Cronobacter sakazakii Isolated in Two Infants in New Mexico, 2008

In the October 30, 2009, edition of its weekly MMWR publication, the Centers for Disease Control and Prevention (CDC) reported on an investigation in November, 2008, when the Cronobacter sakazakii bacteria was isolated in two different infants. As recognized by the CDC, isolation of this organism from human specimens is rare and makes these cases notable. Cronobacter sakazakii (formerly Enterobacter sakazakii) are rare causes of infant septicemia and meningitis, resulting in death in approximately 40% of cases. Since 1958, 120 cases of Cronobacter sakazakii 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. This report provides important additional information regarding this elusive pathogen, and updates the CDC’s recommendations regarding safer PIF preparation, storage, and handling.

The Cronobacter sakazakii bacteria were isolated from two non-hospitalized, unrelated infants in November, 2008, in New Mexico. The CDC and FDA investigators determined that the female infant had been infected with Cronobacter sakazakii, and that the male infant had been colonized with Cronobacter sakazakii, without clear evidence of infection. Ingestion of PIF was the only identified risk factor for Cronobacter sakazakii exposure for the two infants. The two infants had consumed the same brand of PIF but had no other common exposures. The female infant had documented Cronobacter sakazakii infection that led to severe brain injury and hydrocephalus. Although a Cronobacter sakazakii organism was isolated from the male infant at autopsy, the role of that organism in the infant's apparent death from SIDS is unknown.

The two infants had consumed the same brand of formula, but their clinical Cronobacter sakazakii isolates had different Pulsed Field Gel Electrophoresis (PFGE) patterns. None of the samples obtained from the home of the female infant yielded Cronobacter sakazakii. Samples taken from the home of the male infant, however, provided positive results for Cronobacter sakazakii. An opened can of PIF yielded a Cronobacter sakazakii isolate with a PFGE pattern that was indistinguishable from the clinical Cronobacter sakazakii isolate from the male infant. Additionally, the vacuum cleaner filter from the home of the male infant also yielded Cronobacter sakazakii, but with a different PFGE pattern than the PFGE pattern isolated in both the male infant and the open PIF can.

The CDC reaffirmed in this report that prior investigations have found Cronobacter sakazakii 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, no other clear source of Cronobacter sakazakii infection has been identified to date. Accordingly, the report recommended that preparers should be aware that PIF is not sterile and can contain pathogenic organisms, such as Cronobacter sakazakii. The report also recommended that WHO guidelines for preparation of PIF, including reconstitution with water hot enough to inactivate Cronobacter sakazakii, be adopted, for safer PIF preparation, storage, and handling. In the United States and elsewhere, present recommendations are: to breastfeed infants when possible; to use sterile liquid infant formula in high-risk settings (e.g., neonatal intensive-care units and hospital nurseries); and to adhere to the safest available PIF preparation procedures. Interestingly, the CDC report noted that the manufacture of sterile powdered infant formula, perhaps by using irradiation in combination with other techniques, could prevent infant disease. Finally, the CDC stated that further precautions to prevent extrinsic contamination of PIF are needed, including the engineering of PIF packaging to prevent introduction of contaminated hands, scoops, or other items.

The complete report is accessible at MMWR. 2009:58;1179-1183.

Development of a CEN-ISO horizontal standard method

Editor's Note: Another of the presentations follows from last January's 1st International Conference on Cronobacter (Enterobacter Sakazakii) held at University College Dublin.  Dr. Han Joosten from the Nestle Research Center in Switzerland addresses the standard method for detection of Cronobacter.

Biography: Dr. Han Joosten is a Senior Scientist at the Nestle Research Center in Lausanne, Switzerland. At Nestle since 1996, he is responsible for providing scientific guidance on various research projects and early identification of emerging microbiological safety issues. He also provides advice to the business and quality management on analytical methods, hurdle technology, safety assessments and HACCP.
After finishing his studies at the University of Nijmegen in the Netherlands in 1983 Joosten worked five years at NIZO Food Research on the formation of biogenic amines in cheese, obtaining his PhD degree from the University of Wageningen on this subject.
From 1989 to 1991 he worked as a postdoc at the Autonomous University of Madrid on molecular characterization of African Swine Fever Virus. After this he headed the microbiological laboratory of Coberco Research in Deventer, the Netherlands and moved back to Spain in 1994 where he worked for two years at the National Instititute for Agricultural and Food Research (CIT-INIA) in Madrid on a bacteriocin-producing Enterococcus strain.


