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 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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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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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). Below we hear from Professor Peter Kuhnert of the Institute of Veterinary Bacteriology, University of Bern, Switzerland.

Biography:  Peter Kuhnert is working at the University of Bern, Switzerland as an associate professor in bacteriology. He completed a PhD in molecular biology studying gene regulation of the porcine TNF-locus.
As a postdoc at the Weizmann Institute of Science in Israel he focused on the regulation of the human TNF-receptor genes and its role in the immune response. After his return 1994 he switched topics and is since then working at the Institute of Veterinary Bacteriology where he focuses on bacterial virulence, phylogeny and taxonomy.  His work includes pathotyping of E. coli, genotyping of foodborne pathogens with an emphasis on Campylobacter, pathogenesis and virulence mechanisms of Pasteurellaceae as well as work on Mycoplasma hyopneumoniae.

Summary - Multilocus sequence analysis (MLSA) of Cronobacter and related taxa Genetic similarity as determined by DNA-DNA hybridization is still considered the ‘gold standard’ method to determine relatedness between bacterial species. Nevertheless, it is very time consuming and cumbersome to perform and requires cross-hybridization between representatives of a species and related taxa. Moreover, variation between experiments, techniques and laboratories make exchange and comparison of data difficult. Whole genome sequence comparisons could be an alternative to DNA-DNA hybridization however, data handling and the
open question as to what genes should be used for defining genome similarity cannot be neglected. Therefore, for taxonomic purposes investigating as many isolates of a species as possible in order to respect the biodiversity of taxa, a few representative genes indicative for genetic similarity between isolates is the optimal way to go. Recently, we showed that the three genes recN, rpoA, and thdF can be used to estimate whole genome similarity of representatives of the family Pasteurellaceae [Kuhnert & Korczak (2006) Int.J.Syst.Evol.Microbiol 152: 2537-2548]
In the presented work multilocus sequence analysis (MLSA) based on recN, rpoA and thdF genes was done on more than 30 species of the family Enterobacteriaceae with a focus on Cronobacter and the related genus Enterobacter. The sequences provided valuable data for phylogenetic, taxonomic and diagnostic purposes. Phylogenetic analysis showed that the genus Cronobacter forms a homogenous cluster related to recently described species of Enterobacter, but distant to
other species of this genus. Combining sequence information on all three genes is highly representative for the species’ percentage of GC-content used as taxonomic marker. Sequence similarity of the three genes and even of recN alone can also be used to extrapolate genetic similarities between species of Enterobacteriaceae, being an alternative to DNA-DNA hybridization. Finally, the rpoA gene sequence, which is the easiest one to determine, provides a powerful diagnostic tool to identify and differentiate pathogens of this family. The comparative analysis gives important
insights into the phylogeny and genetic relatedness of the family Enterobacteriaceae and will serve as a basis for further studies and clarifications on the taxonomy of this large and heterogeneous family.

Powerpoint: Find it here.

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