Survival characteristics of Cronobacter spp. in model bovine gut and in the environment


01.jun.10
Foodborne Pathogens and Disease. 7(6): 671-675. doi:10.1089/fpd.2009.0449
Catherine Molloy, Claire Cagney, Séamus Fanning, Geraldine Duffy
http://www.liebertonline.com/doi/abs/10.1089/fpd.2009.0449
Abstract
Cronobacter spp. (formally Enterobacter sakazakii) have been linked to illness in infants from contaminated powdered
infant formula. The source of the pathogen remains unclear, and it is believed that farm environments and cattle could
play a role in the transmission of Cronobacter spp. The aim of this study was to establish if the organism would survive
passage through a model of the bovine rumen and abomasum and in bovine feces in the farm environment. Models of
the bovine abomasum and rumen were inoculated with Cronobacter strains (final concentrations of 5.7 and 6.5 log10
CFU/mL, respectively), and survival was examined over time in these environments using an adapted ISO/DTS 22964
culture protocol. Fecal samples were inoculated with Cronobacter (final concentration 6.0 log10 CFU/mL), and survival
on soil and in containers stored outdoors was examined over time. The results showed no significant changes in the
number of Cronobacter in rumen fluid over a 24-h period. Cronobacter were undetectable after 30min of incubation in
the model abomasum. The pathogen survived 105 days in sealed containers and was detectable after 112 days in soil.
This study indicated that Cronobacter spp. are unlikely to be shed in bovine feces as supported by previous
surveillance studies; however, the study did show that the organism survives well in the farm environment.


The Naming Of Cronobacter Sakazakii
http://www.foodsafetynews.com/2009/09/the-naming-of-cronobacter-sakazakii/
by Andy Weisbecker | Sep 27, 2009

"Classifications are theories about the basis of natural order, not dull catalogues compiled only to avoid chaos."  
Stephen Jay Gould, Wonderful Life (1989), 98.

Enterobacter sakazakii, a gram-negative bacillus, is a rare cause of bloodstream and central nervous system infections.  
In 2007, following extensive study, it was proposed that the original taxonomy of Enterobacter sakazakii be revised, to
consist of five new species moved to a new genus, identified as "Cronobacter". (1)  A review of the what, the how, and
the why the change was first proposed, and why it was eventually approved, provides an insight into the related
scientific process of taxonomy at work, involving this notorious neonatal pathogen.

Initially, taxonomy is the science of classifying organisms, identifying and naming species, and organizing them into
systems of classification.  At least 1.7 million species of living organisms have been discovered, and the list grows longer
every year.   Ideally, classification should be based on homology, i.e., the shared characteristics that have been
inherited from a common ancestor.  Until recent decades, the study of homologies was limited to anatomical structures
and pattern of embryonic development.  However, since the birth of molecular biology, homologies can now also be
studied at the level of proteins and DNA. (2)

More specifically, E. sakazakii is a rare, but life-threatening cause of neonatal meningitis, sepsis, and necrotizing
enterocolitis. In general, E. sakazaii kills 40-80 % of infected newborns diagnosed with this type of severe infection. (5)
E. sakazakii meningitis may lead to cerebral abscess or infarction with cyst formation and severe neurologic impairment.
E. sakazakii can cause a variety of infections, though central nervous system infection has been most commonly
described. (6)  For infants, infection typically manifests through signs of sepsis in the first week of life: irritability or
lethargy, temperature instability, and feeding intolerance.  Meningitis often produces overwhelming infection that rapidly
moves through cerebral hemorrhage, infarct, necrosis, liquefaction, and eventually, cyst formation. (7)

E. sakazakii invasive infections occur more frequently in infants than in older children. (9) The neonate's immature
immune system may increase the risk of acquiring an E. sakazakii infection. (10) In a study of E. sakazakii cases over a
47 year period, investigators found that the median age at infection onset was two days and 94% of cases were less
than 28 days old. (11)  

While the reservoir for E. sakazakii is unknown in many cases, a growing number of reports have established powdered
infant formula as the source and vehicle of infection.  In several investigations of outbreaks of E. sakazakii infection that
occurred among neonates in neonatal intensive care units, investigators were able to show both statistical and
microbiological association between infection and powdered infant formula consumption.  These investigations included
cohort studies which implicated infant formula consumed by the infected infants. 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
the survival of an infection with E. sakazakii in infants.  (13)

