For many years we have been working on various detection methods for common and emergent foodborne pathogens such as Campylobacter, Salmonella, Listeria, Yersinia enterocolitica and more recently Cronobacter spp. (Enterobacter sakazakii), ranging from chromogenic agars, immunomagnetic separation and also hygiene assessment via ATP swab techniques. Our studies range from food hygiene and HACCP implementation in industry, through detection methods, to genetic manipulation of common pathogens, and virulence studies with mammalian cells cultures. Microbial diversity is addressed through various techniques; 16S rDNA, 7 loci MLST, rMLST (53 loci), COG-MLST (1865 loci), microarray and more recently whole genome sequence analysis. We are currently moving to microbiome studies of the intestinal flora. Please look at the 'Publications' page to see some of the outputs from our work.
The gold standard for so many detection methods remains the agar plate. Our work has helped develop a number of agars and broths for use within industry. Our emphasis has been on chromogenic agars (ie. DFI) because of their potential for discriminating target organisms within a mixed flora on an agar surface. We also helped develop and validate the immunomagnetic separation techniques for Salmonella and Listeria, as well as improvements for the ATP swabs which overcome the problems of detergent residues. Genotyping including PFGE, 16S rDNA sequencing and multilocus sequence typing (MLST) are used to ensure a diverse range of well characterized strains is used in all method developments.
This is an emergent pathogen that is primarily associated with infant infections including necrotising enterocolitis, bacteraemia and even meningitis. However infections occur in all age groups. The organism is ubiquitous and has been isolated from foods, environment and clinical sources. 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. Our work for the UK Food Standards Agency 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 detection methods. Through the support of Oxoid 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 are also working on the virulence of the organism, improved isolation methods and phylogenetic relationships. 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).
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 ahs over 500 strains listed. The scheme has also shown the lack of reliability in the previous biotyping/phenotyping methods for speciating Cronobacter strains (MolCellProbes). 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 showed 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. We already have the genome sequenced for this particular virulent form of the organism.
In 2012 the significance of C. sakazakii ST4 was substantiated by the analysis of the much publicized cases in the US in 2011, in which the isolates from meningitis cases were in the C. sakazakii ST4 clonal complex, CC4. Also we have announced the formal recognition in Intl J System Evol Microbiol of two new Cronobacter species; C. condimenti and C. universalis. Their recognition was very significantly based on the use of the 7-loci MLST. This scheme has been expanded to cover the whole genus of seven species, and includes the Open Access online curated database and protocols site: www.pubMLST.org/cronobacter/. The clonal complexes were described in our J. Clin. Microbiol. 2012 paper. The prominence of C. sakazakii ST4 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 have now considerably 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 includes describing 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.
Over 100 Cronobacter genomes in the public domain have been collated and can be investigated in the PubMLST database. The user can search for genes of interest using the BLAST facility, or construct their own MLST scheme. We have expand the conventional 7-loci scheme, to rMLST (53 loci), and COG-MLST (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 fifty papers published on the subject, including 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. Plus, the genome sequences of C. sakazakii, C. malonaticus,C. turicensis,C. dublinensis,C. muytjensii,C. universalis,and C. condimenti are published in PLoS ONE (2010 & 2012). 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 eight papers including genome sequencing.
We have also been investigating the bacterial colonisation of nasogastric enteral feeding tubes in neonatal intensive care units (BMC Inf. Dis. 2009) which has revealed the considerable bacterial biofilm formation inside the tubes, and the isolation of Cronobacter from tubes of neonates not fed infant formula. These could pose a risk to neonatal health.
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.
Campylobacter jejuni is recognised as the major cause of foodborne bacterial gastroenteritis. Yet its virulence mechanisms are still poorly understood. Dr Gina Manning is working on the genetics of hyperinvasion strains.
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 University of Northampton, on the virulence and persistence of the organism.
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.
We have an on-going longitudinal study of the neonatal microbiome. Applying Next Generation Sequencing to large number of samples from premature babies on various feeding regimes.
We have used bacteriophage as indicators of possible enteroviruses and there removal during water purification. Overall they are potentially very indicative of the possible presence of Noroviruses (formerly small round structured viruses, or Norwalk-like viruses) which cannot be grown using current tissue culture methods.