Isolation of Psychrotrophic multiple drug resistant Pseudomonas from Pasteurised milk

The present study was taken up with the objective of assessing the microbiological quality of milk processed in an organized dairy plant. The standard plate count and coliform count of all the samples tested were within the limits specified by Prevention of Food Adulteration standards. However microbiological analysis of pasteurized milk revealed a unique bacterial profile characterized by the predominance of Pseudomonas aeruginosa. Assessment of potential of the isolate to multiply at 7oC confirmed its psychrotrophic nature. Antibiogram of the isolate revealed that, of the seven antibiotics tested, the isolate was sensitive only to enrofloxacin. Results are suggestive of the trait of multiple drug resistance in the isolate. Such multiple drug resistant (MDR) bacteria in processed foods are potent biological hazards as there are possibilities for resistance genes to be spread to human beings via food. Isolation of such Multiple drug resistant (MDR) bacteria in pasteurised milk, an essential component of human diet is highly significant from the public health point of view. Presence of Pseudomonas species in processed products implies that post processing contamination can hinder even the quality of properly processed products. The result signifies the importance of appropriate handling practices of processed food to assure safe products at consumer level.


Introduction
antimicrobial agents of human and veterinary importance (Alatossava and Alatossava, 2007).Any further spread Psychrotrophic bacteria have been recognized as of resistance among bacteria in food is likely to have a recurring problem in refrigerated storage and an influence on public health.The European food distribution of perishable food products.Pseudomonas safety authority (2008) has identified food as a source species have been implicated in the spoilage of for the acquisition of Antimicrobial resistant (AMR) processed milk kept under chilled condition because bacteria or bacteria borne antimicrobial resistance by of their capacity to multiply under refrigerated condition humans.AMR bacteria are biological hazards with the production of thermostable proteases and associated with increased human morbidity and lipases (Rajmohan et al, 2002).The Pseudomonas genus mortality.MDR bacteria in processed foods are potent corresponds to a diverse and ecologically significant biological hazards as there are possibilities for group of bacteria that are found in natural environresistance genes to be spread to human beings via food.ments.Such a universal distribution results from the In the context of increasing prevalence of ABR bacteria, capacity of Pseudomonas species to adapt to various the antibiotic sensitivity of a psychrotrophic isolate conditions and degrade wide range of substrates obtained from pasteurized milk was investigated.(Palleroni, 1992).Although Pseudomonas species are eliminated by pasteurization, milk is often spoiled by

