A quick and simple method for quantitative detection of Lactobacillus sakei in fermented sausages... more A quick and simple method for quantitative detection of Lactobacillus sakei in fermented sausages was successfully developed. It is based on Chelex-100-based DNA purification and real-time PCR enumeration using a TaqMan fluorescence probe. Primers and probes were designed in the L. sakei 16S-23S rRNA intergenic transcribed spacer region, and the assay was evaluated using L. sakei genomic DNA and an artificially inoculated sausage model. The detection limit of this technique was approximately 3 cells per reaction mixture using both purified DNA and the inoculated sausage model. The quantification limit was established at 30 cells per reaction mixture in both models. The assay was then applied to enumerate L. sakei in real samples, and the results were compared to the MRS agar count method followed by confirmation of the percentage of L. sakei colonies. The results obtained by real-time PCR were not statistically significantly different than those obtained by plate count on MRS agar (P > 0.05), showing a satisfactory agreement between both methods. Therefore, the real-time PCR assay developed can be considered a promising rapid alternative method for the quantification of L. sakei and evaluation of the implantation of starter strains of L. sakei in fermented sausages.
A new method for the identification of Enterococcus species has been developed. It combines PCR a... more A new method for the identification of Enterococcus species has been developed. It combines PCR amplification of sodA gene and 16S-23S intergenic spacer region with restriction enzyme digestion followed by a melting curve analysis of the restriction fragments (MCARF). All strains analyzed were correctly identified by MCARF. This method was proved to be a reliable enterococcal identification tool.
ABSTRACT Real-time PCR-based methods have been frequently used to detect and enumerate foodborne ... more ABSTRACT Real-time PCR-based methods have been frequently used to detect and enumerate foodborne pathogens. However, these techniques have a major drawback since they cannot differentiate between DNA from live and dead cells. In this study, we developed a propidium monoazide (PMA)-based PCR method to detect and enumerate viable Salmonella cells in the presence of high number of dead cells (up to 108 CFU/g) in cooked ham. Three different specific PCR targets differing in length (95, 285, and 417 bp) were tested. We found that the inhibition effect was dependent on the PCR amplification product length, and only the longer product achieved suppression of 108 CFU/g of heat-killed cells. SYBR® Green and TaqMan® chemistries were compared to develop a highly efficient PMA-quantitative PCR system targeting the 417-bp fragment. Both chemistries showed similar detection (103 CFU/g) and quantification limits (104 CFU/g), but TaqMan® assay showed higher efficiency (98.6 %) than SYBR® Green assay (92.8 %). PMA-quantitative PCR assay developed is a rapid method for the selective detection and enumeration of viable Salmonella cells with further application in postprocessed meat products and safe shelf-life studies.
The bacterial populations of the surface layer and the centre of 15 spoiled vacuum-packed cooked ... more The bacterial populations of the surface layer and the centre of 15 spoiled vacuum-packed cooked ring sausages were characterized. About 95% of the total bacterial population in the surface layer and 55% at the centre were lactic acid bacteria. Another large bacterial group at the centre consisted of Bacillus spp. The lactic acid bacteria on the surface and at the centre were quite similar. Atypical streptobacteria, i.e. homofermentative psychrotrophic lactobacilli, were a major group of lactic acid bacteria in the surface layer of the spoiled sausages. Three main homofermentative groups could be observed on the basis of different carbohydrate patterns. Heterofermentative lactic acid bacteria belonged mainly to genus Leuconostoc. The proportion of leuconostocs in the spoiled sausages was also quite large. They could be divided into three main groups on the basis of different carbohydrate fermentation patterns. The lactic acid bacteria population of spoiled cooked ring sausages thus seemed to be heterogeneous. The strains isolated resembled strains observed by other workers in meat and meat products.
