A study of natural lytic Listeria phages with decontaminating properties for use in seafood processing plants : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology, Institute of Food, Nutrition and Human Health, Massey University, Auckland, New Zealand
therefore it is important to control this pathogen in seafood processing environments.
Sporadic listeriosis outbreaks and seafood recalls indicate that current treatments to
control this pathogen may be inadequate. The ability to adapt to harsh environmental
conditions, develop resistance and form biofilms makes this environmental pathogen
difficult to control using regular disinfectants. Bacteriophages (phages) could serve as
effective alternative biocontrol agents. The main objective of this study was to isolate
and characterize natural lytic Listeria specific phages and examine their effectiveness
against L. monocytogenes under conditions mimicking those found in seafood
Among a group of phages isolated from a seafood waste treatment unit, three
phages (LiMN4L, LiMN4p and LiMN17) were selected based on plaque morphology
and their source. The three phages were distinguished by morphology, efficiency of
plating (EOP) in citrate agar and differences in EOP using different L. monocytogenes
host strains. Three phages which were found as strictly virulent by whole genome
sequence analysis, had broad host ranges at 15 °C and each phage also infected either L.
ivanovii or L. innocua. These phages were unstable at 60 °C for 10 min suggesting
psychrotrophic properties. The three phages showed low burst sizes indicating their
potential suitability as passive biocontrol agents.
Low counts of L. monocytogenes strains (19O9, 19DO3 and 19EO3) in late
exponential phase, metabolically injured/stressed by heat and salt, lysed by the three
phages at 15 oC in 30 min. The results suggested that the three virulent phages may be
good candidates as biocontrol agents against L. monocytogenes under conditions
commonly found in seafood processing plants.
The phages LiMN4L, LiMN4p and LiMN17, used as single phage or a cocktail
of three phages, lysed cells adhered to stainless steel conditioned with soluble fish
protein and on clean stainless steel coupons (SSC). The phage cocktail also eradicated
low cell counts of about 2 log CFU/cm2 adhered to SSC surfaces in the presence of fish
proteins at 15 °C in 15 min and no re-growth of cells was observed from phage infected
surfaces. This study suggested that a biofilm matrix shielded the bacterial cells from
phage infection as three consecutive repeat applications of phages did not efficiently
lyse undisturbed biofilm cells. Biofilm cells, once removed from the surface, showed
similar to sensitivity to that of exponential phase planktonic cells. Therefore, disruption
of the biofilm structure may be required for effective phage treatments.
Phages suspended in phosphate buffered saline survived refrigeration for at least
twelve months and were stable for at least 6 h under likely application conditions such
as ambient temperatures and under fluorescence lighting. The three phages, either
individually or as a cocktail, showed a high lytic efficacy indicating their potential to
serve as bio-decontaminating agents in seafood processing environments.
Appendix 1 removed due to copyright restrictions: Ganegama Arachchi, G. J., Cruz, C. D., Dias-Wanigasekera, B. M., McIntyre, L., Billington, C., Hudson, A., Flint, S. H., Mutukumira, A. N. (2013), Host range and in vitro lysis of Listeria monocytogenes seafood isolates by bacteriophages, Food Science and Technology International, 0:1-13;
Ganegama Arachchi, G. J., Mutukumira, A. N., Dias-Wanigasekera, B. M., Cruz, C. D., McIntyre, L., Young, J., Flint, S. H., Hudson, A., Billington, C. (2013) Characteristics of three listeriaphages isolated from New Zealand seafood environments, Journal of Applied Microbiology, 115(6):1427-1438;
Ganegama Arachchi, G. J., Cridge, A. G., Dias-Wanigasekera, B. M., Cruz, C. D., McIntyre, L., Liu, R., J., Flint, S. H., Mutukumira, A. N., (2013) Effectiveness of phages in the decontamination of Listeria monocytogenes adhered to clean stainless steel, stainless steel coated with fish protein, and as a biofilm, 40:1105-1116