The use of multiplex PCR reactions to characterize populations of lactic acid bacteria associated with meat spoilage

Denaturing gradient gel electrophoresis allowed us to monitor total bacterial communities and to establish a pattern of succession between species in vacuum-packaged beef stored at 2 and 8°C for 9 weeks and 14 days. Species-specific PCR was used to confirm the presence of Lactobacillus sakei and Lactobacillus curvatus. Multiplex PCRs using 16S rRNA-specific primers allowed differentiation between Leuconostoc species. These methods provided the desired information about microbial diversity by detecting the main microorganisms capable of colonizing this ecological niche.Vacuum packaging under chilled conditions has proved very effective in extending the shelf life of perishable foods, such as fresh meat and meat products, and preventing the growth of food-borne pathogens (8). The oxygen supply will be restricted, depending on the gas permeability of the packaging film, and thus has a selective effect on the microbial population (22). Lactic acid bacteria (LAB), such as Lactobacillus spp., Leuconostoc spp., Carnobacterium spp., and Brochothrix thermosphacta, are the main spoilage organisms associated with chilled vacuum-packaged fresh-meat products (6,18,21,26,33). Shortly after vacuum packaging of meat, LAB populations are usually below the routine detection limit (Ͻ10 CFU/g), but they increase during storage (19). Although LAB can cause meat spoilage, a selective growth promotion of LAB capitalizing on their ability to control meat-borne pathogens with a preferential growth of benign strains would minimize their spoilage effects (7,25,32,34).Methods in molecular microbiology, especially those including the sequencing of genes coding for 16S rRNA, have become a very important tool in the study of bacterial communities in meat samples. The trend is toward culture-independent methods, because they are believed to overcome problems associated with selective cultivation and isolation of bacteria from natural samples. Genetic-fingerprinting techniques provide a profile representing the genetic diversity of a microbial community from a specific environment. Denaturing gradient gel electrophoresis (DGGE) is usually employed to assess the structure and dynamics of microbial communities in food samples without cultivation in response to environmental variations (13,14,15,27). Species-specific PCR is a rapid and reliable molecular technique for the characterization of bacterial communities, and it can be also applied in situ without colony isolation (2). The variations in length and sequences of the 16S/23S rRNA intergenic spacer regions of the rRNA operon have proved useful for strain and species identification (2,3,17). In this work, we describe the application of cultureindependent methods to the study of the microbial succession dynamics in vacuum-packaged beef stored at 2 and 8°C for 9 weeks and 14 days, respectively.Bacterial control strains and growth conditions. Lactobacillus sakei CRL1463, Lactobacillus curvatus CRL 1465, and Leuconostoc gelidum CRL 1542 (CERELA culture collection) were used as reference strains. L...

supporting

confidence: 82%

Direct Molecular Approach to Monitoring Bacterial Colonization on Vacuum-Packaged Beef

Fontana

Cocconcelli

Vignolo

2006

“…Several molecular techniques have been applied for the identification of the main bacterial population isolated from meat products, such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins (64), hybridization with rRNA probes (49), restriction fragment length polymorphism analysis of the 16S rRNA gene (67), randomly amplified polymorphic DNA-PCR analysis (4,7), PCR-temperature gra-dient gel electrophoresis (15), and species-specific PCR (83). Cocolin et al (14) reported a PCR-denaturing gradient gel electrophoresis analysis of the 16S rRNA gene V1 region to monitor dynamic changes in the bacterial population during the fermentation of Italian sausages.…”

mentioning

confidence: 99%

Microbial Quality and Direct PCR Identification of Lactic Acid Bacteria and Nonpathogenic Staphylococci from Artisanal Low-Acid Sausages