Listeria monocytogenes is a major concern for the meat processing industry because many listerios... more Listeria monocytogenes is a major concern for the meat processing industry because many listeriosis outbreaks have been linked to meat product consumption. The aim of this study was to elucidate L. monocytogenes diversity and distribution across different Spanish meat processing plants. L. monocytogenes isolates (N = 106) collected from food contact surfaces of meat processing plants and meat products were serotyped and then characterised by multilocus sequence typing (MLST). The isolates were serotyped as 1/2a (36.8%), 1/2c (34%), 1/2b (17.9%) and 4b (11.3%). MLST identified ST9 as the most predominant allelic profile (33% of isolates) followed by ST121 (16%), both of which were detected from several processing plants and meat products sampled in different years, suggesting that those STs are highly adapted to the meat processing environment. Food contact surfaces during processing were established as an important source of L. monocytogenes in meat products because the same STs were obtained in isolates recovered from surfaces and products. L. monocytogenes was recovered after cleaning and disinfection procedures in two processing plants, highlighting the importance of thorough cleaning and disinfection procedures. Epidemic clone (EC) marker ECI was identified in 8.5%, ECIII was identified in 2.8%, and ECV was identified in 7.5% of the 106 isolates. Furthermore, a selection of presumably unrelated ST9 isolates was analysed by multi-virulence-locus sequence typing (MVLST). Most ST9 isolates had the same virulence type (VT11), confirming the clonal origin of ST9 isolates; however, one ST9 isolate was assigned to a new VT (VT95). Consequently, MLST is a reliable tool for identification of contamination routes and niches in processing plants, and MVLST clearly differentiates EC strains, which both contribute to the improvement of L. monocytogenes control programs in the meat industry.
The human-derived potential probiotic strain Lactobacillus rhamnosus CTC1679 was used as a starte... more The human-derived potential probiotic strain Lactobacillus rhamnosus CTC1679 was used as a starter culture in reduced fat and sodium low-acid fermented sausages (fuets) to assess its ability to survive through the gastrointestinal tract (GIT) in a human intervention study consisting of 5 healthy volunteers who consumed 25 g fuet a day for 21 days. Faecal samples were analysed during and after consumption. L. rhamnosus CTC1679 produced a transient colonisation of the human GIT and persisted during the ingestion period of fuet containing L. rhamnosus CTC1679 at levels ca. 8log CFU/g. After 3 days of non-consumption, the strain was still recovered in the faeces of all the volunteers. To evaluate the safety of the nutritionally enhanced manufactured fuets, a challenge test was designed in a separately manufactured batch. L. rhamnosus CTC1679 was able to grow, survive and dominate (levels ca. 10(8) CFU/g) the endogenous lactic acid bacteria (LAB), prevented the growth of Listeria monocytogenes throughout the whole ripening process of the fuets and eliminated Salmonella. After 35 days of storage at 4 °C, L. monocytogenes was not detected, achieving absence in 25 g of the product. The application of high hydrostatic pressure (HHP) treatment (600 MPa for 5 min) at the end of ripening (day 14) produced an immediate reduction of L. monocytogenes to levels <1log CFU/g. After 35 days of storage at 4 °C the pathogen was not detected. Thus, the strain L. rhamnosus CTC1679 is a suitable starter culture for producing safe potentially probiotic fermented sausages.
