With Wal-Mart Stores Inc., Supervalu Inc., Walgreen Co., Kroger Co. and Safeway pulling 12.5-ounce cans of Enfamil Newborn powdered infant formula with the lot number ZP1K7G after a Missouri newborn who consumed it apparently died of Cronobacter sakazakii, Cronobacter sakazakii, formerly, Enterobacter sakazakii is back in the news.
What is Cronobacter sakazakii, formerly Enterobacter sakazakii?
Enterobacter sakazakii was first described as a new bacterial species in 1980, and over a hundred scientific papers have been written about it. From 1980 to 2007 it was known as Enterobacter sakazakii, but in 2007 there was a proposal to reclassify it into a new genus Cronobacter as Cronobacter sakazakii. (1) For simplicity and for the reasons mentioned later, this discussion will continue to use the original name Enterobacter sakazakii. The proposed alternative classification is given as a section at the end and includes a discussion of the problems and the need for future studies.
Enterobacter sakazakii is a Gram-negative rod-shaped bacterium classified in the family Enterobacteriaceae. It has been isolated from three types of infection — devastating meningitis in very young babies (neonates), bacteremia (blood steam infection) in older babies, and a wide variety of infections (or colonization) in older babies, children and adults. The majority of infections reported in the peer-reviewed literature have described neonates—newborn infants, including premature infants, post-mature infants, and full-term newborns—with sepsis, meningitis, or necrotizing enterocolitis. (2) Although it has been isolated from cases of necrotizing enterocolitis, its causative role is unclear. (3)
Reported outcomes in neonatal meningitis are often severe: seizures; brain abscess; hydrocephalus; developmental delay; and death in as many as 40%–80% of cases. Premature infants are thought to be at greater risk than more mature infants, other children, or adults, and outbreaks have occurred in hospital units for newborns. (3)
Although E. sakazakii can cause illness in all age groups, infants are believed to be at greatest risk of infection and E. sakazakii was first implicated in a case of neonatal meningitis in 1958 (before the organism was given a scientific name). Since that time, around 70 cases of E. sakazakii infection have been reported, but it is likely that is the number of cases is significantly under-reported in all countries and the incidence is probably higher. (4)
Experts from the Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO) met in 2004 to summarize information, and develop international guidelines and educational messages regarding E. sakazakii. The meeting confirmed that there is very little known about virulence factors and pathogenicity of this bacterium. Like other bacteria in the family Enterobacteriaceae, such as Escherichia coli, differences in virulence among E. sakazakii may exist, and it is possible that strains are less or non-pathogenic. Work done by Pagotto et al. (2003) was the first describing putative virulence factors for E. sakazakii. Enterotoxin-like compounds were produced by some strains, but not all. Using tissue cultures, some strains produced a cytotoxic effect. Two strains (out of 18 isolates) were capable of causing death in suckling mice by the peroral (administered by mouth) route. (5)
Mortality rates from E. sakazakii infection were once reported to be 50 percent, but this figure has declined to under 20 percent in recent years. Significant morbidity in the form of neurological deficits can result from infection, especially among those with bacterial meningitis and cerebritis (inflammation of the cerebrum of the brain). While the disease is usually responsive to antibiotic therapy, a number of authors have reported increasing antibiotic resistance to drugs commonly used for initial treatment of suspected Enterobacter infection. Long-term neurologic sequelae are well recognized. (5)
Enterobacter sakazakii kills 40%–80% of infected infants. In 2007, the Centers for Disease Control and Prevention (CDC) reviewed literature for reports of E. sakazakii and analyzed 46 cases of invasive infant infection to define risk factors and guide prevention and treatment. Of the 46 cases analyzed, twelve infants had bacteremia, 33 had meningitis, and 1 had a urinary tract infection. Compared with infants with isolated bacteremia, infants with meningitis had higher birth weight (2,454 grams vs. 850 grams, with a probability value, p = 0.002) and longer gestational age (37 weeks vs. 27.8 weeks, p = 0.02), and infection developed at a younger age (6 days vs. 35 days, p<0.001). Among meningitis patients, 11 (33%) had seizures, 7 (21%) had brain abscess, and 14 (42%) died. (3)
Although E. sakazakii has been isolated from all age groups, infants (children less than 1 year) are at most risk. The groups of infants at greatest risk include pre-term infants, low-birth-weight (less than 2.5 kilograms) infants or immunocompromised infants. However, infants who are compromised for any other reason may also be at greater risk of E. sakazakii infection. Infants of HIV-positive mothers are at risk because they may be immunocompromised, and may specifically require powdered infant formula. (4)
