured using method of Muytjens *et al*.
*E. sakazakii* (~ 8 CFU/100 g), *E. cloacae* (48 CFU/100 g) recovered
- Same plasmid, multilocus enzyme profile (formula and patients)

**Notes:**
Important to note that the implicated formula was an elemental formula that required little digestion for absorption.  Premature infants who had diarrhea on other formulas were especially likely to receive the formula.  Thus, most infants who received the formula were highly selected and especially premature.

Method of Muytjens used to culture powdered formula samples: 50 g in 450 ml buffered peptone water; 10 g in 90 ml; 1 g in 9 ml.

## van Acker et al. (2001): Necrotizing enterocolitis associated with E. sakazakii in powdered formula

- 12 cases NEC diagnosed June-July, 1998 in Belgium NICU
- 50 neonates admitted to NICU during this period
- All 12 had birth weights <2,000 g, fed powdered infant formula
- 6/12 neonates had + cultures [blood, anal swab, or stomach aspirate] for *E. sakazakii* vs. 0/38 without NEC (p<0.001)
- 10/12 neonates with NEC had been fed the same powdered formula: Alfaré (semi-elemental formula with low osmolarity)

**Notes:**
A total of 11 E. sakazakii strains from the six culture-positive NEC patients were isolated.

E. sakazakii was isolated from blood culture (1 patient), from anal swabs (5 patients), and from stomach aspirates (3 patients).

## van Acker et al. (2001): Necrotizing enterocolitis associated with E. sakazakii in powdered formula

*                          **E. sakazakii* + (NEC)
-                              Yes         No
-                Yes           6             8          14
- Formula
-                  No            0            36         36
-             Relative risk = undefined; p = 0.0002

**Notes:**
Implicated formula = Alfaré (Nestle) = semielemental formula with low osmolarity.

## van Acker et al. (2001): Necrotizing enterocolitis associated with E. sakazakii in powdered formula

*E. sakazakii* recovered from prepared formula, unopened cans
- Molecular typing (AP-PCR) confirmed partial strain similarity between powdered formula, patient isolates
- No further cases of NEC observed after use of implicated powdered formula stopped
- Inadvertent challenge test:
- Implicated formula stopped on 7/10/98
- Released again on 7/20
- 7/23: new case NEC linked to formula (intrinsic formula contamination shown at same time)

**Notes:**
E. sakazakii recovered from single batch of unopened cans of implicated powder.
Cultures of extra prepared infant formula from milk kitchen revealed E. sakazakii in several Alfaré preparations whereas cultures of powdered formula preparations of other brands were negative or resulted in the isolation of Bacillus spp., coagulase-negative staphylococci, or Acinetobacter spp.  Cultures of the mineral water and the rinsing water (for blender head) remained negative.

Three different profiles of E. sakazakii were found by AP-PCR among the 9 patient isolates (2 patient isolates could not be tested because they had not been stored): Ia, II, and III.  The 14 powdered formula isolates and 4 patient isolates from 3 patients shared profile Ia.

The use of Alfaré was stopped on 10 July 1998, immediately after investigators suspected a possible link to NEC.  However, because initial cultures demonstrated presence of E. sakazakii only in prepared formula and not in Alfaré powder, the formula was released again on 20 July for the feeding of one infant.  This infant developed symptoms of NEC on 23 July and E. sakazakii was isolated from her stomach aspirate and anal swab.  At the same time, further cultures demonstrated the intrinsic contamination of Alfaré powdered formula with E. sakazakii.  From then on, feeding with Alfaré was completely stopped..

## van Acker (2001): NEC-associated with E. sakazakii in powdered formula

- Manufacturer’s quality control data for formula:
- Of 5 samples analyzed: 1 yielded 20 coliforms/g; 4 < 1 coliform/g
- Results fulfilled requirements of Codex Alimentarius: minimum of 4/5 control samples with < 3 coliforms g; maximum of 1/5 with >3 but _<_  20 coliforms/g_ _
- Did not meet Belgian law: < 1 coliform/g in all samples
- Product recalled

## Manufacturer Actions: van Acker (2001): NEC Outbreak

- Production facility in The Netherlands upgraded
- More stringent standards adopted for products (< 0.3 coliform/g, 0 *E. sakazakii* isolates/10 g)
- 1999: implicated powdered formula reintroduced
- As of publication date, no further cases of NEC associated with *E. sakazakii*

**Notes:**
Authors: “From all these elements of our cohort study, we conclude that there is a strong, if not causal, relationship between intrinsic contamination of powdered milk formula with E. sakazakii and the development of NEC.”