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Amoebae as Hosts and Vectors for Spread of Campylobacter jejuni
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Campylobacter jejuni is the leading bacterial cause of gastrointestinal diarrheal disease in humans worldwide. This zoonotic pathogen has a complex epidemiology due to its presence in many different host organisms. The overall aim of this thesis was to explore the role of amoebae of the genus Acanthamoeba as an intermediate host and vector for survival and dissemination of C. jejuni. Earlier studies have shown that C. jejuni can enter, survive and replicate within Acanthamoebae spp. In this thesis, I have shown that C. jejuni actively invades Acanthamoeba polyphaga. Once inside, C. jejuni could survive within the amoebae by avoiding localization to degradative lysosomes. We also found that A. polyphaga could protect C. jejuni in acid environments with pH levels far below the range in which the bacterium normally survives. Furthermore, low pH triggered C. jejuni motility and invasion of A. polyphaga. In an applied study I found that A. polyphaga also could increase the survival of C. jejuni in milk and juice both at room temperature and at +4ºC, but not during heating to recommended pasteurization temperatures. In the last study we found that forty environmental C. jejuni isolates with low bacterial concentrations could be successfully enriched using the Acanthamoeba-Campylobacter coculture (ACC) method. Molecular genetic analysis using multilocus sequence typing (MLST) and sequencing of the flaA gene, showed no genetic changes during coculture. The results of this thesis have increased our knowledge on the mechanisms behind C. jejuni invasion and intracellular survival in amoebae of the genus Acanthamoeba. By protecting C. jejuni from acid environments, Acanthamoebae could serve as important reservoirs for C. jejuni e.g. during acid sanitation of chicken stables and possibly as vectors during passage through the stomach of host animals. Furthermore, Acanthamoeba spp. could serve as a vehicle and reservoir introducing and protecting C. jejuni in beverages such as milk and juice. Validation of the ACC method suggests that it is robust and could be used even in outbreak investigations where genetic fingerprints are compared between isolates. In conclusion, Acanthamoeba spp. are good candidates for being natural hosts and vectors of C. jejuni.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2015. , 50 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1115
Keyword [en]
Amoebae-Bacteria interactions; Campylobacter jejuni; epidemiology; environmental stress; low pH; Acanthamoeba; coculture; intracellular trafficking; bacterial survival; beverages; ACC-method; enrichment; genetic stability; Trojan horse; reservoir; host; vector
National Category
Medical and Health Sciences
Research subject
Infectious Diseases
Identifiers
URN: urn:nbn:se:uu:diva-255804ISBN: 978-91-554-9276-2 (print)OAI: oai:DiVA.org:uu-255804DiVA: diva2:842408
Public defence
2015-09-10, A1:111a, Biomedicinskt Centrum, Husargatan 3, Ing C7:2, Uppsala, 09:00 (Swedish)
Opponent
Supervisors
Funder
Swedish Research Council Formas, 2220-8779-120, 221-2012-1442Medical Research Council of Southeast Sweden (FORSS)Swedish Research Council Formas, 2007-438Swedish Research Council for Environment, Agricultural Sciences and Spatial PlanningThe Crafoord Foundation
Available from: 2015-08-19 Created: 2015-06-18 Last updated: 2015-09-07
List of papers
1. Increase in acid tolerance of Campylobacter jejuni through coincubation with amoebae
Open this publication in new window or tab >>Increase in acid tolerance of Campylobacter jejuni through coincubation with amoebae
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2010 (English)In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 76, no 13, 4194-4200 p.Article in journal (Refereed) Published
Abstract [en]