Aim: To evaluate the biodiversity of lactobacilli from slightly fermented sausages (chorizo, fue... more Aim: To evaluate the biodiversity of lactobacilli from slightly fermented sausages (chorizo, fuet and salchichon) by molecular typing, while considering their safety aspects.Methods and Results: Species-specific PCR, plasmid profiling and randomly amplified polymorphic DNA (RAPD)-PCR were used to characterize 250 lactic acid bacteria (LAB) isolated from 21 low acid Spanish fermented sausages. Lactobacillus sakei was the predominant species (74%) followed by Lactobacillus curvatus (21·2%) and Leuconostoc mesenteroides (4·8%). By plasmid profiling and RAPD-PCR 144 different strains could be differentiated, 112 belonging to Lact. sakei, 23 to Lact. curvatus and 9 to Leuc. mesenteroides. Ion-pair high performance liquid chromatography was used to detect biogenic amine production. Tyramine and phenylethylamine were produced by 14·4 and 12·4% of the isolates, respectively, all belonging to the species Lact. curvatus. The production of tyramine was stronger than that of phenylethylamine. Partial sequencing of the tyrosine decarboxylase gene from Lact. curvatus was achieved. A specific PCR assay to detect the Lact. curvatus tyramine-producers was designed. The disc diffusion test was used to detect antibiotic resistance among the isolates. Most isolates displayed resistance to vancomycin and gentamicin. Only four strains were resistant to most of the antibiotics tested. None of the isolates were resistant to erythromycin.Conclusions: Lactobacillus sakei would be the species of choice for further use as starter culture in fermented sausage production. Strain typing and characterization of biogenic amine production together with antibiotic susceptibility testing for the selection of starter cultures could help to increase the quality and safety of the products.Significance and Impact of the Study: Species-specific PCR, RAPD and plasmid profiling proved to be efficient at typing LAB at species and strain level. Information on biogenic amine production and transferable antibiotic resistance is important in order to avoid selection of strains with undesirable properties as starter cultures.
The manufacturing of fermented sausages is subject to natural contamination processes that can po... more The manufacturing of fermented sausages is subject to natural contamination processes that can potentially carry foodborne pathogens along the process chain and result in contamination of the final product. The aim of this study was to evaluate the occurrence of Salmonella spp. and Listeria monocytogenes at different sampling points during the manufacturing process of fuet, a type of traditional fermented sausage, at 10 small-scale Spanish factories. The presence of both pathogens was studied in the raw materials (19 casings and 19 meat batters), the final products (19 fermented sausages), and the factory equipment (12 mincing, 12 mixing, and 19 stuffing machines, 19 cutting tables, 11 knives, and 12 cold rooms) by using classical microbiological techniques and real-time PCR. Salmonella was not detected in the equipment analyzed or in the final products, but it was detected in the raw materials (23.7% of samples). L. monocytogenes showed higher incidence than Salmonella and was detected in the equipment (11.8% of samples), the raw materials (28.9%), and the final products (15.8%), confirming its ubiquity throughout the manufacturing process of fermented sausages. Five factories were further investigated to study the changes in the distribution of pathogens in the fuet production process over a period of either 2 or 3 years. There was considerable variation in the incidence of both pathogens at different sampling periods, and there was no relation between seasonal variations or geographic location of the factories.
Detection of six species of lactic acid bacteria and six species of gram-positive catalase-positi... more 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
Aims: To determine the biodiversity of enterococci from slightly fermented sausages (chorizo and... more Aims: To determine the biodiversity of enterococci from slightly fermented sausages (chorizo and fuet) at species and strain level by molecular typing, while considering their safety aspects.Methods and Results: Species-specific PCR and partial sequencing of 16S rRNA and sodA genes were used to identify enterococcal population. Enterococcus faecium was the most frequently isolated species followed by E. faecalis, E. hirae and E. durans. Randomly amplified polymorphic DNA (RAPD)-PCR revealed species-specific clusters and allowed strain typing. Sixty strains of 106 isolates exhibited different RAPD profiles indicating a high genetic variability. All the E. faecalis strains carried virulence genes (efaAfs, esp, agg and gelE) and all E. faecium isolates carried efaAfm gene. Enterococcus faecalis showed higher antibiotic resistance than the other species. Only one E. faecium strain showed vanA genotype (high-level resistance to glycopeptides) and E. gallinarum and E. casseliflavus/flavescens isolates showed vanC1 and vanC2/C3 genotypes (low-level resistance only to vancomycin) respectively.Conclusions: E. faecalis has been mainly associated with virulence factors and antimicrobial multi-resistance and, although potential risk for human health is low, the presence of this species in slightly fermented sausages should be avoided to obtain high quality products.Significance and Impact of the Study: The enterococcal population of slightly fermented sausages has been thoroughly characterized. Several relevant safety aspects have been revealed.