Summary - Development of a CEN-ISO horizontal standard method for detection of Cronobacter
The availability of a reliable and internationally accepted reference method for detection of Cronobacter in powdered infant formula is an essential tool to verify compliance with regulatory requirements by public health authorities and manufacturers. ISO-TS 22964:2006 was developed as a temporary solution for this purpose, but shortly after being issued it was decided to prepare a full-fledged horizontal CEN-ISO standard.
A summary will be given of the work done thus far, in particular with respect to the modifications that are envisaged to address the main shortcomings of TS 22964:

  • The scope will be extended to all types of powdered infant formula (incl. soy- based) and infant formula ingredients.
  • It will take into account the latest taxonomical revisions (e.g. definition of the genus Cronobacter and phenotypically related species)
  • It will no longer use yellow pigment production as a confirmation criterion
  • The enrichment broth (mLST) and chromogenic isolation agar (ESIATM) are too selective and need to be replaced by media that will also allow the detection of strains that are very susceptible to commonly used inhibitors of gram positive microorganisms.
  • The main performance characteristics of the new standard will be determined

Based on the results obtained during an extensive comparative/collaborative trial a method based on the utilization of Cronobacter Screening Broth (CSB) in combination with modified DFI agar appears to be the most suitable procedure to be adopted in the new standard.

His POWERPOINT can be found here.

Oxoid Thermofisher Scientist Patrick Duggan Address Culture Media For Isolation and Detection of Cronobacter Species

 Editor's Note: This is another report on the presentations that were made in Dublin earlier this year at the 1st International Meeting on Cronobacter (Enterobacter Sakazakii).  In this segment, we hear from Dr. Patrick Druggan, Oxoid Ltd., Thermo Fisher Scientific, Basingstoke, Hampshire RG24 8PW, United Kingdom.

Biography: Patrick Druggan  is Principle Scientist, Oxoid Thermofisher, Basingstoke, UK.

He received an Honours BSc in Food Science from the University of Strathclyde, Glasgow, UK. He has worked in the diagnostics industry for 22 years. He designed his first chromogenic medium in 1989 while working at Gibco.

Patrick studied part-time for his Ph.D. at the Pharmacy Department of University of Brighton, UK. His thesis was on improvements in the resuscitation of heat-injured Salmonella species from processed food samples.

He synthesized a number of autocytotoxic compounds that could be used during pre-enrichment to inhibit competitive microflora while allowing injured Salmonella spp. to resuscitate and grow.

This invention lead to the development of Inhibigens.™ His skills in chemistry and microbiology have allowed him to design a number of successful rapid biochemical tests and chromogenic culture media, including Druggan-Forsythe-Iversen Agar for the isolation of Cronobacter spp. 

Summary – - Culture media for isolation and detection of Cronobacter species

In 2001 a pre-term infant died of meningitis caused by Enterobacter sakazakii (Cronobacter spp.).  Infant formula milk (IFM) was implicated as a potential source of the infection.

The Food and Drug Administration (FDA) independently develop a method for enumeration of this emerging  pathogen in IFM using culture collections from national bodies that have later been shown to be poorly defined.

This method was introduced in 2002 and has regulatory standing for the import of IFM and skimmed milk powder in to the USA and a number of other countries. The FDA method is a modification of the procedure for the detection of Enterobacteriaceae, with the addition of yellow pigmentation of colonies for presumptive identification of Cronobacter spp.

It should be remembered that the FDA method was developed in a short time due to a public health concern, and this would have put a time constraint and significant pressure on those working on Enterobacter sakazakii (Cronobacter) to get a working method in the field as soon as possible. The FDA method has been shown to have a sensitivity of around 50 percent and a specificity of around 70 percent. Only 75 percent of Cronobacter strains phenotypically express yellow pigmentation, and the low specificity of the method coupled with the recommendation that only five presumptive Enterobacteriaceae colonies are tested from Violet Red Bile Glucose Agar (VRBGA) may explain the poor sensitivity of the method.

