A study by DVM Elias Dahlsten and colleagues under Prof. Hannu Korkeala’s supervision suggests a previously unidentified role for the alternative sigma factor SigK in response and adaptation to low temperature and high salt conditions in Clostridium botulinum. The study was published in Applied and Environmental Microbiology.

C. botulinum ATCC 3502 wild type strain was
evaluated for relative sigK expression levels after cold shock, exposure to hyperosmotic conditions, exposure to acidity, or in optimal growth conditions, using quantitative reverse transcription PCR. Significant induction of sigK expression was observed upon temperature downshift and in hyperosmotic shock, but not in pH downshift or in optimal growth. Insertional inactivation of sigK with the ClosTron system resulted in a phenotype with impaired growth at low temperature and in high salt concentration, but with no difference in growth at optimal temperature or low pH conditions.

To date, SigK of clostridia has been strictly sporulation-associated. In this study, the researchers suggest a hitherto unidentified role for this alternative sigma factor in stress tolerance. Study of mechanisms by which the feared foodborne pathogen C. botulinum senses and responds to various environmental stresses it might encounter as hurdles applied in food preservation is of key importance. Identification of stress-activated genetic machineries of foodborne pathogens might provide biomarkers to exploit in detection of potentially stress-adapted cells, allowing targeted control methods.

The research was carried out at the Finnish Centre of Excellence in Microbial Food Safety Research and supported by the Academy of Finland, the Finnish Graduate School on Applied Bioscience, the European Community’s Seventh Framework Programme FP7/2007-2013 “CLOSTNET”, and the Walter Ehrström Foundation.

The Department of Food Hygiene and Environmental Health and Professor Hannu Korkeala hosted the AntiBotABE meeting on 17-18^th June in Helsinki. Centre Recherche Service Santé Armée supported by VITAMIB coordinates an international consortium on the development of neutralizing antibodies against botulinum toxins A, B, E.

It is a collaborative project under the EU’s 7^th Research Framework Programme in the security research area. The AntiBotABE consortium contains of academic and industrial partners with the expertise to carry out highly innovative research and development in this exciting domain.

Participants from Centre Recherche Service Santé Armée, France, Ministère de la Défense, France, Technische Universität Braunschweig, Germany, Institut Pasteur, France, Health Protection Agency HPA, United Kingdom, Centre National de la Recherche Scientifique, France, LFB Biotechnologies, France, University of Helsinki, Finland and VITAMIB, France shared and evaluated the recent developments in the AntiBotABE project and also experienced the Finnish summer.

>> Read more AntiBotABE

Jere Lindén, DVM will defend the doctoral dissertation entitled “TCDD-induced changes in the expression of selected hypothalamic feeding-regulatory genes and mRNA quantification using reverse transcription–qPCR” in the Faculty of Veterinary Medicine, University of Helsinki, on 8 June 2013 at 12:00. The public examination will take place at the EE building, Walter hall, Agnes Sjöbergin katu 2, Helsinki.

Docent Risto Juvonen, University of Eastern Finland, will serve as the opponent, and Professor Raimo Pohjanvirta as the custos.

The dissertation is also available in electronic form through the E-thesis service.

Contact information:
Jere Lindén
jere.linden@helsinki.fi

The Clostnet conference was the final conclusion for the three-year “Clostnet” international research collaboration funded by the European Union’s 7^th framework program. The project was coordinated by the University of Nottingham. The participants of the project included research groups from several EU countries working with pathogenic or industrially significant bacteria of the genus Clostridium.

DVM Elias Dahlsten’s poster “Role of Two-Component System CBO0365/CBO0366 in Cold Stress of Clostridium botulinum ATCC 3502” received the “Best Poster” award at the conference. The research was conducted under Professor Hannu Korkeala’s supervision; the authors of the poster were DVM Elias Dahlsten, Prof. Miia Lindström, M.Sc. Zhen Zhang, D.Sc. (Tech.) Panu Somervuo and Prof. Hannu Korkeala.

In a study previously published by the authors in “Applied and Environmental Microbiology”, a role for the two-component system CBO0365/CBO0366 in cold tolerance of Clostridium botulinum was shown. The research presented in the poster was a continuation to this study. In this work, the genes under regulation of the aforementioned two-component system were identified by comparing the expression levels of all genes between the wild type, and a mutant strain in which the regulatory component of the system was inactivated, with DNA microarray experiments. Additionally, several genes and associated metabolic routes, found to be significantly differentially expressed between the mutant and wild type strains in the microarray experiments, were shown to play a role in cold tolerance of C. botulinum.  The results of the study will be published shortly in an international peer-reviewed journal.

The research was carried out at the Finnish Centre of Excellence in Microbial Food Safety Research and supported by the Academy of Finland, the Finnish Graduate School on Applied Bioscience, and the Walter Ehrström Foundation.

