Biofilm formation of Vibrio parahaemolyticus : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Microbiology at Massey University, Campus Manawatū, New Zealand

Abstract

Vibrio parahaemolyticus in seafood can cause food poisoning. There is increasing concern with the increase in reports of illness globally believed to be due to climate change affecting sea temperatures. Biofilm formation of V. parahaemolyticus is an additional concern as biofilms are more resistant to cleaning and sanitation than planktonic cells. However, little is known about the biofilm formation of V. parahaemolyticus. Strain variation and the factors determining biofilm formation were investigated in this study with the aim to provide information that can be used to design more effective control strategies.

This study identified two robust biofilm forming strains (PFR30J09 and PFR34B02) from nine V. parahaemolyticus seafood isolates. Comparative genome analysis unveiled 136 unique accessory genes in robust biofilm formers. Protein-protein-interaction analysis showed interactions between UDP-glucose metabolism (Gene ontology (GO): 0006011), cellulose biosynthesis (GO: 0030244), rhamnose metabolism (GO: 0019299) and O antigen biosynthesis (GO: 0009243). Cellulose contributed to robust biofilm formation. Cellulose biosynthesis was identified as being acquired from within the order Vibrionales. The cellulose synthase operons consisting of genes bcsG, bcsE, bcsQ, bcsA, bcsB, bcsZ, bcsC were present in 15.94% (22/138) of V. parahaemolyticus.

Strong biofilm-forming V. parahaemolyticus showed greater resistance to sanitizers of biofilm cells than the weaker biofilm forming cells. The effective concentrations of sodium hypochlorite for inactivating most V. parahaemolyticus biofilm cells were higher than the recommended concentration. Available chlorine of 1176 mg/L inactivated 1.74-2.28 log10 CFU/cm2 of biofilm on stainless steel surfaces and 4704 mg/L inactivated > 7.00 log10 CFU/cm2 of biofilm (to undetectable levels, < 10 CFU/cm2), except for biofilms formed by the strong biofilm formers. Peracetic acid (PAA) at 200 ppm (89.56 mg/L PAA, 471.64 mg/L hydrogen peroxide) inactivated > 5.00 log10 CFU/cm2 of biofilm from stainless steel surfaces (except for those the strong biofilm formers, see Figure 4.4).

RNA sequencing (RNA-seq) identified 74 differentially expressed genes when comparing planktonic and biofilm cells of V. parahaemolyticus. These represented the rearrangement of nucleotide and energy metabolism in biofilm cells. Biosynthesis of secondary metabolites, purine and pyrimidine metabolism, propanoate metabolism, and valine, leucine and isoleucine degradation were deemed essential in the young V. parahaemolyticus biofilms. Genes of purH, purF, pdhA are potential genetic targets for biofilm prevention and control of V. parahaemolyticus.

Understanding V. parahaemolyticus biofilm formation will help to design strategies to overcome the limitations of chemical sanitizers, improving product safety and quality in the seafood industry.