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Research overview

For many years I worked on various detection methods for common and emergent foodborne pathogens such as Campylobacter, Salmonella, Listeria, and especially Cronobacter (previously called Enterobacter sakazakii), ranging from chromogenic agars, immunomagnetic separation and also hygiene assessment via ATP swab techniques. These studies included food hygiene and HACCP implementation in industry, all the way through detection methods and genetic manipulation of common pathogens, to virulence studies with mammalian cell cultures. Understanding microbial diversity was addressed through various techniques, in particular sequence-based techniques of 7 loci MLST, rMLST (53 loci), core genome-MLST (1865 loci), CRISPR-cas arrays and whole genome sequence analysis. More recent research has been microbiome studies of the neonatal intestinal flora and in situ nasogastric feeding tubes. Please browse through the 'Publications' page to see some of the outputs from this work.

Methods Development

The most common initial detection method remains the agar plate, and so our research has helped develop a number of agars and broths for use within industry. The emphasis has been on chromogenic agars (ie. DFI) because of their potential for discriminating target organisms within a mixed flora of closely related organisms on an agar surface. I also helped develop and validate the immunomagnetic separation techniques for Salmonella and Listeria, as well as improvements for the ATP swabs which overcame the problems of detergent residues. Genotyping ensured a diverse range of well characterized strains was used in method development.

Cronobacter (previously known as Enterobacter sakazakii)

This emergent pathogen that is generally associated with life-threatening infant infections including necrotising enterocolitis, bacteraemia and even meningitis. However infections occur in all age groups and the majority of infections are in the adult population. The organism is ubiquitous and has been isolated from foods, environment and clinical sources. Infant cases are mainly C. sakazakii, whereas adult cases are C. malonaticus. The former species is able to use sialic acid as a carbon source, which is a notable virulence trait as sialic acid occurs in gangliosides and mucin (sialic acid utilization). Many, but not all infant cases have been linked to the ingestion of rehydrated infant breast milk substitutes; commonly referred to as powdered infant formulas, PIF. Some cases have occurred in exclusively breast-fed babies, and also to expressed breast milk. Our work for the UK Food Standards Agency on the bacteriocidal preparation of PIF was released on their web site (FSA) in December 2009.

It should be noted that not all strains of Cronobacter produce a yellow pigment, nor grow well at 45C and therefore could be missed when using some 'standardised' detection methods. Through the support of Oxoid Thermo Fisher Scientific we developed a chromogenic agar (Int. J. Food Microbiol. 2004) that was subsequently marketed as Druggan-Forsythe-Iversen (DFI) agar (Oxoid product code CM1055). We also worked on the virulence of the organism, improved isolation methods, phylogenetic relationships and genotyping. Early studies using 16S & hsp60 sequencing showed that isolates identified by commonly used biochemical kits as 'E. sakazakii' were probably more than one species (see paper in J Clin. Microbiol. 2004). There have been a series of taxonomic revisions of this organism, and the genus Cronobacter is now recognised as being composed of 7 species; C. condimenti, C. dublinensis, C. malonaticus, C. muytjensii, C. sakazakii, C. turicensis, and C. universalis.

We have established a 7 loci multilocus sequencing typing scheme (MLST) for all Cronobacter species using strains from a range of sources over a 60 year period. The initial study of just C. sakazakii and C. malonaticus was published in BMC Microbiology (2009) and revealed a strong clonal nature within those species. This scheme has been extended without modification to all seven Cronobacter species, and has revealed the genus evolved ~40 MYA, with C. sakazakii and C. malonaticus differentiating ~11-23 MYA; J. Clin. Microbiol.. The MLST scheme is openly (without registration) available at http://www.pubMLST.org/cronobacter for anyone to use and has over 2000 strains listed, and over 500 whole genomes available for analysis. The MLST scheme has also shown the lack of reliability in the previous biotyping/phenotyping methods for speciating Cronobacter strains (MolCellProbes and BMCMicrobiol). We have also published describing the variation in identification between phenotyping, protein profiles (MALDI-TOF), PCR-probes and DNA-sequence based techniques.

In 2011 we published what may be one of the most important discoveries regarding Cronobacter for further research. We reported in CDC's online journal (Emerging Infectious Disease) that the majority of C. sakazakii meningitis isolates from the past 30 years in 6 countries are one sequence type (ST4). This remarkable observation is highly significant in terms of having a DNA fingerprint for the lineage associated with the majority of the most devastating form of Cronobacter infection. The following year (2012) the significance of C. sakazakii ST4 was substantiated by the analysis of the much publicized US cases of 2011, in which the isolates from meningitis cases were in the C. sakazakii ST4 clonal complex, CC4. A complex complex being closely related sequence types having 3 or less loci different.

