Cronobacter Species Isolation in Two Infants --- New Mexico, 2008

Cronobacter Species Isolation in Two Infants --- New Mexico, 2008

Cronobacter spp. (formerly Enterobacter sakazakii) are rare causes of infant septicemia and meningitis, resulting in death in approximately 40% of cases (1). Since 1958, 120 cases of Cronobacter infection in infants have been reported, an average of fewer than three cases per year worldwide. Powdered infant formula (PIF), which is not sterile, has been implicated repeatedly as a vehicle of Cronobacter infection; consequently, the World Health Organization (WHO) has issued guidelines for safer preparation, handling, and storage of PIF (2,3). This report describes isolation of Cronobacter spp. in two nonhospitalized, unrelated infants (one male and one female) in New Mexico in 2008; one infant developed severe brain injury and hydrocephalus, and the other infant died. An investigation by the New Mexico Department of Health (NMDOH) determined that PIF consumption was the only known risk factor in the two cases, although the sources of the Cronobacter spp. could not be determined. Cronobacter spp. were not isolated from sealed canisters of formula associated with the two infants, and clinical isolates from the infants differed by pulsed-field gel electrophoresis (PFGE). However, a Cronobacter organism was isolated from an opened canister of formula consumed by the male infant and was indistinguishable from an isolate from his postmortem blood culture. Education of formula preparers regarding potential PIF contamination, universal adoption of WHO PIF preparation guidelines, and continued improvement of PIF manufacturing processes might help prevent Cronobacter infection among infants (3).

On November 18, 2008, NMDOH received notification from a hospital laboratory that Cronobacter spp. (formerly Enterobacter sakazakii*) had been isolated from the cerebrospinal fluid of a female infant aged 7 weeks. The isolate was sent to the NMDOH Scientific Laboratory Division for confirmation, a team of investigators from NMDOH and CDC visited the infant's home, and officials from the Food and Drug Administration (FDA) collected PIF samples. Two days later, the Scientific Laboratory Division reported isolation of a second Cronobacter organism from the postmortem blood culture of a male infant aged 7 months. The two infants lived approximately 200 miles apart in different counties of mostly rural southeastern New Mexico.

Case Reports

Case 1. The female infant was born in September 2008 at full term by vaginal delivery without complications. Her mother had received prenatal care. The infant had been fed PIF exclusively since birth. The infant was well until approximately age 3.5 weeks, when a family member noted that she had an axillary temperature of 100.9°F (38.3°C) and took her to a hospital emergency department. Records for this visit noted a normal rectal temperature, normal fontanel, and overall healthy appearance. The infant was discharged without further testing or treatment. However, during the following week, the infant became notably fussier and began vomiting. Her illness progressed during early November, when at age 6.5 weeks she exhibited seizure-like activity. She was admitted to a hospital on November 14. On physical examination the infant was found to have a bulging anterior fontanelle, horizontal nystagmus, a positive Brudzinski sign with neck rigidity, and dehydration. Cerebrospinal fluid cultures collected during this admission yielded a Cronobacter organism; blood cultures were negative for Cronobacter. The infant was placed on intravenous antibiotic therapy, including ampicillin and cefotaxime. She was transferred to a second hospital within 24 hours of this admission for further evaluation and diagnostic testing.

