PCR multiplex for detection of Salmonella Enteritidis, Typhi and Typhimurium and occurrence in poultry meat

Abstract

The occurrence of foodborne diseases is increasing throughout the world. Bacteria of the genus Salmonella are responsible for food poisoning and, in some cases, may be fatal. The aim of this study was to adapt the multiplex PCR technique (mPCR) on the rapid and direct identification of the presence of Salmonella sp. as well as serotypes Enteritidis, Typhi and Typhimurium in poultry carcasses (n = 127) and viscera (n = 73). The implementation of the standard technique using positive controls was successfully adapted. The results of Salmonella sp. detection in refrigerated viscera showed that the mPCR was able to detect Salmonella genus in 2.74% of these samples. Traditional microbiological analysis also identified the same positive samples for Salmonella sp. but was not able to differentiate the serotype. The serotype Enteritidis was detected by mPCR in 1.37% of the samples. Our conclusion was that the mPCR was able to detect the presence of these bacteria in a short period of time and enabled the identification of serotype Enteritidis in one of the samples found positive for Salmonella sp.

Introduction

Salmonellosis is one of the major foodborne diseases. Due to its endemic nature, high morbidity and association with a wide range of foods, this zoonotic disease is of high public health concern (Aarestrup et al., 2007, Alizadeh et al., 2007, Kottwitz et al., 2008). Outbreaks of salmonellosis are rarely reported in Brazil (Kaku et al., 2000). The prevalence of these bacteria in different types of food is thus underestimated (Kottwitz et al., 2008). Salmonella serotype Typhi is human specific (Arya & Agarwal, 2008, Ochiai et al., 2008) and when found in food indicates handling and hygiene failures (Chen et al., 1997, Che et al., 2000). Furthermore, the serotypes Enteritidis and Typhimurium can be isolated both in chickens before slaughter and in humans infected by foodborne diseases (Rabsch et al., 2001). Non-typhoidal salmonellosis is a major cause of human gastroenteritis worldwide (Kornschober et al., 2009). Kaku et al. (2000) reported 211 infected people during an outbreak of Salmonella Enteritidis in a São Paulo city school caused by food containing raw eggs.

It is considered that the presence of Salmonella sp. in chickens makes it unsafe for human consumption (Bjerrum et al., 2005, Agunos et al., 2007). The technological procedures for obtaining chicken carcasses and cuts for consumption are also potential risks for contamination (Deseo & Engeli, 1979, Christensen et al., 1997), especially in evisceration, cooling, packaging and transport stages (Rasschaert et al., 2008, Muth et al., 2009) where microbial growth can occur.

According to the Brazilian Association of Chicken Producers and Exporters (ABEF, 2008), Brazil is the third largest producer of chicken meat, (behind the United States and China) and is the main exporter, followed by the United States and the European Union.

Salmonella sp. microbiological research in poultry meat involves stages of pre-enrichment, enrichment and selective plating as well as biochemical and serological tests (Foley et al., 2007). Negative results are obtained in about 4 days whilst positive confirmations normally take up to 15 days (Blixt et al., 2007, Gallegos-Robles et al., 2009). The conventional method for microbiological diagnosis in the case of this pathogen is also checked by the fact that isolation requires a sufficient number of viable cells (Chaicumpa et al., 1995). Moreover, morphological changes and the biochemical profile of the colonies both increase the uncertainty of this diagnosis (Fach et al., 1999). The multiplex PCR (mPCR) is a variation of reaction chain polymerase (Kumar et al., 2006, Kumar et al., 2008), which involves more than one pair of primers allowing one single reaction to detect more than one type of microorganism along with their serotypes and/or different genes, therefore reducing the diagnosis period (Malkawi and Gharaibeh, 2003).

Considering the importance of this group of bacteria in the processing of chicken, its importance to public health and considering the need of fast and accurate diagnosis methods, we worked towards adapting a multiplex PCR to detect genus Salmonella and serotypes Enteritidis, Typhi and Typhimurium in refrigerated carcasses and chicken viscera. In addition, we were able to estimate the occurrence and frequency of these microorganisms in chicken in Federal District, Brazil.