DETECTION AND CHARACTERIZATION OF LISTERIA SPECIES OF DIFFERENT PATHOGENIC POTENTIAL

Abstract

Listeriosis has been recognized as an emerging food borne bacterial zoonoses and pesky public health hazard. It is a serious invasive disease characterized by neural, visceral, and reproductive clinical entities. The main objective of our investigations was to detect and characterize Listeria species of different pathogenic potential. Examination of listeric infection in clinical cases and food samples with regards to the seropositivity, presence of virulent genes in the Listeria isolates recovered from such cases and pathogenicity profiles of Listeria isolates on the basis of in vitro and in vivo tests, con-elation among these, was done. Atotal of 700 samples were collected from a total no. of 90 buffaloes of (Group A) and 300 samples were collected from (Group B). Overall occurrence of listeric infection was found to be 7.49 %, 11.66 % and 40% in different cases of buffaloes with mastitis, reproductive disorders and slaughtered (Group A) and 7%, 14.24% and 14.72% in bulk tank milk, Ice cream and cheese samples (Group B). In serum samples collected from buffaloes with mastitis and reproductive disorders occurrence of listeric infection was found to be 46% and 44% respectively. Listeria isolates were characterized by biochemical methods such as- Catalase test, Oxidase test, Methyl Red test, Voges-Proskauer test, Nitrate reduction test and Sugar fermentation tests. Differentiation of the pathogenic Listeria species (L. monocytogenes and L. ivanovii) from non-pathogenic listeriae was done by in vitro tests including Phosphatidylinositol-specific phospholipase C (PI-PLC) Assay, DL-Alanine (3-Napthylamide (DLABN) test and haemolysis on sheep blood agar. The pathogenicity of L. monocytogenes and L. ivanovii isolate(s) were tested by in vivo tests namely mice inoculation and chick embryo inoculation. All the Listeria isolates were screened for the presence or absence of IV virulence associated gene(s) by employing the PCR protocols separately standardized for the detection of gene responsible for production of a haemolysin called listeriolysin O (hlyA), phosphatidylinositol-specific phospholipase C(plcA), Act Aprotein (actA), a surface protein called p60 (iap) and positive regulatory factor - PrfA (prfA) in the standard pathogenic strains ofL. monocytogenes procured. Three virulent genes (prfA, picA, hlyA) characterize an array of pathogenic potential among different Listeria isolates. hlyA gene alone can be used as a marker for distinguishing L. monocytogenes and L. ivanovii. L. monocytogenes can be confirmed by the presence ofhlyA gene ofvarious gene combinations. It is present in all gene combinations in L monocytogenes and absent in L. ivanovii. L. ivanovii can be confirmed by the presence ofhaemolytic activity positive in (in vitro PI-PLC assay) and hlyA negative gene combination by PCR assay. Seven haemolytic and pathogenic isolates of L. monocytogenes showing the presence ofall the five virulence-associated genes and five isolates ofL. ivanovii showing the presence ofpicA, prfA and actA genes identical to that of standard L. ivanovii strain respectively were characterized on the basis of their biofilm forming ability. Two maximum biofilm forming Listeria monocytogenes isolates (Z.m.28) and (L.m.33) obtained from dairy food samples were selected and analyzed by SEM on different surfaces namely stainless steel, Polypropylene and PVC coupons. Maximum biofilm formation was observed in case ofPVC followed by PP and SS surfaces. These biofilm cells were much more resistant to sanitizers compared to their planktonic counterparts. Such biofilm cells which were not removed during normal cleaning procedure in a food processing unit could be a source of contamination for foods.