At the second hospital, a computed tomography scan of the head revealed thick-walled hypodense lesions that were identified as abscesses. Within 24 hours of admission to the second hospital the infant was transferred to a third hospital where pediatric specialists in infectious disease and neurosurgery were available. On November 16, at the third hospital, a ToRCH workup (testing for toxoplasmosis, other infections, rubella, cytomegalovirus, and herpes simplex virus) was conducted and was negative. Serial magnetic resonance imaging conducted during November 17, 2008--February 4, 2009, revealed multiple brain abscesses, diffuse brain injury, and hydrocephalus with lateral third and fourth ventricle dilation. Fluid obtained through percutaneous aspiration of an abscess in the right frontal region of the brain yielded a Cronobacter organism that was resistant to cefazolin but susceptible to ceftriaxone, ciprofloxacin, gentamicin, and trimethoprim-sulfa. Treatments administered during hospitalization at the third hospital included intravenous meropenem, vancomycin, gentamicin, acyclovir, baclofen, and phenobarbital and multiple percutaneous aspirates of the left and right ventricles of the brain; placement of a vetriculoperitoneal shunt was not required. An electroencephalogram performed on January 12, 2009, was interpreted as abnormal because of a diffusely slow background. In addition, frequent multifocal epileptiform discharges for multiple areas of cortical hyperexcitability were noted that could have led to localization-related seizures. After 11.5 weeks in the hospital, the infant was discharged on February 6, 2009, with severe brain injury, hypertonicity resulting from central nervous system damage, and hydrocephalus.

Case 2. The male infant was born in April 2008 at 40 weeks' gestation by vaginal delivery without complications. His mother had received prenatal care. The infant was breastfed exclusively until age 6 months and then began transitioning to PIF and age-appropriate foods. Other than a history of mild-moderate eczema that was being treated with oral antihistamines, moderate-dose topical steroids, and topical antibiotics, the infant was healthy, as confirmed by medical records and a visit by a trained new-parent support worker on November 10, 2008. The following day, at age 7 months, he died unexpectedly while taking his usual nap at home. A Cronobacter organism was found in the blood culture obtained during autopsy. However, aside from mild eczema, no congenital or histopathologic abnormalities were noted during postmortem examination. Therefore, the Cronobacter isolate was attributed to postmortem bacterial overgrowth, and the autopsy report listed sudden infant death syndrome (SIDS) as the cause of death.

Epidemiologic and Environmental Investigations

Initial review of the two cases by CDC and FDA investigators determined that the female infant had been infected with Cronobacter spp. and the male infant had been colonized with Cronobacter spp. without clear evidence of infection. Ingestion of PIF was the only identified risk factor for Cronobacter exposure for the two infants. The two infants had consumed the same brand of PIF but had no other common exposures. PIF preparers reported washing their hands before preparing formula and washing bottle components by hand between feedings for the female infant and in the dishwasher for the male infant. Additionally, they reported following the manufacturer's instructions for preparing and handling PIF. In the female infant's household, water used for PIF reconstitution had been boiled, cooled, and then stored in the kitchen in a plastic container. Reconstituted PIF was not reheated, and new bottles were prepared for every feeding (approximately every 3 hours). In the male infant's household, PIF had been reconstituted by using refrigerated bottled water or tap water. The water was not boiled or heated before use.

Investigations were conducted at both infants' homes, which included collecting environmental samples and samples of PIF and other infant food. However, neither the container used to store water for formula preparation nor canisters of formula fed to the female infant before illness onset were available for testing; therefore, the lot numbers of PIF fed to the female infant before illness could not be determined. The PIF collected from the female infant's home had been fed to her after her symptoms began. In addition, NMDOH and FDA collected 30 unopened canisters of the same brand of PIF from retail stores where the parents of the two infants reported obtaining formula. These canisters had the same lot numbers and expiration dates as the formula collected from the infants' homes. Samples from the store canisters were submitted to the NMDOH Scientific Laboratory Division and FDA for testing; no Cronobacter spp. were isolated.

Environmental surface swabs and food and nonfood samples were cultured using standard methods. Opened and unopened canisters of PIF were tested by following the enrichment and isolation methodology for Cronobacter organisms outlined by FDA.† Suspected environmental and clinical isolates were confirmed as Cronobacter spp. through the use of conventional biochemical testing and DNA sequencing. Pulsed-field gel electrophoresis (PFGE) was performed on all Cronobacter isolates by using the standard PFGE methodology CDC employs for Escherichia coli O157:H7.