Campylobacter jejuni is a recognized and common gastrointestinal pathogen in most parts of the world. Human infections are often food borne, and the bacterium is frequent among poultry and other food animals. However, much less is known about the epidemiology of C. jejuni in the environment and what mechanisms the bacterium depends on to tolerate low pH. The sensitive nature of C. jejuni stands in contrast to the fact that it is difficult to eradicate from poultry production, and even more contradictory is the fact that the bacterium is able to survive the acidic passage through the human stomach. Here we expand the knowledge on C. jejuni acid tolerance by looking at protozoa as a potential epidemiological pathway of infection. Our results showed that when C. jejuni cells were coincubated with Acanthamoeba polyphaga in acidified phosphate-buffered saline (PBS) or tap water, the bacteria could tolerate pHs far below those in their normal range, even surviving at pH 4 for 20 h and at pH 2 for 5 h. Interestingly, moderately acidic conditions (pH 4 and 5) were shown to trigger C. jejuni motility as well as to increase adhesion/internalization of bacteria into A. polyphaga. Taken together, the results suggest that protozoa may act as protective hosts against harsh conditions and might be a potential risk factor for C. jejuni infections. These findings may be important for our understanding of C. jejuni passage through the gastrointestinal tract and for hygiene practices used in poultry settings.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-134586 (URN)10.1128/AEM.01219-09 (DOI)000279082800010 ()20453130 (PubMedID)
Available from: 2010-11-29 Created: 2010-11-29 Last updated: 2017-12-12Bibliographically approved
2. The Abundant Free Living Amoeba, Acanthamoeba polyphaga, Increases the Survival of Campylobacter jejuni in Milk and Orange Juice
Open this publication in new window or tab >>The Abundant Free Living Amoeba, Acanthamoeba polyphaga, Increases the Survival of Campylobacter jejuni in Milk and Orange Juice
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Campylobacter jejuni is a common cause of human bacterial diarrhea in most parts of the world. Most C. jejuni infections are acquired from contaminated poultry, milk and water. Due to health care costs and human suffering, it is important to identify all possible sources of infection. Unpasteurized milk has been associated with several outbreaks of C. jejuni infection. Campylobacter has been identified on fresh fruit and other gastrointestinal pathogens such as Salmonella, E. coli O157:H7 and Cryptosporidium have been involved in fruit juice outbreaks. C. jejuni is sensitive to the acidic environment of fruit juice, but co-cultures with A. polyphaga have previously been shown to protect C. jejuni at low pH. In this study we found that co-culture with A. polyphaga  prolonged the C. jejuni survival both in milk and juice. The effect of co-culture was most pronounced in juice stored at room temperature. On the other hand, we found that A. polyphaga did not have any effect on C. jejuni survival during pasteurization of milk or orange juice, indicating that this is a good method for eliminating C. jejuni in these products. In conclusion, amoebae associated C. jejuni in milk and juice might cause C. jejuni infections.

 

 

 

Keyword
juice, unpasteurized milk, Campylobacter, Acanthamoeba
National Category
Medical and Health Sciences
Research subject
Infectious Diseases
Identifiers
urn:nbn:se:uu:diva-255770 (URN)
Funder
Swedish Research Council Formas, 2220-8779-120, 221-2012-1442
Available from: 2015-06-18 Created: 2015-06-18 Last updated: 2015-09-07
3. Campylobacter jejuni Actively Invades the Amoeba Acanthamoeba polyphaga and Survives within Non Digestive Vacuoles
Open this publication in new window or tab >>Campylobacter jejuni Actively Invades the Amoeba Acanthamoeba polyphaga and Survives within Non Digestive Vacuoles
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2013 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 11, e78873- p.Article in journal (Refereed) Published
Abstract [en]

The Gram-negative bacterium Campylobacter jejuni is able to enter, survive and multiply within the free living amoeba Acanthamoeba polyphaga, but the molecular mechanisms behind these events are still unclear. We have studied the uptake and intracellular trafficking of viable and heat killed bacterial cells of the C. jejuni strain 81-176 in A. polyphaga. We found that viable bacteria associated with a substantially higher proportion of Acanthamoeba trophozoites than heat killed bacteria. Furthermore, the kinetics of internalization, the total number of internalized bacteria as well as the intracellular localization of internalized C. jejuni were dramatically influenced by bacterial viability. Viable bacteria were internalized at a high rate already after 1 h of co-incubation and were observed in small vacuoles tightly surrounding the bacteria. In contrast, internalization of heat killed C. jejuni was low at early time points and did not peak until 96 h. These cells were gathered in large spacious vacuoles that were part of the degradative pathway as determined by the uptake of fluorescently labeled dextran. The amount of heat killed bacteria internalized by A. polyphaga did never reach the maximal amount of internalized viable bacteria. These results suggest that the uptake and intracellular survival of C. jejuni in A. polyphaga is bacterially induced.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-212316 (URN)10.1371/journal.pone.0078873 (DOI)000326656200061 ()
Available from: 2013-12-10 Created: 2013-12-09 Last updated: 2017-12-06Bibliographically approved
4. Multilocus Sequence Typing and FlaA Sequencing Reveal the Genetic Stability of Campylobacter jejuni Enrichment during Coculture with Acanthamoeba polyphaga
Open this publication in new window or tab >>Multilocus Sequence Typing and FlaA Sequencing Reveal the Genetic Stability of Campylobacter jejuni Enrichment during Coculture with Acanthamoeba polyphaga
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2013 (English)In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 79, no 7, 2477-2479 p.Article in journal (Refereed) Published
Abstract [en]

Low concentrations of C. jejuni cells in environmental samples make them difficult to study with conventional culture methods. Here we show that enrichment by amoebae co-cultures works well with low concentration samples, and that this method can be combined with molecular techniques without loss of genetic specificity.

National Category
Infectious Medicine
Identifiers
urn:nbn:se:uu:diva-194957 (URN)10.1128/AEM.02918-12 (DOI)000316183500046 ()23377942 (PubMedID)
Available from: 2013-02-20 Created: 2013-02-20 Last updated: 2017-12-06Bibliographically approved

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