Contamination rate, diversity and biofilm formation of the coliforms isolated from raw milk tankers and dairy processing equipment

Coliforms are an important broad class of Enterobacteriaceae that make 10% of the intestinal microbial population and they are not basically pathogen. Microbial evaluations dealing with the detection and enumeration of coliforms as indicator microorganisms are extensively used in dairy industries to show process error, environmental health and secondary contamination of the products (Eden, 2014; Cowan et al., 2004). A biofilm is microbial cells that enclosed in an EPS (extracellular polymeric substance) matrix and attached to surfaces (Srey et al., 2013). The biofilm structure is such that it protects the bacteria against environmental stresses so that biofilm cells are more resistant to antimicrobial agents than planktonic (individualized) bacteria (Rendueles et al. 2013). As a result, biofilms have become a major problem in the milk industry, the processing of fish, poultry, meat and prepared foods (McDonogh 1994). The formation of biofilms, in addition to having health effects, by blocking pipes in heat exchangers and cooling towers in milk processing plants, significantly reduces the amount of heat transfer, and also decomposes and decays the UF membranes used in the production of cheese and reverse osmosis (Kumar et al. 1998). This study aimed to evaluate the contamination rate and biodiversity of coliform bacteria in raw milk tanks and dairy processing equipment as well as to assess the ability of the isolates to produce biofilm. A total of 80 samples, consisting of 30 samples of raw milk tanks, 30 samples of dairy processing, storage and packaging equipment (processing lines of pasteurized milk, sterilized milk, buttermilk, and yogurt) and 20 samples from different surfaces (such as production floor, equipment surface, conveyor belts) were sampled. For this purpose, after cleaning in place (CIP), the specimens were obtained by sterilized wire brush and mechanically removed from the surfaces and transferred to the sterile ringer serum (Percival et al. 2014). The samples were centrifuged for 4 minutes at 4000 g for 5 minutes and the precipitate was cultured on VRBA agar and incubated at 37°C for 24-48 h. The colonies were subjected to screening and confirmation tests (Razavilar 2002). To determine the amount of biofilm produced by the coliform isolates, the quantitative microplate technique was conducted (Srey et al., 2013). According to the results, 8.75% of the samples were contaminated with coliforms. Amongst Enterobacter and Klebsiella had the highest contamination rate of 31.25%; meanwhile Citrobacter showed the lowest contamination rate of 12.5%. The results of ELISA assay showed that among the 16 isolates, 87.5% had the biofilm-forming capability, however 2 (5.12%) Escherichia coli isolates were found biofilm-negative. According to the findings of the study, 87.5% of the isolates (except for the two E. coli isolates) were biofilm producers and produced different biofilm values. Meanwhile, the largest production of biofilms was measured by Citrobacter freundii. Biofilm producing isolates produced different biofilm values. So that E. coli, Enterobacter cloacae, and E. sakazakii produced a low amount of biofilm; meanwhile, medium amounts of biofilm were formed by Klebsiella pneumoniae and K. aerogenes. The ability to form biofilms was not only widely differentiated among different species, but could also be significantly different among isolates of a specific species. Presence of high contaminated specimens (43.75%) on the floor and surfaces of processing plants indicates the high potential of these places for contamination of milk products. The existence of cracks, the presence of residual organic matter –as the sources of the microbes- and the lack of insufficient washing and disinfection of the floor can be the main reasons for the presence of contamination in these areas. In addition to the floors, conveyor belt (37.5%) and raw milk tank drain valve (18.75%) had a high contamination rate. During washing and CIP of tank trucks, it is not possible to have full access to all surfaces of the tanker and consequently is not effectively disinfected; so as biofilm was clearly visible in some of the tankers. To eliminate such biofilms along with the CIP, mechanical methods should be used (Simões et al., 2006). The use of supplemental washing and disinfection techniques, staff training for personal hygiene, more supervision over floor cleaning, replacement of damaged materials, monitoring of CIP and the use of efficient methods for raw milk tankers can greatly reduce coliform contamination.