In 2006, an FAO/WHO expert working group concluded that neonates and infants under two months are at greatest risk. Research has shown that premature infants who develop bacteremia after one month of age and term infants who develop meningitis during the neonatal period are still more at risk. (4)
In the United States, an incidence rate of 1 per 100,000 infants for E. sakazakii infection has been reported. This incidence rate increases to 9.4 per 100,000 in infants of very low birth weight, i.e. less than 1.5 kilograms. (4)
While the source of every E. sakazakii infection was not conclusively shown, a growing number of reports have established powdered infant formula as the source (vehicle) of infection. In several investigations of E. sakazakii outbreaks that occurred among neonates in neonatal intensive care units, investigators were able to show both epidemiological and microbiological associations between infection and the consumption of powdered infant formula. These investigations included cohort studies, which implicated infant formula as the cause of the outbreaks. In addition, there was no evidence of infant-to-infant or environmental transmission; all cases had consumed the implicated formula. The stomach of newborns, especially of premature babies, is less acidic than that of adults: a possible important factor contributing to infection with E. sakazakii in infants. (5)
Limited information was available on the numbers of E. sakazakii organisms that ill patients were exposed to in any of the various outbreaks and it is therefore not possible to develop a dose-response curve for E. sakazakii to establish the “infectious dose.” However, it is possible that a small number of cells present in powdered infant formula can cause illness. (4)
Unfortunately it is not possible to manufacture powdered infant formula to be completely free of E. sakazakii contamination, and even very low levels of contamination are of great concern as an infection risk. In a study of the prevalence of E. sakazakii contamination in 141 powdered infant formulas, 20 were found culture-positive, yet all met the microbiological specifications of the current Codex code for coliform counts in powdered infant formula (less than 3 bacteria per gram). Powdered infant formula that was contaminated with E. sakazakii has been linked to outbreaks. (5)
Outbreaks of E. sakazakii have also occurred in which the investigators have failed to identify lapses in formula preparation procedures. Thus, it seems that neither high levels of contamination nor lapses in preparation hygiene are necessary to cause infection from E. sakazakii in powdered infant formula. Lapses in technique in preparing formula and extended holding at non-refrigerated temperatures can lead to increases in the levels of E. sakazakii contamination at the time of consumption. However, it is not possible to assess the contribution of these factors have on the cases that have been associated with powdered infant formula that contained low levels of E. sakazakii. Thus it must be currently assumed that low levels of E. sakazakii in infant formula (less than 3 cfu/100 g) can lead to infections. (5) These microbiological standards do not consider that E. sakazakii can be present in the form of large pieces of biofilm rather than as individual cells.
The April 12th 2002 issue of CDC’s Morbidity and Mortality Weekly Report described a fatal case of meningitis in an intensive care nursery in Tennessee. The infecting organism was E. sakazakii – an unusual but often fatal invasive pathogen. In the fatal Tennessee case, the infection was traced to powdered infant formula contaminated with E. sakazakii. Other infants in the same nursery were screened for E sakazakii. Of 49 screened infants 10 were infected or colonized (1 proven infection, 2 possible infections, and 7 colonizations). This report showed a direct link to unopened powdered infant formula contaminated with E. sakazakii for the first time. The manufacturer voluntarily recalled the contaminated batch of powdered formula shown to be the source. (6)
In 2004, powdered infant formula was microbiologically linked to two E. sakazakii outbreaks, in New Zealand and in France. The French outbreak involved nine cases, and resulted in the death of two infants. Eight of the cases were in premature infants of low birth weight (less than 2 kg) and one case was in an infant born at 37 weeks and weighing 3.25 kg. The outbreak involved five hospitals, and a review of practices in the hospitals revealed that one hospital was not following recommended procedures for the preparation, handling, and storage of feeding bottles. Four hospitals were storing reconstituted formula for more than 24 hours in domestic-type refrigerators, with no temperature control or traceability. (4)
