Deciphering The Molecular Basis of Environmental Persistence in Campylobacter Using a Systems Approach

Lead Research Organisation: University of Exeter
Department Name: Biosciences

Abstract

Campylobacter jejuni is a bacterium that causes food poisoning in people. They usually catch the disease after eating poultry that has not been cooked properly, in which some bacteria survive. In the UK there are around 50,000 reported cases of gastroenteritis caused by Campylobacter each year - but many more cases go unreported because most people recover naturally after a few days. Even so, disease caused by Campylobacter costs the UK economy an estimated £500million. Surprisingly, the bacteria thrive in the guts of poultry, without causing any disease. The control of disease caused by Campylobacter is likely to require several interlinked control measures, including reducing carriage in poultry flocks, cleaning and decontaminating carcasses as they pass through the food chain and encouraging the appropriate cooking of poultry. It has been shown that most of the poultry on sale in the UK is contaminated with Campylobacter. Therefore, in this proposal we focus on ways of controlling carcass contamination. We have already found that some Campylobacter cells are able to resist killing by antibacterial chemicals, limiting the effectiveness of decontamination methods. In this project we will investigate how Campylobacter cells resist killing, how we might use antibacterial chemicals in more effective ways and how we might change some farming practices to limit carcass contamination.

Technical Summary

Campylobacter jejuni (Cj) remains a leading cause of food borne infection in the UK with most cases arising from the ingestion of contaminated poultry. Cj is notoriously fastidious and survives poorly under laboratory conditions, yet it appears to be ubiquitous in the environment, forming reservoirs of infection. In addition, it is clear that the bacterium can tolerate abiotic stresses encountered during food processing. Recently, we have shown that this remarkable ability of Cj to survive unfavourable conditions is underlined by the formation of metabolically inactive cells (persister cells) that are analogous to the spores of Gram-positive pathogens. Once conditions improve, these persister cells switch back to a normal-growth state and re-establish a colony and potential subsequent infection. Thus, a full understanding and control of food borne infection mediated by Cj would require an understanding of the formation and regulation of the persister cell phenotype. We will undertake an integrated analysis of persister cell formation and its molecular basis in Cj by combining our expertise in experimental characterisation of pathogenic bacteria and in evolutionary theoretical analysis of bacterial behaviour at network level. This integrated, system-level approach will identify the molecular basis and evolution of persistence in Cj. Ultimately, this will enable us to design novel approaches towards reducing Cj levels in the food chain and that are based on knowledge of the behaviour of the persister population in this bacteria.

Planned Impact

Campylobacter is the most common cause of food poisoning in the UK. In 2008 it was responsible for over 300,000 reported cases (England and Wales), resulting in over 15,000 hospitalisations and 76 deaths. Clearly, this has serious health consequences and considerable economic cost - estimated as £583M in 2008. In the UK most cases of C. jejuni infection arise from the ingestion of contaminated poultry. We seek to understand how this bacterium is able to survive in the environment, and in particular during stresses encountered through food processing. This tolerance of abiotic stress is a key reason why food contamination occurs, and we have established that this may in large part be due to the formation of metabolically inactive cells (persister cells) that are analogous to the spores of Gram-positive pathogens. These persister cells may serve as an environmental reservoir and a source of infection, and we intend to provide key information regarding the formation and regulation of the persister cell phenotype, contributing significantly to a full understanding of C. jejuni infection which is likely to lead to effective control of this serious pathogen. Ultimately, this will enable us to design novel approaches towards reducing C. jejuni levels in the food chain - based on knowledge of the behaviour of the persister population in this bacteria. Consequently, we expect that the work will be of considerable interest to four key stakeholder groups, namely representatives from the farming community, meat processors, retailers and government regulatory bodies. We intend to primarily focus our impact activities on the key goal identified in this call, namely to engage and collaborate with industrial partners across the food supply chain, to meet the objective that the outputs of the research are translated to maximum benefit. We have prepared our Pathways to Impact activities so that key stakeholder groups are engaged throughout the project and we will ensure that any potntial improvements to the control of Campylobacter within the food supply chain are disseminated effectively and rapidly. We are confident that our science will lead to tangible and relevant advice which will support the reduction of the prevalence of C. jejuni in the food chain.

Publications

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Description A baseline for our work was to characterise a panel of C. jejuni strains from Asia, to complement our existing panel of N. American and European strains. We sequenced the genomes of 4 C. jejuni strains from Thailand, 4 from Pakistan and 4 from Vietnam. Eight of twelve (67%) Asian C. jejuni isolates possessed a cluster of genes homologous to the type 6 secretion system (T6SS). Our analysis of these genomes and previously reported genome sequences from USA and European strains allowed us to construct a phylogenetic tree based on multi-locus sequence analysis (MLSA) of core conserved genes. The presence of the T6SS was limited to certain MLSA clades.



Strains possessing a complete T6SS cluster could be distinguished by the presence of the hcp gene. Therefore, we used hcp as a proxy for the presence of a functional T6SS in a wider range of C. jejuni from chickens, humans and environmental sources. Most isolates from Europe and N. America lacked the hcp gene. Conversely, the hcp marker was frequently identified in isolates from Thailand, Pakistan and Vietnam. Among the Vietnamese patients in our study, those presenting with bloody diarrhea were more likely to be infected with hcp-positive C. jejuni (32%) than those presenting with non-bloody diarrhea (5%). This suggests a potential correlation between presence of T6SS and bloody diarrhea symptoms, which might be considered the more serious clinical manifestation of the infection given its associated higher rates of hospitalization and antibiotic treatment.