Materials and Methods
these Gram negative rods that gain access as post pasteurization contaminants.
Representative samples of pasteurized milk There is a general consensus that the wide spread were aseptically collected from Kerala Agricultural use of antimicrobial agents has imposed a strong University Dairy Plant twice weekly for ten weeks selective pressure that has contributed multiple drug spread over a period of two months.A total of twenty resistant (MDR) microorganisms.Foodborne bacteria milk samples were analyzed for total viable cells including known pathogens and commensal bacteria coliforms and psychrotrophs as per the standard display an extensive and diverse range of resistance to methods, Harrigan,W.F.(1998).
The psychrotrophic isolates were subjected to the dairy processing unit would have singled out biochemical characterization after activating the psychrotrophs as the major category of spoilage culture in nutrient broth at 37°C for 24 h (Barrow and organism (Alatossava andAlatossava, 2007). Feltham, 1993).To compare the growth kinetics of the The predominanace of Pseudomonas species in isolate at 7°C and 37°C, Calculated quantity of refrigerated pasteurized milk is in accordance with the standardized inoculum of the isolate was added to two findings of Ternstrom et al. (1993) .Stepaniak (1991) conical flasks with100ml sterilized skim milk so as identified Pseudomonas species as the single most have < 10 cells/ml.One flask was kept at 37°C and important detrimental factor determining keeping other at 7°C.The total viable count of the samples were quality of pasteurised milk.Incidence of P.aeruginosa taken at 0,1,3,5,7 and 9 days.
in pasteurized milk has been reported by Kumaresan The psychrotrophic isolate was tested for their and Annal Villi (2008).According to them, post sensitivity to selected antibiotics by disc diffusion processing contamination led to the predominance of method (Bauer et al, 1966).The commercial antibiotic heat sensitive Pseudomonas in pasteurized milk.
Significant contamination by Pseudomonas can occur discs (Ampicillin-25µg,Cefotaxime-10µg,Chloramdue to inadequately sanitized surface of milking phenicol-30µg,Enrofloxacin-5µg Gentamycin-30µg, storage and transport equipments (Mac Phee and oxytetracyclin-30µg, Sulphdiazine-300µg) were Griffith, 2002).Anderson et al. (1990) found that placed on Mueller Hinton Agar plates previously P.aeruginosa could survive within exopolysaccharide seeded with the standardized broth culture of the test masses in the interior walls of piping.Preventing the organism.Plates were incubated at 37°Cfor 48h, after contamination of pasteurized dairy products with which zone of inhibition was measured and interpreted psychrotrophic bacteria is primarily a matter of as per the instruction of manufacturers.samples.The high spoilage potential of Pseudomonas should not exceed 30,000 cfu/ml.The samples analyzed species is not only because of its ability to multiply at in this trial showed an average Total viable count of 2.4 4 refrigeration temperatures but also because of their × 10 cfu /ml.According to PFA, coliforms which are ability to produce thermostable proteases, lipases and considered as index of hygiene should be absent in lecithinases (Sorhaug and Stepaniak, 1997).0.1ml of pasteurized milk.Total viable counts and o The multiplication rate of the isolate at 7 C was coliform counts of all the samples tested in this trial o .
comparable to that at 37 C The viable count after five were within the permitted limits.Results obtained in days of inoculation was almost same in both flasks.this trial are actually suggestive of adequate (Fig. -1)Tolerance to a wide range of temperature is processing.However it was observed that all the suggestive of its potential to survive in diverse range standard plate count plates were characterized by the of habitats.Such organism are of greater importance in predominance of typical small circular entire smooth the spoilage of chilled foods.(Adam and Moss, 2000).and shining translucent colonies.Furthermore, only This result is also supportive of the earlier observation this type of colony was evident in the plates used for of predominance of similar colonies in both TVC and taking psychrotrophic count.Morphological and psychrotrophic plates.biochemical similarity of colonies suggested that all An isolate may be considered as multiple drug the isolates obtained while taking psychrotrophic resistant if they exhibit resistance to more than two count exclusively belonged to the genus Pseudomonas.
classes of antibiotic.As the isolate used in this work Identity of the isolate was confirmed as Pseudomonas was found to be resistant to representatives of more aeruginosa by 16SrRNA sequencing.These observations than five classes of antibiotic, it may be considered as are suggestive of the predominance of mesophilic multiple drug resistant (Fig 2).The trait of multiple psychrotrophs in the samples of processed milk.As drug resistance for psychrotrophs have been previously refrigeration is an integral part of food distribution reported by Alattossava and Alattossava (2007) They system, psychrotrophs are gaining more significance observed that, raw milk psychrotrophic isolates showing in dairy industry.Practices that are being followed in resistance to aminoglycosides and quinolone classes Dean, College of Dairy Science and Technology for were less frequent.However the psychrotrophic providing the facilities.isolate used in this work was found to be resistant to Figure-1.Growth Kinetics of the isolate at 7 C and 37 C Figure-2.Antibiotic sensitivity pattern Identity of the equipment cleaning and sanitation (Beena and Prasad, isolate was further confirmed by 16SrRNA sequencing 2004).Eneroth et al. (1998) reported filling step as the by sending the isolate to Central Institute of Fisheries most critical contamination site for Gram negative and Technology, Cochin.rods, especially Pseudomonas.Same reasons would have