The first cases attributed to this organism occurred in 1958 in England (Urmenyi and Franklin, 1961). Since then, up to
July 2008, around 120 documented cases of E. sakazakii infection, and at least 27 deaths, have been identified from all
parts of the world in the published literature and in reports submitted by public health organizations and laboratories.
(12)

E. sakazakii used to be previously known as a "yellow pigmented Enterobacter cloacae", until 1980.  E. sakazakii was
first defined as a novel species in 1980, when it was introduced as a new species based on differences in DNA-DNA
hybridization, biochemical reactions, and antibiotic susceptibility. The bacteria was named sakazakii in honour of the
Japanese microbiologist, Riichi Sakazaki, when the species was first designated in 1980. (15)  Enterobacter sakazakii
(E. sakazakii) then became identified as one of sixteen distinct species in the genus Enterobacter, within the
Enterobacteriaceae family. (14)  

From the beginning, however, many different biogroups were defined as E. sakazakii, with the existence of these
divergent geno- and biogroups suggesting that E. sakazakii could in fact represent multiple species. (14)  Accordingly,
in 2007, a research group clarified the taxonomic relationship among the various E. sakazakii strains, by using
sophisticated new means of viewing and analyzing the bacteria.  Iverson et al were thus able to distinguish numerous
separate species.  Their work resulted in the proposal of an alternative classification of E. sakazakii into a new genus,
Cronobacter. (16)

The new techniques used by the research group provide clear proof of the substantial advances made in molecular
biology, and included f-AFLP, automated ribotyping, full-length 16S rRNA gene sequencing and DNA-DNA hybridization.
(14)  F-AFLP (fluorescent amplified fragment length polymorphism) is a means to genotype bacteria, by selecting pre-
adapted fragments of DNA and amplifying them to easily detectable and accurately sizeable concentrations.  Automated
ribotyping is a genotyping method that can be used to generate genetic fingerprints of bacterial isolates.  Full-length
16S rRNA gene sequencing provides a means to compare a stable part of the genetic code (the 16S rRNA gene)
amongst different bacteria.  The technique of DNA-DNA hybridization provides genetic comparisons amongst the total
genome of two species.  

E. sakazakii has thus now been reclassified as 6 separate species in the new genus, Cronobacter,  gen. nov., within the
Enterobacteriaceae family. The new species are presently Cronobacter sakazakii; C. turicensis; C. malonaticus; C.
muytjensii and C. dublinensis; the sixth species is identified simply as genomospecies I, as currently it includes only two
representative strains. (19)

The name Cronobacter was appropriately derived from Greek mythology.  E. sakazakii constitutes a microbiological
hazard in the infant food chain, with historic high mortality in neonates.  Accordingly, it was named after the Greek
mythological god, Cronos. (17). Cronos was the son of Uranus (Heaven) and Gaea (Earth), being the youngest of the
12 Titans. He eventually became the king of the Titans, and took for his consort his sister Rhea.  Rhea bore him a
number of children, including Hestia, Demeter, Hera, Hades, and Poseidon.  Cronos, however, had been previously
warned by his parents that he would be overthrown by his own child.  Accordingly, he swallowed all those children.  
When Zeus was born, however, Rhea hid him in Crete, and tricked Cronus into swallowing a stone instead. Zeus grew
up, forced Cronus to disgorge his brothers and sisters, waged war on Cronus, and was victorious. (18)

It was proposed that these species be moved to the new genus, "Cronobacter", in order to facilitate their identification
for the diagnosis of infection and the microbiological monitoring of food products.  (20)  All these species have been
linked retrospectively to clinical cases of infection in either infants or adults, and therefore all these species should be
considered pathogenic. (21)  The correct and more detailed identification of these organisms will improve the
understanding of the broader epidemiology of the members of the new genus. 