The formal recognition in Intl J System Evol Microbiol of two new Cronobacter species (C. condimenti and C. universalis) was significantly based on the use of the 7-loci MLST scheme. This scheme therefore covers the whole Cronobacter genus of seven species, and includes the Open Access online curated database and protocols site: PubMLST Cronobacter. The key clonal complexes were described in our J. Clin. Microbiol. 2012 paper. The prominence of C. sakazakii CC4 is also reflected in isolates from infant formula and environmental samples from milk powdered processing factories (Intl Dairy J.).

We published (2010) the first annotated genome of C. sakazakii in collaboration with Mike McClelland and Sandy Clifton on the genome sequence of C. sakazakii and its comparison with other Cronobacter species. (PLoS ONE). We subsequently expanded that study with the genomic analysis of the entire genus. In our second publication in PLoS ONE (Joseph et al., 2012), we detailed genomic analysis of all 7 Cronobacter species (multiple strains per species). The strains were chosen for sequencing using MLST in order to ensure appropriate coverage of the diverse genus. The paper described previously unreported differences between the seven species, as well as the expected numerous adhesins, type six secretion systems, phage regions and metal resistance genes; as previously described in the two earlier papers by Kucerova et al. (2010 & 2011). See 'Publications' link for details. Now we have so many genomes that we do not publish general genome studies anymore, but the analysis of several hundred at a time such as CRISPR-cas arrays, and the link between particular capsulation genes in meningitic strains.

Over 500 Cronobacter genomes in the public domain have been collated and can be investigated in the open access PubMLST database. The user can search for genes of interest using the BLAST facility etc, or construct their own MLST scheme. We have expand the conventional 7-loci scheme, to ribosomal-MLST (rMLST, 53 loci), and core-genome (based on COG, cg-MLST) using 1865 loci. Four YouTube (and YouKU) videos have been released to help researchers to use the open access database as fully as possible. Please go the the PubMLST web site for the database and YouTube videos; www.pubMLST.org/cronobacter/.

The team has over 90 papers published on the subject of Cronobacter, the first being a risk profile in Trends in Food Science and Technology (2003). The article in Maternal and Child Nutrition (2005) was voted paper of the month by Unicef. We have also published a detailed account of a neonatal intensive care unit outbreak (J. Clin.Microbiol. 2007). The strains from this outbreak have been used in a further 30 papers including genome sequencing (10 year outbreak study).

Prof Steve Forsythe was participant in all three FAO-WHO risk assessment workshops regarding the microbiological safety of infant formula and more recently on infant feeding in famine relief.

See 'Publications' for a full listing of papers, and conference presentations.

Neonatal feeding tubes and intestinal flora

We have also been investigating the in situ bacterial colonisation of nasogastric enteral feeding tubes in neonatal intensive care units. Initial studies (BMC Inf. Dis.) revealed the considerable bacterial biofilm formation inside the tubes, and the isolation of Cronobacter from tubes of neonates not fed infant formula. These tubes can also be colonised by neonatal meningitic E. coli K1 (E. coli K1). We have also shown the near indistingusihable (<30 SNPs) colonisation of neonatal feeding tubes in situ by E. faecalis and E. hormaechei (both encoding for antibiotic resistance) (Nature SciReports).These tubes therefore pose a risk to neonatal health and further management needs to be considered.

We have an on-going longitudinal study of the neonatal microbiome which is applying Next Generation Sequencing to a large number of samples from premature babies on various feeding regimes. To date we have only published a preliminary study of whole genome sequenced strains from both feeding tubes and intestinal contents (ie. poo) which was published in Nature Scientific Reports referred to above. In addition to microbiology the study has used optical coherence tomography to visualise non-destructively the bacterial biofilm within the tubes.

Arcobacter

Arcobacter butzleri and related species are emergent pathogens. A. butzleri is a recognised veterinary pathogen, and is found in the food chain. There have been a few human cases of gastroenteritis due to this organism, but clear epidemiological evidence is still sparse. Regretfully this is partly due to the lack of general awareness and use of appropriate selective media. We have been working in conjunction with various groups on the virulence and persistence of the organism.

Food Surveys

The presence of foodborne pathogens in the food chain is very important in microbial risk assessment. We have undertaken a number of surveys for particular pathogens which serve the dual purpose of assessing the organisms' presence in foods, and also determining the efficacy of novel detection methods being developed. Linked to this are the studies into food hygiene and HACCP implementation in the food industry, particularly small companies. These have largely been published in refereed journals, though further work has been undertaken on consultancy basis.

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