The U. S. Food and Drug Administration (FDA) points out that powdered infant formulas are not commercially sterile products. Powdered milk-based infant formulas are heat-treated during processing, but unlike liquid formula products they are not subjected to high temperatures for sufficient time to make the final packaged product commercially sterile. FDA has noted that infant formulas nutritionally designed for consumption by premature or low birth weight infants are available only in commercially sterile liquid form. However, so-called “transition” infant formulas that are generally used for premature or low birth weight infants after hospital discharge are available in both non-sterile powder form and sterile liquid form. Some other specialty infant formulas are only available as a non-sterile powder. (2)
The FDA has become increasingly aware that a substantial percentage of premature neonates in neonatal intensive care units are being fed non-sterile powdered infant formula products. In light of the epidemiological findings and the fact that powdered infant formulas cannot be manufactured free of dangerous microbial pathogens, FDA recommends that powdered infant formulas not be used in neonatal intensive care settings unless there is no alternative available. If the only option available to address the nutritional needs of a particular infant is a powdered formula, risks of infection can be reduced by:
• Preparing only a small amount of reconstituted formula for each feeding to reduce the quantity and time that formula is held at room temperature for consumption;
• Recognizing differences in infant formula preparation among hospitals—individual facilities should identify and follow procedures appropriate for that institution to minimize microbial growth in infant formulas;
• Minimizing the holding time, whether at room temperature or while under refrigeration, before a reconstituted formula is fed; and
• Minimizing the “hang-time” (i.e., the amount of time a formula is at room temperature in the feeding bag and accompanying lines during enteral tube feeding), with no “hang-time” exceeding 4 hours. Longer times should be avoided because of the potential for significant microbial growth in reconstituted infant formula. (2)
WHO recommends that infants should be exclusively breastfed for the first six months of life to achieve optimal growth, development, and health. Thereafter, to meet their evolving nutritional requirements, infants should receive nutritionally adequate and safe complementary foods while breastfeeding continues for up to two years of age or beyond (WHO/UNICEF, 2003). It is important to support breastfeeding and promote its benefits to infants and young children. (4)
There are, however, instances where breast milk is not available, where the mother is unable to breastfeed, where they have made an informed decision not to breastfeed, or where breastfeeding is not appropriate i.e. when the mother is taking medication that is contraindicated for breastfeeding or when the mother is HIV-positive. Similarly, some very low-birth-weight babies may not be able to breastfeed directly, and in some cases expressed breast milk may not be available at all or available in insufficient quantities. Infants who are not breastfed require a suitable breast-milk substitute, for example, an infant formula prepared in accordance with the present guidelines. WHO Guidelines for infant formula preparation, storage, and handling (2007), in both care settings and at home, are specified at http://www.who.int/foodsafety/publications/micro/powdered infant formula (4)
In January 2006, a second meeting (after the initial one in 2004) of experts from the Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO) took place to summarize information, and develop international guidelines and educational messages. The meeting participants first re-endorsed the recommendations made by the 2004 FAO/WHO meeting on this issue. The additional recommendations made by the expert meeting to member countries included the following:
• Develop prevention strategies for E. sakazakii infections caused by contaminated powdered infant formula that address the different stages of production and preparation and use of powdered infant formula, taking into consideration the risk to infants? Both within and beyond the neonatal period and of any immune status.
• Develop educational messages on the safe handling, storage and use of powdered infant formula, including the health hazards of inappropriate preparation and use; target healthcare workers, parents and other caregivers, in both hospitals and the community, since E. sakazakii infections have occurred in hospital and home settings.
• Review and revise product labels, as appropriate, to enable caregivers to handle, store and use the product safely, and to make clear the health hazards of inappropriate preparation.
• Encourage member countries to establish surveillance and rapid response networks, and facilitate coordinated investigation by clinicians, laboratory workers, and public health and regulatory officials, to enable the timely recognition and cessation of outbreaks of illness associated with E. sakazakii and the identification of contaminated powdered infant formula. (7)
(1) US FDA [U.S. Food and Drug Administration]. Health professionals letter on Enterobacter sakazakii infections associated with use of powdered (dry) infant formulas in neonatal intensive care units. 2002. Available from http://www.fda.gov/Food/FoodSafety/Product-SpecificInformation/InfantFormula/AlertsSafetyInformation/ucm111299.htm.
