Evaluation of various cultural enrichment methods for the detection of selected food borne bacterial pathogens

Aim: The study was conducted to evaluate the performance of different enrichment broths such as Tryptic Soy Yeast extract Broth (TSBYE), Brain Heart Infusion broth (BHI), Nutrient Broth (NB), Luria Broth (LB) and Peptone water (PW) for the detection of Listeria monocytogenes, Yersinia enterocolitica, Staphylococcus aureus and Salmonella enterica Typhimurium. Materials and Methods: The bacterial strains were procured from Institute of Microbial Technology (IMTECH), 0 0 Chandigarh. Growth of these food borne pathogens at two different incubation temperatures (35 C and 37 C) and three different incubation periods (12h., 16h. and 18h.) were studied. Results: The result of the study showed that enrichment in Tryptic soy broth with yeast extract (TSBYE) and incubation at 0 37 C for 18h. is superior for the enrichment of all the organisms under study. Conclusion: TSBYE can be used very effectively as universal enrichment broth in comparison with all other enrichment broths studied for the detection of L. monocytogenes, Y. enterocolitica, S. aureus and S. enterica Typhimurium.


Introduction
responsible for 2,41,148 food borne illnesses worldwide.Contamination of food with staphylococci Surveillance of food borne diseases is of utmost can occur directly from infected food producing animal priority in the public health agenda worldwide.As per or at any stages of food production, processing, World Health Organization report [1] food borne transportation, storage or retailing [10].Salmonellosis diseases are responsible for high level of morbidity and is recognized as a global zoonosis and food borne mortality among the public.Listeria monocytogenes, disease posing public health risk.It is the most Yersinia enterocolitica, Staphylococcus aureus and widespread disease in both developed and developing Salmonella enterica Typhimurium are agents of major countries and contributes to high morbidity and concern because of their association with popular economic loss [11].Salmonella infections have been foods such as meat and meat products, dairy products, associated with the consumption of raw and under fruits and vegetables.cooked meat products [12].Listeria monocytogenes is an emerging food Reliable detection techniques are a prerequisite borne pathogen responsible for both sporadic and for the detection of these pathogenic bacteria in food epidemic cases of Listeriosis associated with a variety and food processing plants.Because the conventional of foods including meat products [2].Listeria culturing technique for detecting pathogens is time monocytogenes is responsible for the highest hospitaliconsuming, results are frequently not available until zation rates (91%) amongst all known food borne the food has been either released to the market or pathogens worldwide [3].Yersinia enterocolitica is an consumed, thus increasing the risk of transmission of important food and water borne pathogen causing a pathogens [13].variety of gastrointestinal problems such as diarrhoea, Though sensitivity of many modern detection abdominal pain and pseudo appendicitis [4,5].There is methods such as PCR have improved significantly, a strong evidence that foods of animal origin especially enrichment protocol is necessary to improve detection pork and dairy products are responsible for Y.
efficiency and to avoid false results because pathogens enterocolitica infections in humans [6].
are often present in very low numbers in food samples Staphylococcus aureus is one of the most rendering the recovery of target organisms difficult common agents of food poisoning outbreaks with [14].Development of multi pathogen detection in a enhanced pathogenicity due to the presence of single assay not only reduces the cost for testing but enterotoxins [7,8].According to CDC report [9], it is also provides data on the presence of different pathogens in a single experiment.Furthermore, multipathogen detection is a rational approach since many foods such as milk and milk products, meat and poultry, Values with different superscripts in the same column differ significantly, p<0.05.sms at different incubation periods viz.12h., 16h.and cfu/ml was achieved within 10 h. while in the present 0 study, an increase of 10 log cfu/ml was achieved in 18h.at 37 C.At 16 h.lowest count was observed for L.
TSBYE within 18 h.monocytogenes, S. aureus and S. enterica Typhimurium in PW and in LB for Y. enterocolitica.Highest bacterial Conclusion count was found in TSBYE followed by BHI and NB It is evident from this study that TSBYE promoted after 18h. of incubation.
the growth of four major food borne pathogens, L. The results showed that variation in bacterial monocytogenes, Y. enterocolitica, S. aureus and S. counts significantly increased from 12h. to 18h.After enterica Typhimurium, significantly.Based on the data 12 h.incubation, mean counts of L. monocytogenes and obtained in this study, TSBYE can be used very effecti-Y.enterocolitica in different enrichment broths did not vely as universal enrichment broth in comparison with show any significant difference.Whereas at 18h. of all other enrichment broths studied for the detection of incubation significant difference was found between these food-borne pathogens by all techniques including TSBYE and other enrichment broths except for Y.
cultural and molecular methods.enterocolitica.

Figure- 1 :
Figure-1: OD values of L. monocytogenes in different broths Figure-2: OD values of Y. enterocolitica in different broths

Table - 1
. Growth of selected bacterial food borne pathogens at various incubation temperatures after 18h incubation in various broths Typhimurium and incubated at 37 C for 24 h.and S. enterica Typhimurium (MTCC 98) were procured After incubation, pink coloured colonies were counted from Microbial type culture collection and Gene bank[16].Selected colonies were then subjected to a series of (MTCC), Institute of Microbial Technology (IMTECH), biochemical tests for confirmation.The entire protocol Chandigarh.Maintenance of pure cultures was carried was repeated for six times.out by regular sub culturing onto Nutrient Agar slants at At 37 C, no significant difference selected as the incubation temperature for further was found between all the counts in NB and BHI.All study.the organisms had maximum bacterial counts in Table-2 represents the growth of different organi- 0S. aureus and S. enterica Typhimurium.incubatedat37C for 48 h.After incubation, grey black to jet black colonies with light coloured margin surroundedMaterials and Methodsby an opaque zone were counted.From selected Bacterial strains: The reference strains of bacterial dilution, 0.1 ml of the inoculum was transferred to pathogens, L. monocytogenes (MTCC 1143), Y.Brilliant Green Sulpha agar plates for isolating S. 0 enterocolitica (MTCC 3234), S. aureus (MTCC 1144) enterica isolation: Five different enrichment broths including 0 dilution was transferred to Polymyxin-Acriflavinobserved for all organisms at 35 C in PW except for S. 0 Lithium Chloride-Ceftazidime-Aesculin-Mannitol aureus.At 37 C, lowest count was observed for L. 0 (PALCAM) agar plates and incubated at 37 C for 48 h.monocytogenes and Y. enterocolitica in PW and in LB After incubation, colonies with graygreen colour with a for S. aureus and S. enterica Typhimurium.sunken centre and halo were counted.Yersinia identi-A significant difference in mean count was found 0 L. monocytogenes.

Media Mean bacterial counts (Log10cfu/ml)± SE time Typhimurium
0Table-2.Growth of different organisms at different incubation periods at 37 C update as before