ORIP Presentation 2012

The role of formate dehydrogenase in the survival of Campylobacter

jejuni in natural water

Kimberly Carter

Mentors: Dr. Gireesh Rajashekara and Dr. Issmat Kassem

Food Animal Health Research Program

Introduction: Campylobacter jejuniMicroaerophilic bacterium and

gastrointestinal pathogenCampylobacteriosis commonly

contracted through undercooked poultry meat, water, and milk

Persists in waterEnters viable but non-culturable state

(VBNC)Forms biofilmsExtensive and complex electron

transport chain Formate dehydrogenase (FdhA)

Does fdhA contribute to C. jejuni persistence in water?

Objectives and HypothesisLittle research has been done to characterize C. jejuni’s survival

mechanisms in water

Our goal: characterize the role of fdhA in Campylobacter jejuni for

persistence in water

Research could lead to techniques to prevent or control infections

contracted through water

Hypothesis: An fdhA deletion mutant strain (ΔfdhA) will exhibit a lower rate of survival compared to the wild type (WT) strain.

Materials and MethodsWater Microcosms

Pond waterOptical Density (OD₆₀₀) 0.500Room temperature Inoculated on MH plates at 0,

4, and 8 hours Incubated plates for 48 hours

in microaerobic conditions

Biofilm AssayGlass tubes filled with water from the microcosms were stained with

1% crystal violet. The stain was dissolved with 80% dimethyl sulfoxide, and the optical density was measured.

Statistical AnalysesStudent t-test: P < 0.05 considered statistically significant

Measurement of formate dehydrogenase activity

After adding formate to the cultures, the optical density of the wild-type increased, but that of the ∆fdhA mutant did not, indicating a loss of formate dehydrogenase activity in the mutant.

WT

∆fdhA

Summary of ∆fdhA phenotypes

The ∆fdhA mutant showed defects in motility, adaption to oxidative stress, biofilm formation, colonization in chicken intestinal cells (PIC), and human intestinal cells (INT-407). It also exhibited an abnormal cell shape.

- Adh: adherence-Inv: invasion-Intra: intracellular survival

Water Microcosm Results

The ∆fdhA mutant showed a decreased ability to survive in water at room temperature compared to the wild-type.

**

Num

ber

of C

. jej

uni C

FU

(lo

g₁₀)

Biofilm Assay Results

There was no significant difference between the ∆fdhA mutant and the wild-type in biofilm formation.

Opt

ical

Den

sity

(O

D55

0)

Conclusions and Future Directions

∆fdhA showed decreased ability to survive in waterHowever, not due to deficient biofilm formation

VBNC? Chemical in water?

Would distilled water or another source of water change the results?

How would adding formate affect the rate of survival?

ReferencesBaffone, W., Casaroli, A., Citterio, B., Pierfelici, L., Campana, R., Vittoria, E., Guaglianone, E. and G. Donelli. 2005. Campylobacter jejuni loss of culturability in aqueous microcosms and ability to resuscitate in a mouse model. IJ Food Micro 107:83-91.Hitchcock, A., Hall, S., Myers, J., Mulholland, F., Jones, M. and D. Kelly. 2010. Roles of the twin-arginine

translocase and associated chaperones in the biogenesis of electron transport chains of the human pathogen Campylobacter jejuni. Microbiology 156:2994-3010.Jackson, N., Davis, B., Tirado, S., Duggal, M., van Frankenhuyzen, J., Deaville, D., Wijesinghe, M., Tessaro, M. and J. Trevors. 2009. Survival mechanisms and culturability of Campylobacter jejuni under stress conditions. Antonie van Leeuwenkoek. 96:377-394.Kassem, I., Zhang, Q., and G. Rajashekara. 2011. The twin-arginine translocation system: contributions to the pathobiology of Campylobacter jejuni. Future Microbiol. 6(11):1-13.Liu, X., Gao, B., Novik, V. and Galán, J. 2012. Quantitative proteomics of intracellular Campylobacter jejuni reveals metabolic reprogramming. PLoS Pathog 8(3):1-12. Rajashekara, G., Drozd, M., Gangaiah, D., Jeon, B., Liu, Z. and Q. Zhang. 2009. Functional characterization of the twin-arginine translocation system in Campylobacter jejuni. Foodborne Path Disease 6(8):935-945.Weerakoon, D., Borden, N., Goodson, C., Grimes, J. and J. Olson. 2009. The role of respiratory donor enzymes in Campylobacter jejuni host colonization and physiology. Micro Pathogenesis 47(1):8-15.Wingender, J. and H. Flemming. 2011. Biofilms in drinking water and their role as reservoir for pathogens.

IJ Hygiene Environ Health 214:417-423.

AcknowledgementsDr. Grewal and all who make ORIP possibleDr. Gireesh RajashekaraDr. Issmat KassemAll personnel in Dr. Rajashekara’s labsAll personnel in the Food Animal Health Research

Program