The food and environmental curriculum was rewarded for the Pedagogic Award of the Year 2013 by the veterinary student association EKY (Bachelor of Veterinary Medicine Association). The 6th year veterinary students (Cursus 64 Chimaera) gave the award in the 67th anniversary of the veterinary student association on the 23rd March, 2013.

Bachelor of Veterinary Medicine Association (EKY)

NOVA PhD Course “Molecular typing and next generation sequencing of food- and waterborne pathogens” is being held from March 4^th to March 8^th 2013 in Helsinki. The course is organized by the Department of Food Hygiene and Environmental Health, Faculty of Veterinary Medicine, University of Helsinki.

The course target group is PhD students registered at any of the NOVA or BOVA member institutions or for other Nordic PhD students. During the course the students get familiar with microbiological typing methods used for analysis of food- and waterborne pathogens and spoilage organisms. Also the potential of next generation sequencing (NGS) as a typing tool will be discussed.

This is the second NOVA PhD course organized by the Department of Food Hygiene and Environmental Heath. The first course, concerning quantitative PCR, took place in March 2012 and was in popular demand and highly valued among the students.

NOVA University Network – The Nordic Forestry, Veterinary and Agricultural University Network – is a platform for efficient and innovative Nordic cooperation.

Read more about NOVA.

NOVA members

Denmark
University of Copenhagen, Faculty of Health and Medical Sciences, HEALTH
University of Copenhagen, Faculty of Science, SCIENCE
University of Aarhus, Faculty of Science and Technology, AU-ST

Finland
University of Helsinki, Faculty of Agriculture and Forestry, HU-AF
University of Helsinki, Faculty of Veterinary medicine, HU-V
University of Eastern Finland, School of Forest Sciences, UEF-F

Iceland
Agricultural University of Iceland, LBHI

Norway
Norwegian University of Life Sciences, UMB
Norwegian School of Veterinary Science, NVH

Sweden
The Swedish University of Agricultural Sciences, SLU

A study by DVM Päivi Lahti and colleagues showed that chromosomal and plasmid-borne enterotoxin gene-carrying (cpe-positive) Clostridium perfringens strains have different epidemiology. The study conducted in Professor Hannu Korkeala’s research group was published in PLoS ONE journal.

PLoS One. 2012;7(10):e46162. doi: 10.1371/journal.pone.0046162. Epub 2012 Oct 19
Comparative Genomic Hybridization Analysis Shows Different Epidemiology of Chromosomal and Plasmid-Borne cpe-Carrying Clostridium perfringens Type A.

Lahti P, Lindström M, Somervuo P, Heikinheimo A, Korkeala H.

Department of Food Hygiene and Environmental Health, Faculty of Veterinary Medicine, University of Helsinki, Helsinki, Finland.

Until recently, only the chromosomal cpe-carrying strains were associated with food poisonings. However, Lahti et al. (2008) found also the plasmid-borne genotypes to be an important cause of food poisonings.

A comparative genomic hybridization (CGH) analysis with a DNA microarray based on three C. perfringens type A genomes was conducted to shed light on the epidemiology of C. perfringens food poisonings caused by plasmid-borne and chromosomal cpe-carrying strains by comparing chromosomal and plasmid-borne cpe-positive and cpe-negative C. perfringens isolates from human, animal, environmental, and food samples. The chromosomal and plasmid-borne cpe-positive C. perfringens genotypes formed two distinct clusters. Variable genes were involved with myo-inositol, ethanolamine and cellobiose metabolism, suggesting a new epidemiological model for C. perfringens food poisonings. The CGH results were complemented with growth studies, which demonstrated different myo-inositol, ethanolamine, and cellobiose metabolism between the chromosomal and plasmid-borne cpe-carrying strains. These findings support a ubiquitous occurrence of the plasmid-borne cpe-positive strains and their adaptation to the mammalian intestine, whereas the chromosomal cpe-positive strains appear to have a narrow niche in environments containing degrading plant material. Thus the epidemiology of the food poisonings caused by two populations appears different, the plasmid-borne cpe-positive strains probably contaminating foods via humans and the chromosomal strains being connected to plant material.

Additional information:
DVM Päivi Lahti
Department of Food Hygiene and Environmental Health
Faculty of Veterinary Medicine/University of Helsinki
paivi.lahti(at)helsinki.fi

In the industrialized world the bacterium Campylobacter jejuni is most commonly the cause of bacterial intestinal disease. In Finland, in 2011, 4262 cases were registered at THL, although the real number of cases is predicted to be much higher. Of the infections in Finland approximately 2/3 are imported from foreign countries.

The symptoms of the disease include diarrhea (sometimes bloody), fever, vomiting and headache. Infections may occur 3-7 days after ingestion of the bacterium. The majority of infections are not part of an outbreak. Therefore, it is often hard to find a source of an infection.