It is also important, however, that this reclassification of species not be detrimental to health protection measures
already in place, and that all these risk organisms continue to be recognized. (22)  As the genus Cronobacter is
synonymous with Enterobacter sakazakii, current identification schemes developed for E. sakazakii remain applicable for
the Cronobacter genus.  The reclassification of E. sakazakii to the new genus Cronobacter will not require the
modification of dedicated culture-based laboratory isolation and detection protocols. All currently valid laboratory
methods will continue to facilitate the recognition of all of the organisms defined within the new taxonomy.  (23)  
Furthermore, the reclassification does not require any change to the regulatory framework currently in place. (24)


REFERENCES:


(1)    Iversen, C., Lehner, A., Mullane, N., Marugg, J., Fanning, S., Stephan, R., and Joosten, H., "Identification of
'Cronobacter' spp. (Enterobacter sakazakii)", Journal of Clinical Microbiology, Nov. 2007, Vol. 45, No. 11, p. 3814-3816.

(2)    Kimball's Biology Pages, http://www.ultranet.com/~jkimball/BiologyPages/ (9/25/2009)

(3)    Dauga, C and Breeuwer, P Taxonomy and Physiology of Enterobacter sakazakii, in ENTEROBACTER SAKAZAKII.
Washington D.C.: ASM Press; 2008:1 (Farber, JM and Forsythe, SJ editors).

(4)    Bowen AB, Braden CR. Invasive Enterobacter sakazakii disease in infants. Emerg Infect Dis [serial on the Internet].
2006 Aug publication. Available from http://www.cdc.gov/ncidod/EID/vol12no08/05-1509.htm.  

(5)    Bowen, AB and Braden, CR Enterobacter sakazakii Disease and Epidemiology. Farber, JM and Forsythe, SJ ed.
ENTEROBACTER SAKAZAKII. Washington D.C.: ASM Press; 2008:104.

(6)    Id.

(7)    Id.

(8)    Id.

(9)    "Enterobacter sakazakii and other microorganisms in powdered infant formula" Microbiological Risk Assessment
Series 6, World Health Organization (2004).

(10)    "Enterobacter sakazakii (Cronobacter spp.) in follow-up formula", Microbiological Risk Assessment Series 15-
FAO/WHO (2008).

(11)    Iversen, C., A. Lehner, N. Mullane, E. Bidlas, I. Cleenwerck, J. Marugg, S. Fanning, R. Stephan, and H. Joosten.
2007. "The taxonomy of Enterobacter sakazakii: proposal of a new genus Cronobacter", 1. BMC Evol. Biol. 7:64

(12)    "Enterobacter sakazakii (Cronobacter spp.) in follow-up formula", Microbiological Risk Assessment Series 15-
FAO/WHO (2008).

(13)    Id.

(14)    Iversen, C., A. Lehner, N. Mullane, E. Bidlas, I. Cleenwerck, J. Marugg, S. Fanning, R. Stephan, and H. Joosten.
2007. "The taxonomy of Enterobacter sakazakii: proposal of a new genus Cronobacter", 1. BMC Evol. Biol. 7:64.

(15)    "Enterobacter sakazakii (Cronobacter spp.) in follow-up formula", Microbiological Risk Assessment Series 15-
FAO/WHO (2008).

(16)    Id.

(17) Iversen, C., A. Lehner, N. Mullane, E. Bidlas, I. Cleenwerck, J. Marugg, S. Fanning, R. Stephan, and H. Joosten.
2007. "The taxonomy of Enterobacter sakazakii: proposal of a new genus Cronobacter", 1. BMC Evol. Biol. 7:64.

(18)    "Cronus." Encyclopædia Britannica. 2009. Encyclopædia Britannica Online. 21 Sep. 2009.

(19)    Iversen, C., Lehner, A., Mullane, N., Marugg, J., Fanning, S., Stephan, R., and Joosten, H., "Identification of
'Cronobacter' spp. (Enterobacter sakazakii)", Journal of Clinical Microbiology, Nov. 2007, Vol. 45, No. 11, p. 3814-3816.

(20)    "Enterobacter sakazakii (Cronobacter spp.) in follow-up formula", Microbiological Risk Assessment Series 15-
FAO/WHO (2008).

(21)    Iversen, C., Lehner, A., Mullane, N., Marugg, J., Fanning, S., Stephan, R., and Joosten, H., "Identification of
'Cronobacter' spp. (Enterobacter sakazakii)", Journal of Clinical Microbiology, Nov. 2007, Vol. 45, No. 11, p. 3814-3816.

(22)    "Enterobacter sakazakii (Cronobacter spp.) in follow-up formula", Microbiological Risk Assessment Series 15-
FAO/WHO (2008).

(23)    Id.