Although the two infants had consumed the same brand of formula, their clinical Cronobacter isolates had different PFGE patterns (Table). None of the samples obtained from the home of the female infant yielded Cronobacter spp. However, PIF from an opened canister and the vacuum cleaner filter from the home of the male infant yielded Cronobacter spp. The clinical Cronobacter isolate from the male infant and the Cronobacter isolate from the PIF canister sample from his home had indistinguishable PFGE patterns; however, the vacuum cleaner Cronobacter isolate from the same home had a different PFGE pattern (Table). Cronobacter spp. were not isolated from any of the unopened canisters of PIF in either home.

In response to the isolation of Cronobacter organisms from these infants, NMDOH issued recommendations regarding infant feeding practices, including breastfeeding when possible and using proper formula preparation procedures. NMDOH also requested notification from the three largest commercial laboratories in New Mexico of any results indicating Cronobacter spp. isolation from infants aged <1 year. In addition, a Health Alert Network message was sent to all laboratories across the state alerting them to these increased surveillance activities. No additional cases were identified.

Reported by: J Baumbach, MD, K Rooney, MPH, C Smelser, MD, P Torres, New Mexico Dept of Health. A Bowen, MD, Div of Foodborne, Bacterial, and Mycotic Diseases, National Center for Zoonotic, Vector-Borne and Enteric Diseases; M Nichols, DVM, EIS Officer, CDC.

Editorial Note:
This report describes the isolation of Cronobacter spp. from two nonhospitalized, unrelated infants in 2008 in New Mexico. Isolation of this organism from human specimens is rare and makes these cases notable. The female infant had documented Cronobacter infection that led to severe brain injury and hydrocephalus. Although a Cronobacter organism was isolated from the male infant at autopsy, the role of that organism in the infant's apparent death from SIDS is unknown. Isolation of Cronobacter spp. in association with SIDS has not been reported previously.

Previous investigations have found Cronobacter spp. cultured from prepared formula, unopened PIF containers, and the environment where PIF was reconstituted, clearly implicating PIF as the source of outbreaks. Other than an improperly prepared intravenous nutrition solution implicated in one outbreak (5), no other clear source of Cronobacter infection has been identified. Cronobacter spp. rarely are isolated from human intestines (6), and mother-to-child transmission has not been documented. The isolation of a Cronobacter organism from the vacuum cleaner filter in the male infant's household indicated the organism's presence in the home environment. However, the vacuum isolate differed by PFGE pattern from the clinical isolate, demonstrating that at least two distinct strains of Cronobacter spp. were present concurrently in the household. Although Cronobacter spp. have been isolated from household vacuum cleaners previously (7), the source of the Cronobacter organisms in such situations and the implications for human health are unknown. Isolation of Cronobacter organisms with indistinguishable PFGE patterns from the male infant's clinical specimen and an opened PIF canister in his home suggest contaminated PIF as a source of Cronobacter spp. for this infant. Because Cronobacter spp. were not isolated from unopened canisters of the PIF associated with the two cases in New Mexico, the PIF associated with the male infant might have been contaminated extrinsically. Canisters of PIF consumed by the female infant before her illness began were not available for testing, and the source of her infection could not be determined.

Although isolation is rare, the extent of Cronobacter infection has not been well enumerated in the United States; formal surveillance and reporting systems exist only in Minnesota, where a single infant case has been identified since 2005. A national FoodNet§ survey in 2002 estimated the annual incidence of invasive Cronobacter infection at one per 100,000 infants aged <1 year and at 8.7 per 100,000 low birthweight infants (<2,500 g [5.5 lbs]).¶ Despite limited surveillance for this infection in the United States and elsewhere, at least 12 outbreaks of severe Cronobacter infection have been observed worldwide since 1958, generally among infants hospitalized in intensive-care units (2). Although sporadic cases of community-acquired Cronobacter infection have been reported, outbreaks of community-acquired disease have not. However, enhanced surveillance might produce a more accurate estimate of the Cronobacter disease burden and insight into risk factors; accordingly, FoodNet is expected to soon begin piloting surveillance for Cronobacter infections in the United States.