Three bacteriocinogenic, non-aminogenic and non-virulent Enterococcus strains (Enterococcus faeci... more Three bacteriocinogenic, non-aminogenic and non-virulent Enterococcus strains (Enterococcus faecium CTC8005, Enterococcus devriesei CTC8006 and Enterococcus casseliflavus CTC8003) were used as starter cultures in low-acid fermented sausages to assess their competitiveness and their bioprotective potential against Listeria monocytogenes and Staphylococcus aureus. The inoculated strains were successfully monitored by RAPD-PCR. All the strains were able to grow, survive and dominate the endogenous enterococci population and avoided the growth of Enterobacteriaceae. E. devriesei CTC8006 and E. faecium CTC8005 particularly inhibited the growth of L. monocytogenes during the whole ripening process. S. aureus was not affected by the inoculated bacteriocinogenic enterococci strains.
ABSTRACT The aim of this study was to compare the performance of different pre-PCR treatments for... more ABSTRACT The aim of this study was to compare the performance of different pre-PCR treatments for the detection of Listeria monocytogenes and Salmonella spp. by real-time PCR in spiked ready-to-eat meat samples (cooked ham, dry-cured ham and fermented sausage). Three different pre-PCR treatments were assayed: (i) PrepMan® Ultra Sample Preparation Reagent, (ii) DNA purification using DNeasy Tissue Kit and QIAcube automated sample preparation system, and (iii) BAX® system. The analysis range was 2 to 2 × 106 CFU/ml. PCR was done in quintuplicate and the experiment was repeated five times. The ‘probability of detection’ was used to compare the efficiency of the pre-PCR treatments and to calculate the theoretical limit of detection of each treatment.The best pre-PCR treatment was DNA purification using DNeasy Tissue kit and QIAcube; it showed the highest detection probability and the lowest limit of detection for each food type and pathogen assayed, followed by PrepMan and BAX treatments. Probability of detection showed differences among food matrices and between L. monocytogenes and Salmonella detection probability. Despite being an expensive method, DNeasy Tissue kit would be the recommended treatment to reduce the risk of false negative results and to improve detection of foodborne pathogens in RTE-meat products by real-time PCR.
A quick and simple method for quantitative detection of Lactobacillus sakei in fermented sausages... more A quick and simple method for quantitative detection of Lactobacillus sakei in fermented sausages was successfully developed. It is based on Chelex-100-based DNA purification and real-time PCR enumeration using a TaqMan fluorescence probe. Primers and probes were designed in the L. sakei 16S-23S rRNA intergenic transcribed spacer region, and the assay was evaluated using L. sakei genomic DNA and an artificially inoculated sausage model. The detection limit of this technique was approximately 3 cells per reaction mixture using both purified DNA and the inoculated sausage model. The quantification limit was established at 30 cells per reaction mixture in both models. The assay was then applied to enumerate L. sakei in real samples, and the results were compared to the MRS agar count method followed by confirmation of the percentage of L. sakei colonies. The results obtained by real-time PCR were not statistically significantly different than those obtained by plate count on MRS agar (P > 0.05), showing a satisfactory agreement between both methods. Therefore, the real-time PCR assay developed can be considered a promising rapid alternative method for the quantification of L. sakei and evaluation of the implantation of starter strains of L. sakei in fermented sausages.
A new method for the identification of Enterococcus species has been developed. It combines PCR a... more A new method for the identification of Enterococcus species has been developed. It combines PCR amplification of sodA gene and 16S-23S intergenic spacer region with restriction enzyme digestion followed by a melting curve analysis of the restriction fragments (MCARF). All strains analyzed were correctly identified by MCARF. This method was proved to be a reliable enterococcal identification tool.