(2) Bowen AB, Braden CR. Invasive Enterobacter sakazakii disease in infants. Emerg Infect Dis 2006; 12(8): 1185-1189. Available from http://www.cdc.gov/ncidod/EID/vol12no08/05-1509.htm.
(3) WHO [World Health Organization], FAO [Food and Agriculture Organization of the United Nations]. Guidelines for safe preparation, storage and handling of powdered infant formula. 2007. Available from
(4) WHO [World Health Organization]. Enterobacter sakazakii and other microorganisms in powdered infant formula. MRA Series 6, 2004. Available from http://www.who.int/foodsafety/publications/micro/mra6/en/index.html.
(5) CDC [Centers for Disease Control and Prevention]. Enterobacter sakazakii infections associated with the use of powdered infant formula-Tennessee, 2001. MMWR, Morb Mortal Wkly Rep 2002; 51(14):298-300. Available from http://www.cdc.gov/mmwr/preview/mmwrhtml/mm5114a1.htm.
(6) WHO [World Health Organization]. Enterobacter sakazakii and Salmonella in powdered infant formula: Meeting report. MRA Series 10, 2006. Available from http://www.who.int/foodsafety/publications/micro/mra10/en/index.html.
(7) Iversen C, Lehner A, Mullane N, et. al. The taxonomy of Enterobacter sakazakii: proposal of a new genus Cronobacter gen. nov. and descriptions of Cronobacter sakazakii comb. nov. Cronobacter sakazakii subsp. sakazakii, comb. nov., Cronobacter sakazakii subsp. malonaticus subsp. nov., Cronobacter turicensis sp. nov., Cronobacter muytjensii sp. nov., Cronobacter dublinensis sp. nov. and Cronobacter genomospecies I. BMC Evol Biol 2007; Apr 17;7:64.
Note: Reclassification of Enterobacter sakazakii
Both the original 1980 study and subsequent studies noted heterogeneity in the species E. sakazakii. Recently Iversen and co-workers proposed that E. sakazakii be reclassified in a new genus Cronobacter which was defined to have four named species, one unnamed species, and five named subspecies (7):
Cronobacter sakazakii Cronobacter sakazakii subspecies sakazakii Cronobacter sakazakii subspecies malonaticus Cronobacter dublinensis Cronobacter dublinensis subspecies dublinensis Cronobacter dublinensis subspecies lactaridi Cronobacter dublinensis subspecies lausanensis Cronobacter muytjensii Cronobacter turicensis Cronobacter genomospecies 1 (a distinct species, but unnamed)
This proposal was based on sound scientific evidence and is generally being accepted by the scientific community. However, the new proposed raises several issues and problems. The first problem is that only a few laboratories in the world have the expertise and resources to identify bacterial isolated to this precise level. Almost all hospital laboratories use commercial identification systems, which cannot identify strains to the level of species/subspecies as given above. Even Dr, Matthew Arduino’s laboratory at CDC does not identify strains of the Enterobacter sakazakii – Cronobacter group to the species and subspecies level.
The second problem is that all of the paper that have been written about E. sakazakii need to be re-evaluated in the light of the new knowledge of the study of Iversen and co-workers. Until these problems are solved, precise names should be used that do not overstate what was actually found by laboratory identification methods. For example, the statement “Enterobacter sakazakii was isolated from the intestine of a fruit fly caught in the kitchen of the case.” would be replaced by one of the following statements: “An isolate of Cronobacter species was isolated from the intestine of a fruit fly caught in the kitchen of the case.” or “An isolate of the Enterobacter sakazakii – Cronobacter complex was isolated from the intestine of a fruit fly caught in the kitchen of the case.” All isolates of the Enterobacter sakazakii – Cronobacter complex need to be studied by experts who can identify them precisely. This would lead to corrections in the literature and correction about our knowledge of this group. One theoretical result of this needed retrospective analysis would be:
“The organism that Farmer isolated in 1978 from his dog’s water bowl (CDC strain designation: 1167-78) and originally identified as E. sakazakii biogroup 3 has been retested. The revised identification of this strain is Cronobacter dublinensis subspecies lactaridi* based on the following criteria … .” Or whatever the revised identification turns out to be. Many additional studies of this complex group of organisms are needed to define its ecology, epidemiology and role in human infections. Until these studies are done, all laboratory results and literature reports should be interpreted critically and with due caution.