Infants throughout the world consume PIF, some exclusively. PIF preparers should be aware that PIF is not sterile and can contain pathogenic organisms (e.g., Cronobacter spp.). Preparers also should be aware that PIF can be contaminated extrinsically (although mechanisms for such contamination are not well defined) and that bacteria can multiply rapidly in reconstituted PIF. Consequently, WHO has developed guidelines for preparation of PIF, including reconstitution with water hot enough to inactivate Cronobacter organisms (3). Universal adoption of these guidelines can aid in implementation of safer PIF preparation, storage, and handling. In the United States and elsewhere, recommendations are to breastfeed infants when possible, use sterile liquid infant formula in high-risk settings (e.g., neonatal intensive-care units and hospital nurseries), and adhere to the safest available PIF preparation procedures (8,9). Manufacturing sterile powdered infant formula, perhaps by using irradiation in combination with other techniques, could prevent infant disease (10). Additionally, further precautions to prevent extrinsic contamination of PIF are needed. Engineering of PIF packaging to prevent introduction of contaminated hands, scoops, or other items might help prevent additional infant disease.

References
Bowen AB, Braden CR. Invasive Enterobacter sakazakii disease in infants. Emerg Infect Dis 2006;12:1185--9.
Food and Agriculture Organization of the United Nations/World Health Organization (FAO/WHO). Enterobacter sakazakii (Cronobacter spp.) in powdered follow-up formulae. Rome, Italy: Food and Agriculture Organization of the United Nations/World Health Organization; 2008. Microbiological risk assessment series, No. 15.
Food and Agriculture Organization of the United Nations/World Health Organization (FAO/WHO). Guidelines for the safe preparation, storage and handling of powdered infant formula. Geneva, Switzerland: World Health Organization; 2007. Available at http://www.who.int/foodsafety/publications/micro/pif2007/en. Accessed October 23, 2009.
Iversen C, Mullane N, McCardell B, et al. Cronobacter gen. nov., a new genus to accommodate the biogroups of Enterobacter sakazakii. Int J Syst Evol Microbiol 2008;58(Pt 6):1442--7.
Santos M, Silva C, Marangoni D, Pinto M, Moreira B. Detection of Enterobacter sakazakii sepsis outbreak in four hospitals in Rio de Janeiro, Brazil. Presented at Fourth Decennial International Conference on Nosocomial and Healthcare-Associated Infections, Atlanta, GA; March 5--9, 2000.
Kim J, Cho S, Park Y, et al Surveillance of stool samples for the presence of Enterobacter sakazakii among Korean people. Yonsei Med J 2008;49:1017--22.
Kandhai M, Reij M, Gorris L, et al. Occurrence of Enterobacter sakazakii in food production environments and households. Lancet 2004;363:39, 40.
CDC. Enterobacter sakazakii infections associated with the use of powdered infant formula---Tennessee, 2001. MMWR 2002;51:298--300.
Drudy D, Mullane NR, Quinn T, et al. Enterobacter sakazakii: an emerging pathogen in powdered infant formula. Clin Infect Dis 2006;42:996--1002.
Lee JW, Oh SH, Kim JH, et al. Gamma radiation sensitivity of Enterobacter sakazakii in dehydrated powdered infant formula. J Food Prot 2006;69:1434--7.
* In 2008, the taxonomy of Enterobacter sakazakii was updated, resulting in creation of five novel species belonging to a new genus, Cronobacter, which is contaxic with E. sakazakii (4). Most laboratories do not yet have the capability to differentiate between the new Cronobacter species.

† Available at http://www.fda.gov/food/scienceresearch/laboratorymethods/ucm114665.htm.

§ The Foodborne Diseases Active Surveillance Network (FoodNet) of CDC's Emerging Infections Program collects data from 10 U.S. states on diseases caused by enteric pathogens transmitted commonly through food.

¶ Available at http://www.who.int/foodsafety/publications/micro/es.pdf.

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