Antibiotic Resistance and Genetic Diversity of Listeria spp. Not Showing 16S rDNA Sequence Similar to Known Listeria Strains Isolated from Pekin Ducks and Their Environmental Sample

Introduction: Listeria spp., particularly L. monocytogenes, are responsible for rare foodborne infections albeit very high mortality especially in immune-challenged individuals. The isolation of strains of Listerianot showing 16S rDNA sequence similarity to known strains highlights a possible new risk and potential for the emergence of new foodborne pathogens resulting from human activity. Resistance of foodborne pathogens to multiple antibiotics is of concern worldwide and makes it difficult to treat infected patients when needed, while molecular typing methods provide useful information for tracing the source of infection.

Purpose: The objectives of this study was to determine the antibiotic resistance, presence of plasmids and hemolysin gene and to characterize new Listeriastrains isolated from ducks and their environmental sample using RAPD, REP and ERIC to determine their genetic relatedness.

Methods: A total of 531 ducks and their environmental samples were examined between 2009 and 2010 of which 5 were positive for unknown Listeria spp. These five positive Listeria spp. not showing DNA homology with known species, confirmed by sequencing at Eurofins MWG, were examined for their resistance to 15 antimicrobial agents using the disc diffusion method. They were also examined for the presence of plasmids (using Promega wizard^® plus minipreps DNA purification system by following manufacturer's instructions). Detection of the hlyAgene, ERIC, REP and RAPD typing were done following procedures published in peer-revewed journals.

Results: 0.94% (5/531) of ducks and duck environmental samples examined in Penang, Malaysia between 2009 and 2010 were positive for a potential novel Listeria sp. They were isolated from fecal swabs (3 isolates), cloacal swab (1 isolate) and pond water (1 isolate). The five isolates were all resistant to cefotaxime, nalidixic acid and tetracycline but susceptible to ampicillin and nitrofurantoin. Four out of five of the isolates were also resistant to cephalothin, gentamicin and streptomycin. MAR index ranged from 0.2 to 0.8. All the isolates harbored plasmids and the hlyAvirulence-associated gene. Plasmid sizes ranged from 2.1-24 kb. Each genotyping method (RAPD, REP, and ERIC) grouped the isolates into four similar types. Typing results indicated greater genetic diversity among the 5 isolates.

Significance: Our results suggest that these Listeria isolates from ducks may be pathogenic due to the presence of the hlyA virulence gene and its multi-antibiotic resistance profile. It is possible that these represent a novel species. This report also draws attention to the emerging importance of this foodborne pathogen and to the need for monitoring changes in antibiotic resistance patterns so that efficient risk and control management strategies can be developed.