The bacterium is carried by a lot of animal sources, which usually do not develop clinical symptoms. Especially chickens carry a lot of C. jejuni, although compared to the rest of Europe; Finnish chickens carry very few campylobacters. In addition to chickens C. jejuni has also been found from cows and geese. Cats and dogs could also carry the bacterium.
Finally, C. jejuni can be found in the environment, especially in water.

In order to find a link between the sources of human infections and the human infections itself certain methodology called multilocus sequence typing (MLST) can be used. This method distinguishes the isolates on a genetic level and generates sequence data. These sequences are translated to a numerical output, a sequence type (ST). The sequence data can be analysed and modeled mathematically (for example by Bayesian modeling, such as BAPS) which groups the isolates, from which we can estimate the relative source attribution to human infections of each source.

Human and broiler strain genotypes frequencies evolved in different directions

De Haan and colleagues used the MLST methodology to group C. jejuni isolated from human infections, acquired in the Helsinki-Uusimaa region, and those of potential sources including chickens, cows, geese and natural water bodies. “We looked how the genetic pool of C. jejuni changed over the years and compare these changes in different hosts. We first looked at the human and chicken C. jejuni isolates and how their populations changed in a 10-year time period (1999-2006). We found many common C. jejuni types between the two hosts, but we also found opposite trends of some types between the two hosts. For example, in the human host ST-677 became less relevant for infection toward the end of the study period, whereas in chickens this type was found in increasing numbers each year.” De Haan tells about the study. “At the end of our study period, the total overlap of similar types between the human and chicken C. jejuni isolates had decreased 20%.

Source attribution of human infections

By making use of MLST and BAPS we determined that both poultry and water seemed to be the most likely sources of infection when the disease was acquired in Finland. However, especially in the C. jejuni isolates from water some strange types appeared as well, indicating that not all the C. jejuni present in water seems to be able to infect humans.”

The types from the bovine and the wild bird C. jejuni isolates were mostly found in a different group from the humans, chickens and waters. Part of the human C. jejuni isolates did cluster with the bovines, although this association was not as strong compared to those of the chickens and water.

Strains with a different genetic makeup can cause infections

De Haan looked at potential virulence properties (FspA, GGT, AnsBs and FucP) to see if these could be found in their C. jejuni strain collection. A statistically significantly association of FspA1 with patient isolates was found. Also, statistically significantly associations of FspA with certain sequence types was found and it is possible that FspA plays a role in the adaptation to and colonization of different hosts; this was especially prominent in the CC ST-48. The other examined properties showed a heterogeneous distribution among different sources and STs. This suggests that their role in adaptation, colonization and pathogenesis is not critical.

MSc Astrid de Haan academic dissertation was presented, with the permission of the Faculty of Veterinary Medicine, Univerity of Helsinki, for public examination in Helsinki on 28th of September 2012.

Title of thesis is “Molecular typing and source attribution of Finnish Campylobacter jejuni isolates” and it belongs to the field of environmental health.

The opponent was professor Dr. Birgitta Duim (Utrecht University, Netherlands) and the custos was professor Marja-Liisa Hänninen.

An electronic version of the dissertation.

A study by DVM Annukka Markkula and colleagues showed that DEAD-box RNA helicases have a marked role in growth of Listeria monocytogenes under heat, alkali, ethanol, and oxidative stress conditions. The study conducted in Professor Hannu Korkeala’s research group was published in valued Applied and Environmental Microbiology journal.

Appl Environ Microbiol 2012, 78:6875-6882.

Markkula A, Lindström M, Johansson P, Björkroth J and Korkeala H.

Department of Food Hygiene and Environmental Health, University of Helsinki

Roles of four putative DEAD-box RNA helicase genes in growth of Listeria monocytogenes EGD-e under heat, pH, osmotic, ethanol and oxidative stress conditions

In the study the growth of single DEAD-box RNA helicase gene deletion mutant strains were examined under heat, acid, alkali, osmotic, ethanol and oxidative stress conditions. The growth of one of the deletion mutant strains was restricted under 3.5% ethanol compared to that of the wild-type L. monocytogenes EGD-e strain. The maximum growth temperature of the mutant strain was increased by 0.6˚C. The growth of another mutated strain was impaired at 42.5˚C, and restricted under pH 9.4 and in 5 mM H₂O₂. Deletion of either of two remaining DEAD-box RNA helicase genes had no effect on the growth of L. monocytogenes under any growth condition examined.

A previous study by Annukka Markkula and colleagues has shown that DEAD-box RNA helicases have a marked role in growth of L. monocytogenes under cold stress. The importance of DEAD-box RNA helicases in cold stress tolerance in other organisms has also been reported. The role of these proteins in other stresses is almost unknown. The DEAD-box RNA helicases are found in most living organisms and are involved in various aspects of RNA metabolism from transcription to RNA decay. The functions of helicases under various stresses are unknown.

The research was carried out at the Finnish Centre of Excellence in Microbial Food Safety Research and supported by the Finnish Graduate School on Applied Bioscience and the Walter Ehrström Foundation.