ABSTRACT Real-time PCR-based methods have been frequently used to detect and enumerate foodborne ... more ABSTRACT Real-time PCR-based methods have been frequently used to detect and enumerate foodborne pathogens. However, these techniques have a major drawback since they cannot differentiate between DNA from live and dead cells. In this study, we developed a propidium monoazide (PMA)-based PCR method to detect and enumerate viable Salmonella cells in the presence of high number of dead cells (up to 108 CFU/g) in cooked ham. Three different specific PCR targets differing in length (95, 285, and 417 bp) were tested. We found that the inhibition effect was dependent on the PCR amplification product length, and only the longer product achieved suppression of 108 CFU/g of heat-killed cells. SYBR® Green and TaqMan® chemistries were compared to develop a highly efficient PMA-quantitative PCR system targeting the 417-bp fragment. Both chemistries showed similar detection (103 CFU/g) and quantification limits (104 CFU/g), but TaqMan® assay showed higher efficiency (98.6 %) than SYBR® Green assay (92.8 %). PMA-quantitative PCR assay developed is a rapid method for the selective detection and enumeration of viable Salmonella cells with further application in postprocessed meat products and safe shelf-life studies.
The bacterial populations of the surface layer and the centre of 15 spoiled vacuum-packed cooked ... more The bacterial populations of the surface layer and the centre of 15 spoiled vacuum-packed cooked ring sausages were characterized. About 95% of the total bacterial population in the surface layer and 55% at the centre were lactic acid bacteria. Another large bacterial group at the centre consisted of Bacillus spp. The lactic acid bacteria on the surface and at the centre were quite similar. Atypical streptobacteria, i.e. homofermentative psychrotrophic lactobacilli, were a major group of lactic acid bacteria in the surface layer of the spoiled sausages. Three main homofermentative groups could be observed on the basis of different carbohydrate patterns. Heterofermentative lactic acid bacteria belonged mainly to genus Leuconostoc. The proportion of leuconostocs in the spoiled sausages was also quite large. They could be divided into three main groups on the basis of different carbohydrate fermentation patterns. The lactic acid bacteria population of spoiled cooked ring sausages thus seemed to be heterogeneous. The strains isolated resembled strains observed by other workers in meat and meat products.
Listeria monocytogenes is a major concern for the meat processing industry because many listerios... more Listeria monocytogenes is a major concern for the meat processing industry because many listeriosis outbreaks have been linked to meat product consumption. The aim of this study was to elucidate L. monocytogenes diversity and distribution across different Spanish meat processing plants. L. monocytogenes isolates (N = 106) collected from food contact surfaces of meat processing plants and meat products were serotyped and then characterised by multilocus sequence typing (MLST). The isolates were serotyped as 1/2a (36.8%), 1/2c (34%), 1/2b (17.9%) and 4b (11.3%). MLST identified ST9 as the most predominant allelic profile (33% of isolates) followed by ST121 (16%), both of which were detected from several processing plants and meat products sampled in different years, suggesting that those STs are highly adapted to the meat processing environment. Food contact surfaces during processing were established as an important source of L. monocytogenes in meat products because the same STs were obtained in isolates recovered from surfaces and products. L. monocytogenes was recovered after cleaning and disinfection procedures in two processing plants, highlighting the importance of thorough cleaning and disinfection procedures. Epidemic clone (EC) marker ECI was identified in 8.5%, ECIII was identified in 2.8%, and ECV was identified in 7.5% of the 106 isolates. Furthermore, a selection of presumably unrelated ST9 isolates was analysed by multi-virulence-locus sequence typing (MVLST). Most ST9 isolates had the same virulence type (VT11), confirming the clonal origin of ST9 isolates; however, one ST9 isolate was assigned to a new VT (VT95). Consequently, MLST is a reliable tool for identification of contamination routes and niches in processing plants, and MVLST clearly differentiates EC strains, which both contribute to the improvement of L. monocytogenes control programs in the meat industry.
