Occurrence and Characteristics of Listeria spp. in Fish from East Coast Malaysia and Pathogenicity Assessment of Bile-stressed Listeria monocytogenes Serotype 1/2b and Atypical Haemolytic Listeria innocua

An intracellular foodborne pathogen, L. monocytogenes is a ubiquitous Gram-positive bacterium. It has been a limelight in the food industry as it can withstand diverse environmental conditions and has become a major threat to public health due to the emergence of antibiotic-resistant isolates. Even though L. monocytogenes is recognised as the causative agent of human listeriosis, few cases of L. innocua infection have been reported. The success of listeriosis depends on bacterial survival along the gastrointestinal tract where they need to encounter low pH of the stomach and high bile concentration in the small intestine. Bile tolerance ability has been studied extensively in L. monocytogenes, but little is known about atypical haemolytic L. innocua. In this study, 125 freshwater fish, 164 marine fish, and 41 samples of fish contact surfaces were screened for Listeria spp. Specific Listeria spp. and their characteristics have been identified using multiplex PCR. 12 (3.64 %) out of 330 samples harboured Listeria spp. including L. monocytogenes (41.67 %), L. innocua (41.67 %), and unidentified Listeria spp. (16.67 %). Four (80.0%) and one (20.0%) isolates of L. monocytogenes belonged to serotypes 1/2b and 4b, respectively. Interestingly, not only in pathogenic L. monocytogenes, but all Listeria spp. isolates also possessed all virulence genes tested; inlA, inlB, inlC, inlJ, hlyA, actA, plcA and plcB. They also exhibited haemolytic activity on sheep blood agar. Two of the isolates displayed resistance to tetracycline. Screening of tet genes identified tetM in the plasmid of both tetracycline-resistant isolates. The intraspecies acquisition of tetM gene was confirmed using DNA sequencing. In this study, bile-stressed L. monocytogenes serotype 1/2b and atypical haemolytic L. innocua strains were compared for their survival, morphological changes, susceptibility to antibiotics, virulence profiles, as well as their pathogenic potential using 9-day-old chicken embryos. Our findings indicate that exposure to 1 % bile for 6 hours impaired the growth of both strains and significantly altered their cell length, width, and shape. Additionally, bile increased the antimicrobial sensitivity of L. monocytogenes strain against 10 tested antibiotics. The chicken embryo model showed 100 % mortality due to L. monocytogenes and bile-stressed L. monocytogenes within 36 hours and 48 hours, respectively. In comparison, L. innocua and bile-stressed L. innocua resulted in only 20 % embryo mortality in 60 hours and 36 hours, respectively. The virulence of Listeria strain in the presence of bile correlated with the expression of certain virulence genes; inlA, actA and stress response genes; sigB, murA. L. innocua showed an increase in virulence under bile stress and it even can resist antibiotics better than pathogenic L. monocytogenes. The findings of this study collectively suggest that both L. monocytogenes and L. innocua strains isolated from raw fish and water samples have the potential to cause foodborne zoonotic listeriosis, and the presence of multidrug-resistant isolates is alarming. It also provides information about the contamination status of this important source of protein diet in East Coast, Malaysia which may be useful for control measure implementation.