Poultry are a well-documented reservoir of human Campylobacter infection and we found that Campylobacter strains harboring the hcp marker were significantly associated with chickens in Asia. Large numbers of poultry are imported into North America and Europe from low-income countries, including Thailand. This may introduce T6SS-positive Campylobacter genotypes into the food chains of importing countries, posing a potential emerging threat to public health.



This work is currently in press; James W. Harrison, Tran Thi Ngoc Dung, Fariha Siddiqui, Sunee Korbrisate, Habib Bukhari, My Phan Vu Tra, Nguyen Van Minh Hoang, Juan Carrique-Mas, Juliet Bryant, James I. Campbell, David J. Studholme, Brendan W. Wren, Stephen Baker, Richard W. Titball, and Olivia L. Champion. Identification of Possible Virulence Marker from Campylobacter jejuni Isolates. Emerging Infectious Disease 20(6), June 2014 in press.



Subsequently we selected a panel of Asian, N. American and European strains for our analysis of responses to stresses. We have found no differences in the responses of strains to antibiotic (or acid) stress. Therefore we selected one strain (C. jejuni 1168) for subsequent studies on the formation of persister cells in response to antibiotic stress. Using different dosing regimens we have determined the survival patterns of C. jejuni 1168 and used this data to construct a mathematical model describing the survival pattern. This model has subsequently been used to investigate persister cell formation in other bacterial species.



We have devised methods for sorting persister cells based on their metabolic activity. This has allowed us to collect different populations of persister cells and determine their proteomic makeup.
Exploitation Route Our findings suggest that poultry imported into the UK from low-income countries, including Thailand may introduce T6SS-positive Campylobacter genotypes into the food chains, posing a potential emerging threat to public health. Further work is required to investigate this possibility
Sectors Agriculture, Food and Drink,Healthcare

 
Description Pakistan 
Organisation COMSATS Institute of Information Technology
Department Department of Bio Sciences
Country Pakistan 
Sector Academic/University 
PI Contribution Genome sequencing and genome analysis of Campylobacter jejuni isolates. Hosting of visiting PhD student, Fariha Siddiqui March-Sep 2012. Training on molecular methods such as PCR typing of strains. This has enabled Fariha to return to Pakistan with the methods and techniques which can now be applied more widely. It has also enabled her to submit a publication arising from her work; Fariha Siddiqui, Olivia Champion, Muhammad Akram, David Studholme, Brendan W. Wren, Richard Titball, Habib Bokhari. Molecular Detection Identified a Type Six Secretion System in Campylobacter jejuni from Various Sources but not from Human Cases. J. Appl. Microbiol. In press
Collaborator Contribution Provision of strains of Campylobacter jejuni from cases of disease in Vietnam. Placement of PhD student at Exeter
Impact Identification of possible virulence marker from Campylobacter jejuni isolates. Harrison JW, Dung TT, Siddiqui F, Korbrisate S, Bukhari H, Tra MP, Hoang NV, Carrique-Mas J, Bryant J, Campbell JI, Studholme DJ, Wren BW, Baker S, Titball RW, Champion OL.Emerg Infect Dis. 2014 Jun;20(6):1026-9. doi: 10.3201/eid2006.130635. Fariha Siddiqui, Olivia Champion, Muhammad Akram, David Studholme, Brendan W. Wren, Richard Titball, Habib Bokhari. Molecular Detection Identified a Type Six Secretion System in Campylobacter jejuni from Various Sources but not from Human Cases. J. Appl. Microbiol. In press Training of Fariha Siddiqie on molecular methods such as PCR typing of strains. This has enabled Fariha to return to Pakistan with the methods and techniques which can now be applied more widely to strain identification
Start Year 2012
 
Description Vietnam 
Organisation Wellcome Trust
Department Wellcome Trust Major Overseas Programme in Ho Chi Minh City Vietnam
Country Viet Nam 
Sector Charity/Non Profit 
PI Contribution Genome sequencing and genome analysis of Campylobacter jejuni isolates Dr Olivia Champion was invited to the Enteric Infections Unit at the Welcome Trust Oxford University Clinical Research Centre (OUCRU), Ho Chi Minh City, Vietnam from 14 September 2012 for two weeks. The aim of Olivia's visit was to teach researchers in the Enteric Infections Unit how to culture, passage and store Campylobacter jejuni as previous attempts in the Unit to work with this fastidious human pathogen had failed. Olivia successfully transferred microbiological skills to the researchers in the Unit, who are now carrying out research to understand the role of C. jejuni as an aetiologic agent of diarrheal disease in Vietnam
Collaborator Contribution Provision of strains of Campylobacter jejuni from cases of disease in Vietnam
Impact Harrison JW, Dung TT, Siddiqui F, Korbrisate S, Bukhari H, Tra MP, Hoang NV, Carrique-Mas J, Bryant J, Campbell JI, Studholme DJ, Wren BW, Baker S, Titball RW, Champion OL. Identification of possible virulence marker from Campylobacter jejuni isolates. Emerg Infect Dis. 2014 20(6):1026-9. doi: 10.3201/eid2006.130635.
Start Year 2012