The human-derived potential probiotic strain Lactobacillus rhamnosus CTC1679 was used as a starte... more The human-derived potential probiotic strain Lactobacillus rhamnosus CTC1679 was used as a starter culture in reduced fat and sodium low-acid fermented sausages (fuets) to assess its ability to survive through the gastrointestinal tract (GIT) in a human intervention study consisting of 5 healthy volunteers who consumed 25 g fuet a day for 21 days. Faecal samples were analysed during and after consumption. L. rhamnosus CTC1679 produced a transient colonisation of the human GIT and persisted during the ingestion period of fuet containing L. rhamnosus CTC1679 at levels ca. 8log CFU/g. After 3 days of non-consumption, the strain was still recovered in the faeces of all the volunteers. To evaluate the safety of the nutritionally enhanced manufactured fuets, a challenge test was designed in a separately manufactured batch. L. rhamnosus CTC1679 was able to grow, survive and dominate (levels ca. 10(8) CFU/g) the endogenous lactic acid bacteria (LAB), prevented the growth of Listeria monocytogenes throughout the whole ripening process of the fuets and eliminated Salmonella. After 35 days of storage at 4 °C, L. monocytogenes was not detected, achieving absence in 25 g of the product. The application of high hydrostatic pressure (HHP) treatment (600 MPa for 5 min) at the end of ripening (day 14) produced an immediate reduction of L. monocytogenes to levels <1log CFU/g. After 35 days of storage at 4 °C the pathogen was not detected. Thus, the strain L. rhamnosus CTC1679 is a suitable starter culture for producing safe potentially probiotic fermented sausages.
Aim: To evaluate the biodiversity of lactobacilli from slightly fermented sausages (chorizo, fue... more Aim: To evaluate the biodiversity of lactobacilli from slightly fermented sausages (chorizo, fuet and salchichon) by molecular typing, while considering their safety aspects.Methods and Results: Species-specific PCR, plasmid profiling and randomly amplified polymorphic DNA (RAPD)-PCR were used to characterize 250 lactic acid bacteria (LAB) isolated from 21 low acid Spanish fermented sausages. Lactobacillus sakei was the predominant species (74%) followed by Lactobacillus curvatus (21·2%) and Leuconostoc mesenteroides (4·8%). By plasmid profiling and RAPD-PCR 144 different strains could be differentiated, 112 belonging to Lact. sakei, 23 to Lact. curvatus and 9 to Leuc. mesenteroides. Ion-pair high performance liquid chromatography was used to detect biogenic amine production. Tyramine and phenylethylamine were produced by 14·4 and 12·4% of the isolates, respectively, all belonging to the species Lact. curvatus. The production of tyramine was stronger than that of phenylethylamine. Partial sequencing of the tyrosine decarboxylase gene from Lact. curvatus was achieved. A specific PCR assay to detect the Lact. curvatus tyramine-producers was designed. The disc diffusion test was used to detect antibiotic resistance among the isolates. Most isolates displayed resistance to vancomycin and gentamicin. Only four strains were resistant to most of the antibiotics tested. None of the isolates were resistant to erythromycin.Conclusions: Lactobacillus sakei would be the species of choice for further use as starter culture in fermented sausage production. Strain typing and characterization of biogenic amine production together with antibiotic susceptibility testing for the selection of starter cultures could help to increase the quality and safety of the products.Significance and Impact of the Study: Species-specific PCR, RAPD and plasmid profiling proved to be efficient at typing LAB at species and strain level. Information on biogenic amine production and transferable antibiotic resistance is important in order to avoid selection of strains with undesirable properties as starter cultures.
The manufacturing of fermented sausages is subject to natural contamination processes that can po... more The manufacturing of fermented sausages is subject to natural contamination processes that can potentially carry foodborne pathogens along the process chain and result in contamination of the final product. The aim of this study was to evaluate the occurrence of Salmonella spp. and Listeria monocytogenes at different sampling points during the manufacturing process of fuet, a type of traditional fermented sausage, at 10 small-scale Spanish factories. The presence of both pathogens was studied in the raw materials (19 casings and 19 meat batters), the final products (19 fermented sausages), and the factory equipment (12 mincing, 12 mixing, and 19 stuffing machines, 19 cutting tables, 11 knives, and 12 cold rooms) by using classical microbiological techniques and real-time PCR. Salmonella was not detected in the equipment analyzed or in the final products, but it was detected in the raw materials (23.7% of samples). L. monocytogenes showed higher incidence than Salmonella and was detected in the equipment (11.8% of samples), the raw materials (28.9%), and the final products (15.8%), confirming its ubiquity throughout the manufacturing process of fermented sausages. Five factories were further investigated to study the changes in the distribution of pathogens in the fuet production process over a period of either 2 or 3 years. There was considerable variation in the incidence of both pathogens at different sampling periods, and there was no relation between seasonal variations or geographic location of the factories.
