translated from EA italian journal march 2011

DETECTING SALMONELLA IN HYDRIC ENVIRONMENTS

ADVANTAGES OF REAL-TIME PCR

Detection of Salmonella in hydric environments is usually performed using traditional microbiological methods that are excessively long and complex. To optimize response times and limit operational steps, the Biology Department of Hera Laboratories has introduced use of the Polymerase Chain Reaction (PCR) technique. A study of this method has demonstrated the high degree of sensitivity and specificity of this technique.
The Biology Department of Hera (Holding Energia Risorse Ambiente) Laboratories performs microbiological testing of water quality during the various phases of the Integrated Water Service [service formed pursuant to the Italian «Galli Law» 36/1994 to implement an integrated reform of water services in Italy]. Of these various tests, testing for Salmonella is particularly important, as the presence of this bacteria in hydric environments
indicates fecal contamination. Salmonella (Enterobacteria family) is a genus that includes aerobic and facultative
anaerobic, gram-negative, C8-estarase positive, non-lactose fermenting microorganisms. This genus includes species that are pathogenic to humans, which may cause infections with serious symptoms, such as typhoid fever. These pathogens are spread to humans in water and contaminated food products. Quality characteristics for surface water used to produce drinking water are defined in table 1/A of Annex 2 of Legislative Degree 258/2000: «Supplementary and corrective provisions of Legislative Decree 11 May 1999, no. 152, regarding water safeguard from pollution, according to article 1, paragraph 4 of Law 24 April 1998, no. 128». The guidelines require the absence of Salmonella spp. in 5,000 ml for category A1 water, which has been physically treated and disinfected, as well as the absence of this bacteria in 1,000 ml for category A2 water, which has
been both physically and chemically treated and disinfected. The absence of Salmonella is also the limit value set for waste water by Ministerial Decree no. 185/2003, which sets out requirements for the reuse of waste water: «Regulation containing technical standards for the reuse of waste water to implement article
26, paragraph 2, of Legislative Decree 11 May 1999, no. 152». With regard to drinking water, detection of disease-causing enterobacteria, which includes Salmonella, is considered as supplementary in Legislative Decree no. 31/2001 Implementation of EC Directive 98/83/EC relative to the quality of water destined 
for human consumption». Salmonella must be consistently absent in 1,000 ml. For many years, the Biology Department of Hera Laboratories used traditional microbiological methods to detect Salmonella, including the ISS A 011A method - Istisan reports 07/5 for drinking water [1] and the Apat Irsa Cnr 7080 method for waste and surface water [2]. Both methods are extremely long and complex: they require a pre-enrichment step,
followed by a selective enrichment step, extraction and biochemical and/or serologic identification of suspect colonies. A positive Salmonella detection requires approximately 6 -7 days. The number of «man-hours» in the lab is significantly high, and because said analysis involves continuous hands-on activity, technicians are required to work on weekends and holidays. These constraints gave rise to the need to assess an alternative reliable, fast, sensitive and easy-to-use method that would limit involvement of human resources. Of the various alternate biomolecular methods based on the study of specific genetic sequences of the target pathogen, we selected the Polymerase Chain Reaction, download the pdf file to read the article...