Aymerich

Martín

Garriga

et al. 2003

225

Detection of six species of lactic acid bacteria and six species of gram-positive catalase-positive cocci from low-acid fermented sausages (fuets and chorizos) was assessed by species-specific PCR. Without enrichment, Lactobacillus sakei and Lactobacillus curvatus were detected in 11.8% of the samples, and Lactobacillus plantarum and Staphylococcus xylosus were detected in 17.6%. Enriched samples allowed the detection of L. sakei and S. xylosus in all of the samples (100%) and of Enterococcus faecium in 11.8% of the sausages. The percentages of L. curvatus, L. plantarum, Staphylococcus carnosus, and Staphylococcus epidermidis varied depending on the sausage type. L. curvatus was detected in 80% of fuets and in 57% of chorizos. L. plantarum was found in 50% of fuets and 100% of chorizos. S. epidermidis was detected in only 11.8% of fuets, and S. carnosus was detected in only 5.9% of chorizos. Lactococcus lactis, Staphylococcus warneri, and Staphylococcus simulans were not detected in any sausage type. From a microbiological point of view, 70.6% of the samples could be considered of high quality, as they had low counts of Enterobacteriaceae and did not contain any of the food-borne pathogens assayed.

“…Currently, the widely accepted strategy formulated to improve methods of Carnobacterium strain identification includes sequence analysis of the 16S rRNA gene and construction of genus-and/or species-specific oligonucleotide probes for use with a multiplex and multistep PCR (1,4,8,32,42). Each technique has several advantages and disadvantages.…”

Section: Discussionmentioning

confidence: 99%

Differentiation of Closely Related Carnobacterium Food Isolates Based on 16S-23S Ribosomal DNA Intergenic Spacer Region Polymorphism

Hristova

Dousset

Cam

et al. 2002

A novel strategy for identification of Carnobacterium food isolates based on restriction fragment length polymorphism (RFLP) of PCR-amplified 16S-23S ribosomal intergenic spacer regions (ISRs) was developed. PCR amplification from all Carnobacterium strains studied always yielded three ISR amplicons, which were designated the small ISR (S-ISR), the medium ISR (M-ISR), and the large ISR (L-ISR). The lengths of theseLactic acid bacteria (LAB) have been the focus of extensive research due to their value in the food-processing industry (14,27). One of the most recent taxonomic additions to the LAB group is the genus Carnobacterium. Members of this genus are widespread in nature, and the habitats of these organisms range from foods, such as poultry, meat, cheese, and seafood, to fish intestines and anoxic Antarctic lake water (2,10,16,26,29). The four species of Carnobacterium isolated from food are Carnobacterium divergens, C. piscicola, C. gallinarum, and C. mobile (6). Most research on the genus Carnobacterium has been focused on the production of bacteriocins and the regulation of metabolic enzymes (9, 28). In the last few years, many ways to differentiate Carnobacterium species have been developed. The phylogenetic interrelationships of species in the genus Carnobacterium have been investigated by using numerical taxonomic matrices and sequencing of the 16S rrn segments encoding mature rRNAs (19,41). The Carnobacterium species form a phylogenetically coherent group, which is quite distinct from all other LAB. However, the number of polymorphic sites in the 16S rRNA of Carnobacterium species is rather low, since some species have the same sequence (the sequences of C. piscicola and C. gallinarum are 98% identical) and other species exhibit very high degrees of sequence similarity (the sequences of C. divergens and C. mobile are 96% similar) (41). Thus, it is difficult to define specific 16S RNA rrn sequences that can be used for differentiation of these closely related species. Carnobacteria have been identified at the genus level by nucleic acid hybridization by using 16S rRNA-targeted genus-specific probes (32). Species-specific primers have been designed by using the domains that exhibit low homology in the 16S ribosomal DNA (rDNA) sequences of species of Carnobacterium (1, 4). However, the PCR primers used in these studies were not specific enough to differentiate Carnobacterium spp. from other genera of LAB. Data obtained with the randomly amplified polymorphic DNA PCR technique enable only C. divergens to be differentiated from other Carnobacterium species (20).In view of this, a study of the more variable sequences in the rRNA operon of phylogenetically closely related Carnobacterium species could be useful. In most prokaryotes, the ribosomal genes form an operon with the order 5Ј-16S-23S-5S-3Ј, and the genes are separated by two intergenic spacer regions (ISRs). ISRs, especially those located between the 16S and 23S rDNAs, are thought to be under less evolutionary pressure and, therefore, to provide great...