Detection of six species of lactic acid bacteria and six species of gram-positive catalase-positi... more 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
Aims: To determine the biodiversity of enterococci from slightly fermented sausages (chorizo and... more Aims: To determine the biodiversity of enterococci from slightly fermented sausages (chorizo and fuet) at species and strain level by molecular typing, while considering their safety aspects.Methods and Results: Species-specific PCR and partial sequencing of 16S rRNA and sodA genes were used to identify enterococcal population. Enterococcus faecium was the most frequently isolated species followed by E. faecalis, E. hirae and E. durans. Randomly amplified polymorphic DNA (RAPD)-PCR revealed species-specific clusters and allowed strain typing. Sixty strains of 106 isolates exhibited different RAPD profiles indicating a high genetic variability. All the E. faecalis strains carried virulence genes (efaAfs, esp, agg and gelE) and all E. faecium isolates carried efaAfm gene. Enterococcus faecalis showed higher antibiotic resistance than the other species. Only one E. faecium strain showed vanA genotype (high-level resistance to glycopeptides) and E. gallinarum and E. casseliflavus/flavescens isolates showed vanC1 and vanC2/C3 genotypes (low-level resistance only to vancomycin) respectively.Conclusions: E. faecalis has been mainly associated with virulence factors and antimicrobial multi-resistance and, although potential risk for human health is low, the presence of this species in slightly fermented sausages should be avoided to obtain high quality products.Significance and Impact of the Study: The enterococcal population of slightly fermented sausages has been thoroughly characterized. Several relevant safety aspects have been revealed.
Three bacteriocinogenic, non-aminogenic and non-virulent Enterococcus strains (Enterococcus faeci... more Three bacteriocinogenic, non-aminogenic and non-virulent Enterococcus strains (Enterococcus faecium CTC8005, Enterococcus devriesei CTC8006 and Enterococcus casseliflavus CTC8003) were used as starter cultures in low-acid fermented sausages to assess their competitiveness and their bioprotective potential against Listeria monocytogenes and Staphylococcus aureus. The inoculated strains were successfully monitored by RAPD-PCR. All the strains were able to grow, survive and dominate the endogenous enterococci population and avoided the growth of Enterobacteriaceae. E. devriesei CTC8006 and E. faecium CTC8005 particularly inhibited the growth of L. monocytogenes during the whole ripening process. S. aureus was not affected by the inoculated bacteriocinogenic enterococci strains.
ABSTRACT The aim of this study was to compare the performance of different pre-PCR treatments for... more ABSTRACT The aim of this study was to compare the performance of different pre-PCR treatments for the detection of Listeria monocytogenes and Salmonella spp. by real-time PCR in spiked ready-to-eat meat samples (cooked ham, dry-cured ham and fermented sausage). Three different pre-PCR treatments were assayed: (i) PrepMan® Ultra Sample Preparation Reagent, (ii) DNA purification using DNeasy Tissue Kit and QIAcube automated sample preparation system, and (iii) BAX® system. The analysis range was 2 to 2 × 106 CFU/ml. PCR was done in quintuplicate and the experiment was repeated five times. The ‘probability of detection’ was used to compare the efficiency of the pre-PCR treatments and to calculate the theoretical limit of detection of each treatment.The best pre-PCR treatment was DNA purification using DNeasy Tissue kit and QIAcube; it showed the highest detection probability and the lowest limit of detection for each food type and pathogen assayed, followed by PrepMan and BAX treatments. Probability of detection showed differences among food matrices and between L. monocytogenes and Salmonella detection probability. Despite being an expensive method, DNeasy Tissue kit would be the recommended treatment to reduce the risk of false negative results and to improve detection of foodborne pathogens in RTE-meat products by real-time PCR.
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