Assuming the prevalence of Cronobacter spp. in IFM is around 2 percent, the FDA method will fail to detect around 50 percent of batches contaminated with Cronobacter, while around 95 percent of rejected batches will not contain this organism.

This high rate of failure has lead many stakeholders to question the usefulness of the FDA method. This presentation reviews developments in culture media since the release of the FDA method in 2002, with specific emphasis on media that have improved the specificity of methods for Cronobacter spp. The unique phenotypic trait of this emerging pathogen that aid and hinder design of methods is discussed.  

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Dublin's Professor Fanning Speaks About Molecular Identification Methods for Cronobacter spp.

Editor’s Note: We next are going to hear from Professor Seamus Fanning of University College Dublin, addressing Molecular identification methods for Cronobacter. He also spoke at the Dublin conference on Cronobacter.


Biography:  Seamus Fanning is the Professor of Food Safety & Zoonoses and the Director of the Centre for Food Safety, University College Dublin. Professor Fanning is an editor of Research in Microbiology and a member of the editorial board of the Journal of Food Protection.

Professor Fanning received an Honours BSc in Biochemistry from NUI, Cork, where he also completed his PhD in Microbiology and Molecular Genetics. Current research interests include the application of molecular methods to Food Safety to control zoonotic microorganisms associated with human disease. A significant part of this work relates to the characterization of the genetic mechanisms contributing to the emergence of multiple drug resistance (MDR); the role of membrane bound efflux pumps in MDR and virulence and how these phenotypes are regulated at a local and global level.
Also, in the past few years the UCD Centre for Food Safety has published several papers describing the detection and characterization of Cronobacter. Professor Fanning is a member of the Microbiology Sub-Committee of the Food Safety Authority of Ireland (FSAI), the Scientific Advisory Committee of safe food and was recently appointed by the European Food Safety Authority (ESFA) to a working group to provide expert opinion on the emergence of antibiotic resistance in food. He also served as a member on the FAO/WHO expert panel on Enterobacter sakazakii in follow-up formula.


Summary: Molecular identification methods for Cronobacter spp.


Historically the ancestry of the genus Enterobacter can best be described as nebulus and confusing. In the 1970’s and 1980’s considerable movement of species, originally assigned to this genus occurred, and these re-designations arose because of initial misplacements, based on older phenotypic and morphological approaches to describing taxonomy.
Currently the genus Enterobacter comprises a large and heterogenous group of organisms within the Enterobacteriaceae family being accounted for by 16 distinct species. Enterobacter sakazakii (E. sakazakii) is one of these species and the only member of the genus recognised as a food-borne pathogen. Following a revision of Enterobacter taxonomy, a new genus Cronobacter was devised which is synonymous with E. sakazakii. Cronobacter consists of a least five distinct species and an additional genomospecies, Cronobacter sakazakii (C. sakazakii), C. dublinensis, C. malonaticus, C. muytjensii, C. turicensis and C. genomospecies I. A further three sub-species of C. dublinensis are also recognised. Correct identification of these organisms is important in order to improve our understanding of the broader epidemiology of the members of this new genus.
In recent years there have been rapid improvements in the provision of microbiologically-based culture approaches to isolate and identify these organisms. A number of molecular identification methods have also been proposed, however the recent recognition of multiple species that share less than 70 percent DNA-DNA similarity has important implications for the sensitivity and specificity of these methods. In this paper, three examples of the application of molecular-based detection strategies for the identification of Cronobacter will be presented.
These will include strategies to identify the genus, specific targets that are thought to be related to pathogenicity and the development of a molecular-based approach to begin to define the O-serotypes of C. sakazakii. Although by no means complete, these examples will illustrate some of the current and future challenges to enable a more refined and reliable molecular-based approach to the identification of all Cronobacter spp.
The development of appropriate molecular methods will facilitate not only a rapid identification of an isolate, but in addition complement the more traditional microbiological-based methods.


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