Simultaneous Detection of Pseudomonas fragi , P. lundensis , and P. putida from Meat by Use of a Multiplex PCR Assay Targeting the carA Gene

Abstract

Species-specific primers and a multiplex PCR assay were developed for the simultaneous identification and differentiation of Pseudomonas fragi, P. lundensis, and P. putida based on the coamplification of different portions of the small subunit of the carbamoyl phosphate synthase gene (carA). The carA multiplex PCR was used to detect the presence of the three Pseudomonas species from beef, chicken, and pork samples and proved to be effective in showing their evolution during the storage of meat.Several species of the genus Pseudomonas are very often recognized as the principal causative agents of the spoilage of fresh foods stored aerobically. Members of the Pseudomonas fluorescens group, along with the psychrotrophic P. fragi, P. lundensis, and P. putida, are usually involved in spoilage of milk, meat, and fish, even during storage at low temperatures. These bacteria are often isolated from spoiled meat (10,12,18).The molecular detection and identification of microorganisms is widely used in food microbiology. However, only limited information is now available on the molecular detection of spoilage bacteria, and the development of appropriate strategies for their rapid identification and monitoring is needed. Some molecular approaches, such as ribotyping, PCR amplification of the 16S-23S rRNA gene spacer region, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, have been exploited for the analysis of the diversity of Pseudomonas isolates from foods (4,13,19,21).The molecular identification of Pseudomonas is often difficult and controversial. The sequence analysis of the 16S rRNA gene is widely employed for the identification of bacteria; however, this region is not satisfactorily discriminating between the species of Pseudomonas. Phylogenetic studies have highlighted that inferred phylogenies based on the 16S rRNA gene lack resolution at the intrageneric level because of its low rate of evolution (1,15,20). In recent studies on the spoilage-related microbiota of beef, we also realized that it was difficult to achieve an unequivocal identification of Pseudomonas at the species level, even though variable regions of the 16S rRNA gene were analyzed (7,17). Several authors have evaluated the use of alternative sequences for the identification and phylogenetic studies of Pseudomonas spp. For this purpose, the sequences of the carA, recA, gyrB, fliC, and rpoD genes of Pseudomonas species have been determined (2,11,20). The sequences of the carbamoyl phosphate synthase gene small subunit (carA) of several Pseudomonas spp. of environmental origin have been determined by Hilario et al. (11). However, the carA sequences for species of food interest, such as P. fragi and P. lundensis, were not considered.In this study, the carA gene sequence was used as a target in order to design species-specific primers to selectively and simultaneously detect P. fragi, P. lundensis, and P. putida from meat.carA gene sequencing and primer design. The Pseudomonas strains used in this study are listed in Table 1. They were cul...

Processing Environment and Ingredients Are Both Sources of Leuconostoc gelidum, Which Emerges as a Major Spoiler in Ready-To-Eat Meals

“…a typical contaminant of fresh produce and meat carcasses (27,54). Additionally, among the species of LAB, Pseudomonas was highly abundant in two spoiled end products (Fig.…”

Mesophilic and Psychrotrophic Bacteria from Meat and Their Spoilage Potential In Vitro and in Beef

“…For the numbering and isolation, a nonselective medium such as PCA was used, which is commonly used for the cultivation of mesophilic and psychrotrophic bacteria from foods (29). A considerable microbial diversity, including enterobacteria, pseudomonads, and carnobacteria, was found in both mesophilic and psychrotrophic groups of microorganisms, including taxa often recognized as spoilage agents (4,14,15,21,25,33,56,58). C. maltaromaticum and C. divergens, recognized as dominant spoilers of meat (35), were isolated at both 30 and 7°C.…”

“…and Enterobacteriaceae (21,24). Species of Pseudomonas are particularly involved in the spoilage of meat stored at chill temperatures (15,30,33). In particular, the microflora of vacuum packaged chillstored meat is characterized in most cases by psychrotrophic lactic acid bacteria (4,10,23,48,54,55).…”

Antibiotic Multiresistance Analysis of Mesophilic and Psychrotrophic Pseudomonas spp. Isolated from Goat and Lamb Slaughterhouse Surfaces throughout the Meat Production Process

“…The spread of antimicrobial resistance genes throughout the food chain increases the resistance gene pool from which both pathogens and commensals can pick up resistance determinants, including those that pose a potential threat to public health and ecological balance (21,58,59). The main microorganisms recovered from hides, carcasses, butchered meat, and meat processing plant surfaces (60,61) include a wide spectrum of Gram-negative bacteria, with P. fluorescens and the psychrotrophic P. fragi, P. lundensis, and P. putida being the most relevant spoilage agents of fresh meat stored aerobically (42,(62)(63)(64)(65). In the present study, MDR pseudomonads (100% of strains isolated in this study) were isolated from all slaughterhouse zones (77%), except the freezing tunnel, and from the end products (23%), represented mainly by P. lundensis (65%) and P. putida (17%), followed by P. fragi (8%), P. fluorescens (6%), and P. alkylphenolia (4%).…”